The ability to direct cell behavior has been central to the success of numerous therapeutics to regenerate tissue or facilitate device integration. Collagen often serves as a design basis for bioactive materials due to its putative role in regulating cell adhesion and phenotype, which occurs in part through α1β1 and α2β1 integrin adhesion signals it presents to cells. These integrins are involved in an array of cell activities including angiogenesis, cell migration, adhesion, and proliferation. However, all collagen-containing products on the market today utilize materials from slaughterhouses with the associated disadvantages of animal-derived materials (e.g. disease risks, purification concerns, and batch-to-batch heterogeneity). In addition, there is no means to optimize the molecular composition of the collagen to guide regeneration. We propose to circumvent these limitations by generating novel bioactive materials using a collagen-mimetic protein engineered to have enhanced therapeutic action and improved scale-up potential. Initial sequence design was based on the collagen-like protein, Scl2 in Streptococcus pyogenes. Whereas native collagen has numerous binding sites for integrins present on a wide range of cells, the Scl2 protein acts as a biological blank slate that only displays the selected receptor-binding sequences programmed in by site-directed mutagenesis. We used site directed mutagenesis to introduce human integrin binding sites into this protein and have provided evidence that human integrin binding sites function within the engineered protein bind and activate α1β1/α2β1. This “Designer Collagen” has the following advantages: 1) it is a triple helical protein, 2) allows the introduction of multiple and specific biological cues, 3) is produced with consistent batch-to-batch properties, 4) is relatively resistant to enzymatic degradation, 5) is suitable for large-scale purification, and 6) is non-cytotoxic and non-immunogenic. To generate robust materials based on this technology, the collagen-mimetic protein was conjugated into a poly(ethylene glycol) (PEG) based hydrogel to generate bioactive hydrogels. This platform technology is currently being explored in several tissue engineering applications including chronic wound dressings and vascular grafts. It also provides a unique opportunity to investigate the contribution of collagen binding integrins in a variety of regenerative processes and disease pathogenesis.

Laser processing of hydrogels and other biomaterials enable biomedical researchers and engineers to fabricate and rapidly iterate through complex microenvironments with different inherent mechanical cues. Bio-compatible/bio-reactive gels have a range of differing properties that affect their ablation, such as coefficient of thermal expansion, hydration and Young&’s modulus. This necessitates a system for generating laser paths that takes into account these differences to produce statistically similar shapes in different materials. Laser path generation is further complicated when constructs require high depth to lateral feature aspect ratios and uniform ablation for precision channel depths.
To quickly generate laser path routines, we have created user-friendly stand-alone computer software, STEVE (simulated tool-path evaluation and visualization environment), which enables users, with no computational knowledge, to design and fabricate reproducible and disposable smart-gel constructs for research applications. The user selects 2D shape profiles from a preexisting library or creates a new 2D profile via CAD and specifies the depth and surface features in different regions. After the construct is digitally defined, the user specifies the gel composition and hydration and laser paths are iteratively generated and evaluated, incorporating properties of the specified material.
This software relies on physical simulation of laser-gel interaction and ablation for a library of popular bio-compatible/bio-reactive gels of varying composition and hydration. The ablation simulations have been experimentally confirmed by the implementation of an automated test pattern system and subsequently imaging the results. Test patterns were imaged with a confocal microscope and an optical profilometer and the surface roughness was measured using a bio-AFM and an optical profilometer.
Simulation was done in two regimes: ablation from a single laser pulse and the effects of 200 Hz laser processing. In order to iteratively generate laser paths we optimizes high frequency laser processing simulation, linearized the system and implemented finite element method.
Using the STEVE platform and accurate laser-gel interaction modeling, has allowed for the automatic generation of laser tool-paths to create “sunburst” and “horse race” microenvironment constructs in Agarose, Geletin and Cross-Linked Gelatin. The “sunburst” and “horse-race” constructs are being used for pluripotent stem cell differentiation and migration studies. The machined sunburst is a central circle (260 mu;m radius, 580 mu;m depth) with six protruding channels (10 / 20 mu;m wide, 630 mu;m depth) and the horse-race pattern is an asymmetric barbell repeated six times (1000 mu;m lateral separation). The constructs&’ surface roughness was measured using an optical profilometer and bio-AFM.

Cryogels are micro- to macroporous hydrogels where the pore space, filled with fluid, is that occupied by ice crystals during the fabrication at sub-zero temperatures. The large pore size makes them ideal candidates as tissue engineering scaffolds, as it provides room for the accommodation of cells while presenting them with a biocompatible, extracellular matrix-mimicking substrate. As a consequence of their gel-liquid structure, their mechanical response becomes important at two scales: at the macro-scale where the material should possess similar properties to that of the target tissue and be suitable for handling; at the micro- to nanoscale where it can affect the attachment and consequent diversification of the seeded cells.
Smart cryogels can be made to respond to an external stimulus, such as a change in temperature or pH, by swelling or de-swelling. The effect of a change in bulk volume on the mechanical properties of the gels is the object of the present study. Smart, pH responsive cryogels made from a polyvinyl alcohol-polyacrylic acid blend were utilized: their morphology as a function of swelling state was observed by confocal microscopy. The materials were tested mechanically through multi-scale indentation in a poroelastic framework, in order to probe the properties of the gels at the bulk scale, as well as at the scale of single cells. All results were compared to those for continuous gels made from the same polymers.
We show that contrary to conventional gels, where the modulus of elasticity is reduced with swelling, the stiffness of cryogels can increase in the swollen state. This unconventional phenomenon is explained as a result of gel fraction and nanoscale intrinsic properties of the gel making up the porous structure. It was observed that it depends on the concentration of the gels investigated, and is expected to be found in other two-phase materials. The results presented picture these materials as cellular scaffolds with enhanced mechanical properties, showing a larger modulus of elasticity compared to continuous gels with the same polymer content, while maintaining a microporous environment favorable for cell seeding.

Several natural materials, like bone and seashell, represent an optimal compromise between their properties, such as durability, mechanical properties, degradability, etc. A key feature is the combination of organic and inorganic components in such a way that they form hierarchical structures with carefully engineered interfaces [1]. Since few materials possess individually all the features required for their application, researchers, basing on “biomimetic” approaches, try to mimic nature and synthesise smart materials given by the combination of different components where the interactions between the domains of the dissimilar phases are reduced to the nanometer scale. These co-networks of inorganic-organic components are called “hybrids”. The challenge is to synthesise via sol-gel Class II hybrids, which also contain covalent bonds between SiO₂ and polymeric chains [2]. These represent new materials where the two components act as a single phase, enhancing the toughness of inherently brittle bioactive glass scaffolds used in the substitution of the osteochondral tissue. Polycaprolactone (PCL) is a synthetic polyester widely used for biomedical applications because of its biocompatibility and biodegradability. However, since it lacks functional groups and has very low solubility, PCL needs to be functionalised with a coupling agent to be covalently bonded to silica.
Herein new Class II silica/PCL hybrids using glycidyloxypropyltrimethoxysilane (GPTMS) as coupling agent were developed by introducing GPTMS-functionalised PCL into the traditional sol-gel process using tetraethylorthosilicate (TEOS) as silica source. The covalent coupling was confirmed through solid-state NMR. Bulk samples (cylinders or thin disks) in a wide range of inorganic/organic ratio (2- 65% SiO₂) were produced. Thanks to the interpenetration of PCL chains within silica network, the hybrids showed high flexibility with an elastomeric behavior in compression and in tension, more evident at higher PCL contents: for instance, samples containing 20% SiO₂ were compressed to more than 30% of their initial height and they were able to recover the deformation when the load was released. The fast gelation due to the covalent bonding with GPTMS allowed the tuning of the viscosity of the forming gel in order to print 3D-porous structures directly from the sol. 3D-printed scaffolds, characterised with micro-CT, still maintained great flexibility (i.e. scaffolds containing 30% SiO₂ achieved 1.8 MPa in stress at 25% of strain). Hydrophobic nature of PCL prevented the hybrids from swelling in wet environment and good cell response was observed. The great versatility of SiO₂/PCL hybrid compositions combined with the processing via 3D-printing, that allows the tailoring of shape, porosity and dimensions of the final scaffold, leads to a material with great potential in tissue regeneration.
[1] C. Sanchez et al., Chem. Soc. Rev., 2011, 40, 696-753
[2] B.M. Novak, Adv. Mater., 1993, 5, 422-33

Cardiac and skeletal muscle tissues represent two structurally similar, yet functionally very different types of striated muscles. In this presentation, I will describe our recent studies with engineering of highly functional 3D cardiac and skeletal muscle tissues made using a mixture of fibrin-based hydrogel matrix and primary human muscle cells or human cardiomyocytes derived from pluripotent stem cells. These studies collectively demonstrate that specific combinations of hydrogel composition, dynamic biochemical culture environment, boundary conditions imposed on cells, and supporting non-myogenic cell types can uniquely advance structural and electromechanical maturation of engineered human striated muscles to a level approaching that of adult native tissues. Such maturation levels can be achieved without the use of exogenous biophysical or growth factor stimulation, signifying the potential for commercialization and translation. I will further present examples of how these in vitro tissue-engineered systems can be utilized for physiological and pharmacological studies and modeling of human disease.

The native extracellular environment constantly undergoes cycles of stiffening and softening to control cell fate during cases of disease, wound healing, and development. Although traditional cell culture substrates have static moduli, recapitulating these dynamic matrix cues would enable better in vitro models and potentially better scaffolds for regenerative medicine. Furthermore, these changes in substrate stiffness should be in situ, reversible, and non-invasive to best probe their effect on cell behavior. Current dynamic matrices often rely on stimuli that introduce altered charge or redox state of the cellular microenvironment, which can lead to unintended changes in phenotype. To address these issues and provide a complementary system, we present a strategy to reversibly control extracellular matrix stiffness using photoresponsive azobenzene-containing hydrogels.
We show that azobenzene provides a mechanism to control crosslinking density - and therefore modulus - with light in both covalently bound poly(ethylene glycol) (PEG) hydrogels and in supramolecularly-assembled hyaluronic-acid (HA) hydrogels. By irradiation with either 365 nm or 405 nm light (both 10 mW/cm² for 5 min), we modulate the isomeric structure of the azobenzene from cis to trans, respectively. First, in the covalent gels, isomerization leads to a conformational change in the crosslinker and an overall softening of the hydrogel that depends on the amount of azobenzene incorporated. The azobenzene unit was chosen carefully to maximize the lifetime of the cis state (half-life of 9 hours at 37°C or 162 hours at room temperature), and when the stimulus is removed, there is minimal change in the modulus of the hydrogel. Second, in the supramolecular gels, azobenzene functions as a guest in a cyclodextrin complex that is dissociated upon isomerization, which decreases hydrogel modulus. Due to the dynamic nature of these associative crosslinks, the modulus exhibits viscoelastic behavior, and the magnitude of the modulus change upon isomerization depends on the relative elasticity of the hydrogel. Using this platform, the modulus can be switched by up to 2-fold in a biologically relevant range (starting modulus between 100 Pa - 1000 Pa). Encapsulated cells demonstrate high viability (85% - 95%) in these hydrogels after three days. Due to the tunability and non-invasive properties of the stimulus, these innovative materials should be broadly applicable to examining the effect of modulus changes on many different cell functions and cell types.

Poly(ε- caprolactone) (PCL) is a biodegradable, biocompatible polymer that has been proven suitable for a number of biomedical applications ; PCL is frequently used for tissue engineering scaffolds, absorbable sutures, contraceptive devices, and long-term drug delivery systems. PCL formed into fibers of the micro- or nano-scale has been found advantageous to other PCL formations for the mentioned applications. These PCL nanofibers are commonly produced by various electrospinning techniques using a suitable PCL/solvent system.
Most previous studies on PCL electrospun nanofibers utilized high-voltage direct current electrospinning and organic solvents with varied levels of toxicity. Alternatively, acetic acid was proposed as a moderately good, “green” PCL solvent due to its chemical stability and because it can be removed from the fibers without leaving potentially harmful residues.
Good crystallinity and improved mechanical strength of PCL fibers was achieved for this polymer/solvent system. However, the reliable electrospinning of PCL/acetic acid solution required PCL concentrations above 15% and resulted in fiber diameters larger than 1 micrometer.
In this study, PCL nanofibrous materials with fiber diameters in the range from 100 nm to 2 micrometers were prepared for the first time from PCL/acetic acid solutions with polymer concentrations in the range of 5% to 20% w/v by using an uncommon high-voltage alternating current electrospinning method. This method has recently emerged as an effective approach to significantly increase efficiency of the nanofiber manufacturing process. Increase of up to 3 orders of magnitude in the nanofiber generation rates has been observed.
The PCL nanofibers in this study were investigated using SEM, FTIR, and X-Ray Diffraction to establish the relationships between the fiber structure and morphology, the rheological properties of PCL/acetic acid solution, and the AC-electrospinning process parameters. The results were compared with the PCL materials prepared by common dc-electrospinning and centrifugal spinning methods. It has been found that PCL nanofibrous materials produced by ac-electrospinning seem to exhibit better controlled structures and new morphological features, such as aligned fiber bundles. The potential of the developed approach for PCL nanofibrous tissue engineering scaffolds applications has been discussed.
This work was supported in part by the National Science Foundation award OISE-1261154.

The role of surgically implanted biomedical devices in modern medicine is rapidly expanding but their efficacy is often compromised by host recognition and subsequent foreign body responses. Herein, we demonstrate that by tuning the geometry of implanted materials we can reduce their host recognition. In rodent and non-human primate animal models, materials prepared as spheres 1.5 mm and above in diameter significantly abrogated foreign body reactions and fibrosis when compared to their non-spherical or smaller-sized spherical counterparts. Remarkably, these findings translated across a broad spectrum of materials including hydrogels, ceramics, metals, and plastics. To highlight the implications of these findings we studied the effect of hydrogel capsule geometry on the survival of encapsulated pancreatic islet cells. In a xenogeneic treatment model of transplanting encapsulated rat islets into streptozocin (STZ) induced diabetic C57BL/6 mice, islets prepared in 1.5 mm alginate capsules were able restore blood glucose control in diabetic mice for up to 180 days, a great than 5-fold longer duration than transplanted grafts encapsulated within conventionally sized 0.5 mm diameter alginate capsules. Our study of probing immune cells recruited to implanted materials implicates the limited activation of macrophages, a key driver of host recognition, as central to these truncated foreign body responses. Combined these findings suggest that the simple tuning of the size and geometry of biomedical devices can significantly improve their in vivo biocompatibility.

Bioactive glass is known to be more bioactive than other bioceramics[1]. Although bioactive glass scaffolds were made with porous structure and compressive strength that resembles bone, they were quite brittle for the sites under cyclic loading[2]. Organic-inorganic class II hybrids are of great interest because their molecular level co-networks of SiO₂ and organic polymer can provide synergetic mechanical properties while displaying a congruent biodegradation[3]. Methacrylate based hybrids, such as copolymers of methyl methacrylate (MMA) and 3-(trimethoxysilyl)propyl methacrylate (TMSPMA) have been investigated in bone scaffold materials due to their self-hardening mechanical property and bioactivity. However, past studies have not examined well-defined polymer architectures in the hybrid system.
In this study, reversible addition-fragmentation chain transfer (RAFT) technique was used to synthesize P(MMA-co-TMSPMA). Linear, randomly branched, and star architecture copolymers were synthesized in a comparable molecular weight, and they were made into class II hybrids via sol-gel process. The star copolymer had the largest hydrodynamic radius which resulted in modulus of toughness 9.62 fold greater than 70S30C bioactive glass, and Young&’s modulus 1.6 fold lower than the other hybrids. Additionally, all the hybrids passed ISO standards for cytotoxicity, and pre-osteoblast cells (MC3T3) were able to adhere on the surface. Introducing star polymers to organic-inorganic hybrid could give a higher tailorability in the mechanical properties towards bone scaffold materials.
[1]Oonishi, H.; Hench, L. L.; Wilson, J.; Sugihara, F.; Tsuji, E.; Kushitani, S.; Iwaki, H., Comparative bone growth behavior in granules of bioceramic materials of various sizes. J Biomed Mater Res 1999,44 (1), 31-43.
[2]Jones, J. R.; Ehrenfried, L. M.; Hench, L. L., Optimising bioactive glass scaffolds for bone tissue engineering. Biomaterials 2006,27 (7), 964-973.
[3]Jones, J. R., Review of bioactive glass: From Hench to hybrids. Acta Biomaterialia 2013,9 (1), 4457-4486.

PLGA-PEG-PLGA (poly (D,L-lactic acid-co-glycolic acid)-b-poly (ethylene glycol)-b-poly (D,L-lactic acid-co-glycolic acid)) is one of the PEG-derived thermoresponsive block copolymers whose aqueous solution transfers from sol to gel by increasing temperature. In the previous studies, it was found that the PLGA-PEG-PLGA with the PLGA/PEG block ratio of R=1.8 or lower did not form a hydrogel. In fact, for the maximum utilization of highly biocompatible PEG, the ratio R should be kept low. Hence to develop a hydrogel composed of PLGA-PEG-PLGA with low R, our group suggested an aqueous nanocomposite solution of PLGA-PEG-PLGA with synthetic clay in the form of nano-disk (laponite).
In this study, we investigated the thermoresponsive sol-gel transition behavior and the mechanical properties of aqueous laponite/PLGA-PEG-PLGA solution. We synthesized four different PLGA-PEG-PLGAs with different low R<1.3. The block molecular weights of each polymer were 0.5k-1.0k-0.5k (R=1.0), 0.8k-1.5k-0.8k (R=1.1), 1.8k-3.0k-1.8k (R=1.2), and 3.0k-6.0k-3.0k (R=1.0). It was found that every prepared aqueous laponite/PLGA-PEG-PLGA solution with different R immediately transferred from sol to gel in response to the temperature regardless of the molecular weight. Moreover all the sol-gel transition occurred by adding laponite even below the physiological temperature of 370C.
The rheological characterization of the solution was carried out by the dynamic mechanical analysis (DMA). It was found that the storage and the loss moduli largely depended on the PLGA-PEG-PLGA molecular weight and the nanocomposite concentration. The storage modulus of laponite/PLGA_0.5k-PEG_1.0k-PLGA_0.5k hydrogel could reach the value of 600 Pa, which is an expedient modulus value for general biomedical applications, while that of laponite/PLGA_1.8k-PEG_3.0k-PLGA_1.8k hydrogel showed only 170 Pa. The DMA results confirmed that the PLGA-PEG-PLGAs with shorter PEG in the nanocomposite hydrogel with laponite possessed higher viscoelastic properties. The cryo-transmission electron microscopy (cryo-TEM) and the small angle X-ray scattering (SAXS) were carried out for the microstructural analysis. The results revealed the highly versatile gel feature of the aqueous laponite/PLGA-PEG-PLGA with the prospective thermoresponsive sol-gel transition.

Tendons are connective tissues whose main function is force transmission between muscles and bones, allowing body movement. Thus, tendon injuries lead to substantial morbidity, pain and disability, affecting not only athletes, but also active working people and elder population. Tissue engineering efforts have been focused on translating tendon architecture and functionality into biomimetic materials. The present study aims at developing microengineered hydrogel fibers by combining microfabrication techniques with polyelectrolyte interactions. For this purpose, methacrylated hyaluronic acid (MeHA) and chondroitin sulfate (MeCS), which are negatively charged, were combined with chitosan (CHT) that is positively charged. Hydrogel fibers were obtained by injecting polymer solutions (either MeHA or MeHA/MeCS and CHT) in separate microchannels that join at a y-junction, with the materials interacting upon contact at the interface. Hydrogel fibers were photocrosslinked by UV light and then extracted. Morphological analysis by scanning electron microscopy showed alignment of the developed microfibers, resembling the hierarchical structure of tendon. Cell behavior was evaluated through encapsulation of human tendon derived cells (hTDCs) obtained from surplus samples during orthopedic surgeries. hTDCs were homogeneously distributed along the fibers, remaining viable after 7 days in culture, and were able to produce extracellular matrix components found in tendon tissue, such as collagen type 1 and tenascin.
The combination of microfabrication technology with both physical and chemical crosslinking mechanisms allowed the development of biomimetic structures with parallel fibers being formed towards the replication of tendon tissue architecture that enable a proper functionality of tendon derived cells, evidencing their potential in tendon tissue engineering applications.
Acknowledgements
The authors would like to thank Portuguese funds through FCT - Fundaccedil;atilde;o para a Ciecirc;ncia e a Tecnologia in the framework of FCT-POPH-FSE, the PhD grant SFRH/BD/96593/2013 of R.C-A, to the project RL3 -TECT -NORTE-07-0124-FEDER-000020 cofinanced by ON.2 under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF) and the European Research Council for the project ComplexiTE grant agreement ERC-2012-ADG 20120216-321266.

A key tenet of bone tissue engineering is the development of scaffold materials that can support cell growth and stimulate stem cell differentiation for bone regeneration. Bioactive glasses (BGs) by sol-gel processing have several appealing characteristics as scaffolds for hard-tissue engineering, but are limited by their inherent brittleness. Dispersion of micro or nanoscale BGs in polymers has been shown to improve modulus, and crack resistance of scaffolds. However, their application is flawed as the BGs are generally covered by the polymer matrix. For example, conventional bioactivity tests, where the scaffolds were immersed in simulated body fluid (SBF), showed that hydroxyapatite (HAP) formation only occurred on the surface of BG particles. A confluent HAP layer was formed over 21 days when the polymer phase degraded and the BG particles exposed. This indicated that bonding to the bone would be slow in vivo. Inspired by nature&’s toughening mechanisms, we designed a new polyhedral oligomeric silsesquioxane (POSS)-derived hybrid glass (PHG) that has covalent interactions on the molecular scale between the inorganic POSS cage and organic phase. These features allow “elastic deformation” of the inorganic POSS cage in limited scale. The final product is a bulk hybrid material with toughness (3.56 ± 0.25 MPa.m^1/2) similar to natural bone (2.4-5.3 MPa.m^1/2). PHG exhibited excellent bioactivity by promoting the formation of plate-like hydroxyapatite on its surface in simulated body fluid and showed good cell adhesion. PHG also can be a platform to guide adipose tissue-derived mesenchymal stem cells differentiation and mineralization. The key structural features of this material can be used to guide the design of bio-inspired composites with unique toughness, which would be of great benefit to hard tissue engineering.

Chitin is a widely available biopolymer which is biocompatible as well as has low immune response. When blended with carbon nanotubes (CNTs), it results in a biopolymer with excellent dual characters of being biocompatible and electrically conductive. This is extremely useful for cells like neurons which are highly sensitive of electrical impulse1. This research aims to investigate the biocompatibility of chitin nanotubes based electrically conducting composites for neurons adhesion with assisted plasma treatment.
Chitin CNTs composite based films were prepared by dissolving chitin and CNTs in ionic liquid as a solvent2. The chitin CNT films were then dried in air before plasma treatment. The chitin and CNT composite films were subjected to plasma treatment using a radio frequency plasma reactor (PlasmaPrep5, Germany) at room temperature. Plasma treated samples were cut into small pieces using a biopsy punch and placed on 24 well tissue culture plates. Neurons at the density of 2.5x106 per well were added to the well and plates were observed at 7 and 14 days by live/dead cell assays under fluorescent microscope.
AFM studies showed that the plasma modified chitin nanotube film has the nanotubes appear on the surface due to erosion of the top layer of the composite after plasma treatment which resulted in good electrical conductivity of these films. The plasma modified chitin nanotubes showed excellent biocompatibility of neurons with the neurons still alive after 14 days. The chitin nanotubes plasma treated films also retained the capacity of post synaptic potential after 21days as evident by PSD 95 staining. The plasma treated chitin nanotube bio scaffolds thus prepared has the potential to be used for electrical stimulation of neurons.
REFERENCES
1. Cellot et al. Nat Nanotechnol 2009; 4:126-33.
2. Singh et al. Green Chem 2013; 15: 1192-202.

Titanium oxide is well known biocompatible material besides its application in different diverse fields. Its coatings are generally applied on body implants to prevent adverse biological effects and different substrates (Si & quartz) coated with thin titanium oxide film are used for different biological studies and development of some biosensors. Large numbers of research articles are available on these studies. Properties of titanium oxide thin film are modified by process parameters and/or incorporation of dopants in to the films. What actually these dopants at micro or nano level in the TiO₂ to change its biological property is still an issue to be explored in more depth. It has reported by some authors that it is surface morphology of the film which is responsible for biological properties of the surface where as other group of researchers rely on work function of the dopant atom to justify the role of dopant in titanium oxide film. Conflicting results appear in the literature. Being a very important issue we tried to enhance the understanding this topic.
We prepared glass substrates coated with thin films of TiO₂ undoped as well as doped with Ni, Ag, Mo, La and Ru. We studied biological carried out osteoblast cell line studies and biosensor response properties using different enzymes adsorbed on the surface these substrates. Large variation in properties exhibited by these substrates has been observed. We are making efforts to link our observations to the nature of dopants and surface morphology of the substrate at micro/nano level. XRD, SEM and AFM investigations of all the substrates have been carried out and some definite clues appears other basic investigations; XRD, XPS and spectral response is being carried out to support these findings.
Keywords: Biocompatible material, dopants, surface morphology, osteoblast cell, Titanium oxide, Biosensor

Autologous tissues (e.g. adipose tissue) and synthetic materials are routinely used to restore soft tissue defects caused by tumor extirpation, trauma, aging, or congenital malformation. However, these materials and repair options suffer from severe drawbacks such as donor-site morbidity and fibrous encapsulation. Hydrogels have been proposed as a scaffold material for restoring the soft tissue defects due to their hydrated nature and tunable mechanical properties. However, they often lack the mechanical strength necessary to maintain the integrity of the tissue repair site, while maintaining the porosity and pore size to allow efficient cell infiltration and integration with host tissue following the reconstruction. Here, we report an electrospun fiber-hydrogel composite for reinforcing the mechanical strength via interfacial bonding as well as facilitating cell migration by the extracellular matrix-mimetic structure of the composite. The fiber-hydrogel composite was prepared by crosslinking thiolated hyaluronic acid (HA) with poly(ethylene glycol) diacrylate (PEGDA) in the presence of poly(ε-caprolactone) (PCL) electrospun fibers that were surface grafted with maleimide groups to enable the formation of interfacial bonding between PCL fibers and thiolated HA, thus bonding solid fibers with hydrogel network. The average length of the PCL fiber fragments was controlled by cryo-milling time and intensity. In the composite, PCL fibers were evenly dispersed throughout the hydrogel. Scanning electron microscopy revealed that composite resembles the structure of the ECM of decellularized native fat tissue. The compression strength and shear moduli, as well as the porosity of the composite, could be adjusted by tuning the density of interfacial bonding sites, diameter of the fibers, and amount of fibers loaded. Moreover, the interfacial bonding was found to be the most important factor for tuning the mechanical strength of the composite. The composite can be prepared with a similar elastic modulus as HA hydrogel at much lower hydrogel pore size, allowing higher degree of cell migration in 3D. We have shown that human adipose-derived stem cells spheroids (hASCs) cultured inside the composite with 1.9 kPa elastic modulus exhibited higher cell migratory capacity through the fibrous structure, in contrast to those seeded in the HA hydrogel at the same modulus. This 1.9-kPa fiber-hydrogel composite implanted into the fat pad of SD rats yielded better tissue integration by mediating more infiltrating endogenous cells from the surrounding tissue, whereas mild fibrosis and lack of cell integration was observed following implantation of the HA hydrogel with the same modulus. These results suggest that the fiber-hydrogel composite with tunable mechanical properties is a promising new material to promote soft tissue reconstruction.

Thanks to their favorable properties, conducting polymers are becoming reference materials for the interfacing of electronics and biology, and promising candidates for tissue engineering applications. Indeed, they not only provide a “soft” and biocompatible scaffold for physical support, but they can also be used to transmit simultaneous mechanical, electrical and chemical stimuli to the cells. This ability occurs along with other changes in the surface properties of these materials, such as roughness, elasticity, wettability and electrical conductivity. Moreover, the physical and bio-chemical features of these materials can be modulated by functionalization through the incorporation of appropriate doping molecules, which include either inorganic or biological elements.
Recently, conducting polymers were proved to influence cell behavior, in terms of cell adhesion and growth, by a change in their oxidation state. The cell-substrate interaction involves many different parameters, both physical (surface roughness, surface energy, stiffness, electrical conductivity), chemical (pH, oxidation state) and biological (extra-cellular matrix formation, protein conformation), but the way these parameters are related to each other and to the oxidation state itself is still not clear. Gaining a better understanding of the processes that control cell adhesion is crucial in order to use conducting polymers as a new tool in basic research, medical diagnostics, and regenerative engineering.
Among conducting polymers, poly(ethylene dioxythiophene) (PEDOT) is one of the most promising materials for bioelectronics applications, thanks to its good electrical conductivity, chemical stability and biocompatibility. In order to improve these properties, PEDOT is usually doped with anionic dopants. We polymerized PEDOT thin films using either non-biological dopants, as poly(styrenesulfonic acid) (PSS) and dodecylbenzenesulfonic acid (DBSA), or biological molecules which are major components of the extracellular matrix, as hyaluronic acid (HA) and chondroitin sulfate (CS). The oxidation state of the obtained films was electrochemically switched and they were subsequently characterized by cyclic voltammetry, contact angle measurements and atomic force microscopy (AFM) as a function of the applied potential. Despite the increasing interest towards this material, less is known about the effects induced on PEDOT by the exposure to electrolyte solutions like buffer solutions or cell culture media, which represents the standard environment in biological applications. By using liquid-mode AFM, we observed local modifications in terms of swelling, morphology, elastic modulus and electrical conductivity in-situ on PEDOT films during oxidation and reduction in phosphate buffer saline solution.

Tissue engineering is one of the most promising technologies to regenerate patients&’ defective tissues and organs. 3D scaffolds based on various kinds of materials have been attempted for total or partial replacement of organs and tissues. Fabricating implantable scaffolds in traditional method, however, has certain limitation to mimic complex organ structures such as globular kidney, tripod shaped trachea and tubular blood vessels.
Previously, paper-based biotechnology has been utilized for many fields such as 3D cell culture, high throughput biochemical assays and a point-of-care (POC) diagnostic system. Macroporous structures of paper, especially, allow the mass transport of nutrients, the diffusion of oxygen for cell cultures, and enhancing its gradient formation. Especially, the high flexibility of paper enables the paper origami to form 3D scaffolds. A variety of shaped scaffolds could be designed with computer-aided design (CAD) and paper origami-based method.
In this report, we fabricated a robust paper-based hydrogel-laden scaffold for implantation. Firstly, the surface of paper substrates was modified conformally with functional polymer thin films via a vapor-phase fabrication method, initiated chemical vapor deposition (iCVD). Polymer film including a specific functional group could be deposited onto the substrates without damaging to its surface microstructure due to the mild process condition of the solvent-free iCVD process. The surface of paper was conformally coated with poly(styrene-co-maleic anhydride) (PSMa) via iCVD followed by immobilization of poly-L-lysine (PLL) and deposition of Ca²⁺. This bio-functionally modified paper could be folded freely into complex 3D tissue-like structure by an origami-based method. Cell-encapsulated alginate hydrogel could be formed on this bio-functionally modified paper due to the Ca²⁺ diffusion into the alginate solution and a strong adhesion between the alginate hydrogel and paper substrates was achieved due to an electrostatic interaction between the PLL and alginate hydrogel. The biocompatibility of the paper scaffold was also confirmed. The cell-laden paper scaffold was also suitable for mass transport of nutrients and oxygen. Folded paper scaffolds with rabbit articular chondrocyte-encapsulated alginate hydrogel were cultured in vivo for 6 weeks through its biochemical and histology test. In detail, rabbit&’s native trachea was removed and replaced successfully with a cylindrical hydrogel-laden scaffold with rabbit chondrocytes. These findings demonstrated that the simple and bottom-up method to fabricate implantable paper scaffold in 3D through paper origami has broad application in the transplantation of hydrogel-laden paper scaffolds for origami-based tissue engineering.

Ceramic-based porous materials are promising candidates for manufacturing bone tissue models to be used in in-vitro testing in replacement of animal models prior to clinical trials.
The bioactivity of the constituent material will promote cell and tissue growth in a bioreactor. However, mechanical reliability and biomechanical compatibility are also required for a reliable and functional tissue model.
It is therefore of great relevance to achieve the capability to predict mechanical properties of ceramic porous scaffold in order to design a manufacture biomechanically reliable tissue substitutes.
In this context, the aim of this work is to achieve a mechanical characterization of the elastic properties of the scaffold by means of small scale experimental testing and micro-CT based finite element modeling.
An experimental SiO2-based bioactive glass (CEL2; molar composition: 45% SiO2, 3% P2O5, 26% CaO, 15% Na2O, 7% MgO and 4% K2O) was selected to produce the porous samples.
Three-dimensional (3-D) scaffolds were produced by the sponge replication method, that was shown to be very effective to obtain porous ceramics with a highly-interconnected 3-D network of open macropores. Small cubic blocks (10.0 mm × 10.0 mm × 10.0 mm) of a commercial open-cells polyurethane sponge were coated with CEL2 and a sintering process produces the final scaffold.
In order to characterize the elastic properties of the whole scaffold, the knowledge of the mechanical properties of the constituent materials is the starting point. This was achieved through nanoindentation experiments performed with different characteristic lengths (different contact area, from few hundreds of nanometer to tens of microns). This allowed us to assess the mechanical role of the micro- and nano-porosity of the solid structure resulting from the sintering process. The indentations were performed on the solid phase of the 3D scaffolds.
The overall elastic properties of the porous scaffold were obtained by means of CT-scan based finite element models of the samples. Voxel-type finite element meshes were obtained for the porous samples. The full effective elastic tensor of the porous structure was achieved by applying standard homogenization techniques, which determine the local stress and strain fields within the struts of the samples resulting from a macroscopically homogeneous state of strain; the elastic properties assigned to the solid part are those obtained through the nanoindentation experiments.
The effective elastic tensor for different "Volumes Of Interests" has been determined and the “porosity-elastic modulus“ relationship was established. Comparison with experimental data validated the model.
The results show that reliable predictions of the overall elastic properties of a porous glass-ceramic scaffold can be obtained through the above procedure, which proves to be effective to assess the mechanical properties of different kinds of tissue models such as healthy and aged or diseased tissues.

Regenerated Bombyx mori silk fibroin (SF) has excellent biological and mechanical properties, including biocompatibility, programmable biodegradability, and remarkable strength and toughness. Diverse and adaptable properties of Sf are possible by varying the structural form using different processing conditions. One of the important physical forms for biomaterials is the formation of hydrogels, which has been extensively studied for a variety of polymers. The sol-gel transition depended on the concentration of the protein, temperature, and pH. In the SF hydrogel, random coil to β-sheet(physical cross-linking) structural transitions were noted during the process of hydrogelation. Due to the β-sheet formation, SF exhibits relatively slow degradation in vitro and in vivo, compared to collagen and many other biopolymers.
Tissue engineering has potential to address this need through the combination of biomaterials, growth factors, and cells. Highly porous scaffolds are generally used as the substrate for anchorage dependent cells and to facilitate nutrient and metabolite distribution to guide cell growth leading to new bone tissue formation. For bone tissue engineering, biodegradable synthetic polymers such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and copolymers of poly(DL-lactic-glycolic acid) (PLGA), and biodegradable naturally derived polymers such collagen and fibrin.
Hydroxyapatite (HAP) has been investigated for bone replacement since this material mimics natural bone mineral features. HAP has been studied extensively in cell culture and possesses osteoconductivity.
In this study, the synthesis and characterization of bone-like mineral HAP into highly porous biodegradable silk fibroin scaffold with via chemical cross-linking reaction of SF by gamma-ray (g-ray) were investigated. These 3-D SF scaffolds had different secondary structures, elasticity and nanostructure compared with β-sheet induced hydrogels.The effect of degradation rate, elasticity and mineralization on osteogenic responses of osteoblast was assessed with respect to bone tissue engineering.

Guided Assembly-based Biolithography (GAB) is a powerful replica-molding technology that allows the transfer of on-demand functional topographies to the surface of bacterial cellulose films. The approach is based on standard fermentation of glucose into cellulose in a static culture of Acetobacter Xylinum. In particular, at the interface of a microstructured PDMS mold preferential nanofiber patterns are created by the physical confinement of bacterial motion. In this way, the cellulose nanofibers are assembled in a three-dimensional network which closely replicates the geometry imposed by the mold. GAB shows also the capability of controlling assembly process at the nanoscale with a microscale softlithographic mold, yielding directional alignment of individual nanofibers. Additionally, GAB structures possess memory of the transferred geometrical features upon dehydration and rehydration of the cellulose substrates. The fidelity of this facile and affordable method is assessed by scanning electron (SEM) and atomic force microscopy (AFM).
Rational design of the patterns on the cellulose allows direct and efficient control on cellular response, promoting or demoting adhesion, spreading and migration. The interaction of surface-structured bacterial cellulose substrates with human fibroblasts and keratinocytes is reported to illustrate the efficient control of cellular activities which are fundamental in wound healing and tissue regeneration.
Additionally, the deployment of surface-structured bacterial cellulose substrates in model animals as skin wound dressing or body implant proved the high durability and low inflammatory response to the material over a period of 21 days, demonstrating beneficial effects of the surface structure on skin regeneration.
Altogether, the possibility to control specific cell and tissue responses by a simultaneously non-inflammatory and micro-structured material has huge potential in the field of soft tissue repair and medical implant dressing.

Over the last couple of decades there has been increasing interest in using nanoindentation techniques to determine the mechanical properties of soft and biological materials [1]. This has allowed the measurement of mechanical properties from small tissue samples such as biopsies and histological sections. However, measurement of the mechanical behaviour of highly compliant and hydrated materials via traditional nanoindentation is challenging due to their high time-dependence as well as to their low elastic moduli [2]. In addition, samples are often mounted as thin sections on much stiffer glass substrates, which make interpretation of test data difficult [3]. In this work, an iterative finite element analysis methodology coupled with an optimization algorithm was developed to determine the viscoelastic material parameters of highly compliant materials such as PDMS and pHEMA hydrogels with varying cross-linker concentrations (0.5 and 1.0%) and thicknesses varying from 5 to 3000 µm.
This was done by performing nanoindentation tests using a conospherical indenter and trapezoidal loading profiles with different loading rates and maximum loads to account for the specimens&’ time dependency. By using the experimentally obtained time vs displacement and load vs displacement indentation curves along with the iterative FEA methodology, a set of material parameters could be determined for the specimens. This methodology allowed determination of the material parameters whilst enabling removal of the effects caused by the samples&’ thickness and the underlying substrate used for experimentation. Iterative non-linear optimization algorithms were used, leading to good agreement between the experimental and FEA obtained loading-unloading curves. Typical convergence was achieved in 5-12 iterations.
1. Oyen, M., Nanoindentation of biological and biomimetic materials. Experimental Techniques, 2013. 37(1): p. 73-87.
2. Galli, M., et al., Viscoelastic and poroelastic mechanical characterization of hydrated gels. Journal of Materials Research, 2009. 24(03): p. 973-979.
3. Akhtar, R., et al., Nanoindentation of histological specimens: Mapping the elastic properties of soft tissues. Journal of Materials Research, 2009. 24(3): p. 638-646.

Introduction: Tracheal damage, abnormality or absence can result from tumors or Congenital High Airway Obstruction Syndrome. No optimal, routine treatment has been established, despite numerous attempts with natural and artificial prostheses¹. Achieving closely matched, anisotropic mechanical properties in an engineered replacement is important for graft patency and functionality. Construct mechanics must be sufficient to maintain a patent airway under loading, yet a mismatched stiffness may induce fatal obstruction or bleeding². During respiration the trachea may extend 46% in healthy neonates³. Abnormal longitudinal properties can lead to ongoing respiratory problems and impede normal lung ventilation⁴. These observations make a strong case for the development of a patterned trachea replacement that can adequately approximate the longitudinal extensibility and lateral rigidity conferred to native trachea by its cartilage and smooth muscle composition⁵.
Methods: 2-Hydroxyethyl methacrylate is crosslinked with triethylene glycol dimethacrylate via photopolymerization in custom molds to create 2D and tubular 3D gels with horizontal bands. Hard and soft bands of varying thickness are described by hard:soft ratios, resulting in increasing percentages of hard regions. Constructs were evaluated for longitudinal tensile modulus, radial compression yield behavior, cyclic radial compression at physiological rates, and compliance under physiologically relevant transmural pressures.
Results: Constructs show an increase in elastic modulus with increase in % hard regions, where tensile loading is applied perpendicular to the banded pattern. This corresponds to a decreasing yield strain, an important metric of extensibility. Trachea constructs stretch longitudinally during respiration, and should function in the elastic region to avoid material failure. In tubes subjected to radial compression, higher deformation and load at yield indicate superior resistance to collapse. Patterned 2:1 (Hard:Soft) tubes outperform solid hard gels in both of these metrics, although their longitudinal properties are not as advantageous as those of other patterns. The 1:2 pattern has significantly greater longitudinal extensibility, but a similar deformation at yield in the radial direction to the other patterned constructs. The 1:1 4mm pattern has greater longitudinal extensibility than the 1:1 2mm pattern, yet not significantly different radial yield properties.
Conclusions: Hydrogels can be patterned to achieve physiologically relevant construct-level anisotropic properties. Patterned gels outperformed solid hard gels in their ability to recoil under cyclic loading at physiologically relevant respiratory rates
References: 1)Kunisaki, SM JPediatr Surg, 2006. 41(4):p.675-82; 2) Kawaguchi, S Biomaterials, 2001. 22(23):p.3085-90; 3) Harris, RS Thorax, 1959 14p.201-10; 4) Inglis, AF Arch Otolaryngol Head Neck Surg, 1992. 118(4):p.436-8; 5) Teng, Z J Biomech. 45(9):p.1717-23

The objective of this investigation was to study the adhesion and viability of bone marrow derived mesenchymal stem cells (BMSC) at the cell-biomaterial interface in the direct culture with four Mg-4Zn-xSr alloys (x = 0.15, 0.5, 1.0, 1.5 wt. %; designated as ZSr41A, B, C, and D respectively) in vitro. The direct in vitro culture method, in which cells are seeded directly onto the surface of the samples and characterized for both direct contact and indirect contact, was established by our lab to understand cellular interactions with biodegradable metals. Additionally, a systematic comparison on the degradation of the ZSr41 alloys in the direct culture with multiple cell types in their respective media along with the biological performance compared with other magnesium (Mg) alloys was included in this study. Results from this study showed a significantly faster degradation rate for alloys ZSr41A and ZSr41B after the 72 hr incubation with BMSCs. Although BMSCs adhered and remained viable on the surfaces of all ZSr41 alloys, ZSr41A and ZSr41B showed a significantly lower amount of viable BMSCs adhered to their surfaces. In contrast, BMSCs adherent to the culture plate surrounding the samples were largely unaffected by the solubilized degradation products from the ZSr41 alloys. Results from the comparative study showed that: (i) the degradation rates of the ZSr41 alloys were higher when compared with Mg-xZn-0.5Ca alloys, (ii) the degradation rates of Mg-based materials in different culture media might be most affected by media buffer capacity (i.e. HCO₃^- concentration), and to a lesser extent, D-glucose concentration, (iii) BMSCs were more robust than other cell lines to screen the cytocompatibility of Mg-based biomaterials, and (iv) mean percent BMSC viability at the cell-substrate interface is likely inversely proportional to alloy degradation rate. This study presented a clinically relevant in vitro model for screening bioresorbable alloys and provided useful design guidelines for determining the degradation rate of Mg-based biomaterials.

The aim of this work was to develop novel hydrogel scaffolds using biocompatible polymers and the influence of silane crosslinker and poly (vinyl alcohol) PVA concentration on CS/PVA scaffolds were studied. Blends of chitosan (CS) and (PVA) were prepared and a novel approach was used to crosslink these blends to give network structure. Fourier transform infra-red spectroscopy confirmed the presence of incorporated components and formation of siloxane linkage between CS and PVA in hydrogel scaffolds. The mechanical properties that the tensile strength was increased with increase in the percentage of crosslinker while elongation at break was decreased and opposite response was observed with the increase in PVA amount. The surface properties showed the hydrophilic behavior of scaffolds. The structural analysis by XRD showed an increase in the crystallinity of scaffolds attributed to the improved interactions between crosslinker, CS and PVA. The cytotoxicity of the hydrogel scaffolds revealed that these materials were nontoxic, viable to the cells, helpful in the growth of the cells and can be used for tissue engineering and other biomedical applications. The properties of the developed material were quite promising for tissue engineering applications. Furthermore, it has the flexibility to be used into different forms such as: hydrogel, films, and the scaffolds.

Although transplantation of autologous cells has been tested by injection of cell suspensions using a syringe, limited regeneration results were observed due to low cell viability of the injected cells, and restricted cell integration into the host tissue. To address these issues, a new approach would be to locally deliver the cells using engineered, cell-loaded substrates. This flexible polymeric nanosheets has nanometer thickness and can be aspirated and injected into tissues through a conventional syringe needle with minimal invasive delivery.
In this study, we developed microfabricated poly (lactic-co-glycolic acid) (PLGA) nanoribbons with different thicknesses using spin coating and micropatterning techniques, in order to generate freestanding ribbons loaded with murine skeletal myoblasts (C2C12). We anticipate that the injectable nanoribbons that has a structure like italian pasta “Fettuccine” inspirited bundle structure of muscle tissue and it can be a useful tissue engineering technique for local delivery of cells while maintaining the cellular organization, viability, and functionality.
The morphology of PLGA nanoribbons was imaged using field emission scanning electron microscopy (FE-SEM). It was observed that the center of the nanoribbons became deformed due to the solvent evaporation, whereas the structure of their walls was maintained.
We cultured C2C12 cells on freestanding nanoribbons. Prior cell seeding, the nanoribbons were covered with poly(L-lysine) to promote cell adhesion and also to modify their surfaces for preventing their agglomeration when submerged in culture medium. Individual cell-loaded nanoribbons could be aspirated and injected through a syringe needle with different gages (18, 23, 25 G) due to their highly flexible structure. In addition, the flexibility reduces the mechanical stress on the attached cells. The cell stability and viability was evaluated on ribbons with different thickness by using a live/dead staining. Fluorescent microscopy analysis following the staining showed that the C2C12 cells adhered to the nanaoribbons, remained viable and retained cellular activity.
Myotubes or multi-nucleated fibers generated from the fusion of myoblasts, play a crucial role in myogenesis. After seeding C2C12 myoblasts with a cell density of 2*10⁶ cell/ml and getting confluency on the nanoribbons, we used a cell differentiation medium to induce myotubes formation for 8 days of culture. To quantify the effect of cell alignment on the assembly of myotubes and also possibility of myotubes formation on freestanding nanoribbons, myotubes were stained using immunofluorescence at day 8 of myogenesis. We investigated the formation and alignment of myotubes on each single nanoribbon with elongated and multinucleated structure and peripheric position of their nuclei, which is a feature of mature generated myotubes. These highly flexible, injectable and cell-loaded nanoribbons will be beneficial for tissue engineering.

Favorable bio-material interactions are pivotal to the successful utilization of medical implants, particularly titanium-based implants. Every year, millions of people undergo surgery to replace faulty implants. In part, this is due to the incomplete comprehension of the critical biomolecular recognition events and the interfacial interactions that take place at bio-material interfaces. Predicting and controlling bio-interfacial properties is extremely challenging without a molecular-level understanding of how biomolecules interact with material surfaces, and the factors that influence these interactions. Here, we utilize cutting-edge computational techniques such as Replica Exchange with Solute Tempering (REST)¹ and Metadynamics to study, at the negatively-charged aqueous rutile TiO₂ (110) interface, the adsorption of amino acids and titania-binding peptides (namely Ti1, QPYLFATDSLIK, and Ti2, GHTHYHAVRTQT)² and calculate their binding free energies. Our results revealed a stronger affinity between charged amino acids and the titania surface compared to uncharged amino acids.³ In addition, peptides Ti1 and Ti2 are found to have a similar free energy of adsorption but different binding mechanisms. While Ti2 reveals an enthalpic binding, Ti1 shows an entropically-driven binding mechanism.⁴ Moreover, the effect of using Ca²⁺ counterions, in contrast to Na⁺, on the adsorption of both peptides at aqueous titania is also studied. Finally, the impact of His protonation state on the adsorption of Ti2 is evaluated. Our results provide valuable insights into the complex interplay between peptide sequence, structure, and binding at the aqueous TiO₂ interface.
Terakawa, T., Kameda, T. and Takada, S. J. Comput. Chem. (2010) 32, 1228-1234.
Puddu, V., Slocik, J.M., Naik, R.R. and Perry, C. Langmuir (2013) 29, 9464-9472.
Sultan, A.M., Hughes, Z.E. and Walsh, T.R. Langmuir (2014) 30, 13321-13329.
Tang, Z., Palafox-Hernandez, J.P., Law, W.C., Hughes, Z.E., Swihart, M.T., Prasad, P.N. and Walsh, T.R. ACS Nano (2013) 7, 9632-9646.

Inspired by the essential role of reactive oxygen species (ROS) on various physiological events, extensive studies utilizing ROS have emerged. It is important to note, however, that current systems employ external stimuli such as light or electrical energy to produce ROS, thereby limiting their practical usage. Moreover, because ROS exert concentration-dependent effects on cells, controlling the amount of ROS in an application-specific manner is pivotal. In this regard, a system that can generate ROS in a controlled manner in the absence of external stimuli is highly desirable for potential biological applications.
Here, we utilize biocompatible metals to construct a novel electrochemical system that can spontaneously generate H₂O₂ without any external light or voltage. Corrosion of metal (anode) transferred electrons to nanostructured Ti (cathode) in an energetically-favorable manner, and spontaneous generation of H₂O₂ by an oxygen-reduction-reaction (ORR) was revealed at the Ti cathode via combined spectroscopic and electrochemical analyses. Cyclic voltammetry curves and discharging-voltage profiles showed that metal corrosion and ORR occur at the anode and cathode, respectively, and the overall discharging voltage was stably maintained around 0.8 V for 12 h at the constant discharging current density of 0.2 mA/cm². The faradaic efficiency for H₂O₂ generation was measured as 65 % with DPD/POD method, and Koutechky-Levich plots based on rotating-disk-electrode analysis confirmed the formation of H₂O₂ via a two-electron reduction pathway in O₂-saturated phosphate buffer saline (PBS). By simple electric connection between the anode and nanostructured Ti cathode with a metal conduit, H₂O₂ was generated in the absence of external applied voltage or current, and the released amount of H₂O₂ can be easily controlled from 1 mu;M to 500 mu;M by varying the discharging time.
Interestingly, after 8 h of cultivation, the controlled release of H₂O₂ (~ 10 mu;M ) from our system noticeably enhanced in-vitro angiogenesis even in the absence of growth factors. Enhanced capillary formation, human umbilical vein endothelia cells (HUVECs) proliferation was found comparable to positive control with growth factors. Contrary to the positive results observed at low levels of H₂O₂, HUVECs exposed to high levels of H₂O₂ (~100 mu;M) instead reduce cell numbers due to the oxidative stress of H₂O₂. The high levels of H₂O₂ generated from our system had an anti-bacterial effect and significant decrease in survival rate of E.coli was observed. By controlling the released amount of H₂O₂ with discharging time, our system exhibited bi-functionality toward angiogenesis and antibacterial effect. Ultimately, we take advantage of the corrosion of biocompatible metals for the generation of electrons and ROS, and envision that our approach can be further extended to triggering other redox-reactions in biological systems.

Nephrotoxicity is a major side effect of drugs, accounting for 19% of failures in phase III trials. These failures stem from extensive use of animal models in preclinical testing that are not sufficiently predictive of the human response. To address this challenge, we have recently introduced a new bioprinting method for creating 3D multi-material, cell-laden architectures with embedded epithelial networks. Our convoluted proximal tubule model consists of open 3D networks of channels circumscribed by human proximal tubule cells and perfused at physiological shear stresses. To date our bioprinting methods have resulted in proximal tubules surrounded by stromal cells that remain viable greater than 30 days. Our recent efforts to create these three-dimensional, biomimetic, multi-compartmental human proximal tubule models that reproduce renal tubule physiology and in vivo response to nephrotoxic drugs will be highlighted.

In tissue engineering, it is desirable to use a suitable scaffold for providing cells with a conducive growth environment with optimal growth factor cocktails and proper stiffness. Biocompatible and biodegradable materials, polycaprolactone (PCL) nanofibers and alginate hydrogels, play a significant role in both designing controlled release matrix for bioactive molecules and tissue growth. Although prolonged release of bioactive molecules is readily achievable using these polymer materials independently as a matrix, it is not seen how to release various bioactive molecules at a different rate over a different length of time. In this study, we fabricated a multilayer nanofiber-hydrogel scaffold using alternating layers of electrospun PCL nanofiber and alginate hydrogel, and evaluated its controlled release property. Adenosine triphosphate (ATP), encapsulated in the designated hydrogel layers, was used as a mock drug for the release study. We demonstrated that ATP release from the exposed top layer of the scaffold has dramatically high burst release and shorter release time compared to that from deeper layers in the scaffolds. Such a differential release property of designated layers can be employed to achieve releasing of multiple drugs at different rates over a different length of time. Beside the drug release, we also modulate the stiffness of each layer of the scaffold with a differential hydroxyapatite coating to achieve the gradient stiffness of the scaffolds with the increase of layer numbers.

Injectable hydrogels have been attracting great interest for therapeutic cargo delivery due to their ability to be surgically implanted in a minimally invasive way. Many potential applications of injectable hydrogels are limited by their gelation kinetics. If the gelation is too fast, delivery may fail due to a clogged needle. If too slow, material may flow away from the desired site as a result of incomplete gelation. Shear-thinning, self-healing hydrogels are a promising approach to address this issue; however, most shear-thinning hydrogels are mechanically weak and have fast erosion rates. To overcome these challenges, we designed an injectable hydrogel using a novel combination of dynamic covalent crosslinking and physical crosslinking. Ex situ, rapid gelation occurs through the formation of dynamic covalent hydrazone bonds by simply mixing two components: hydrazine-modified elastin-like polypeptide (ELP) and aldehyde-modified hyaluronic acid (HA). This shear-thinning network provided significant mechanical protection to encapsulated chondrocytes during injection and rapidly recovered after injection to maintain a homogeneous dispersion of cells within a 3D environment. In situ at physiological temperature, the network was further reinforced and stiffened due to the thermoresponsive ELP. The mechanical properties of these ELP-HA hydrogels were easily tuned by the stoichiometry of the functional groups to achieve storage moduli ranging from ~50 to ~5000 Pa. Primary chondrocytes encapsulated within the hydrogels remained highly viable after injection and showed positive staining for cartilage tissue markers (type II collagen and sGAG) after 3 weeks of in vitro culture. These data demonstrate the potential application of these injectable ELP-HA hydrogels for tissue regeneration applications.

While amphibians regenerate lost appendages spontaneously, mammals generally scar over the injury site via wound repair. Inspired by the spontaneous healing trait of amphibians, we are interested in developing biomaterials that facilitate pro-regenerative interventions in mammals. In this talk we will describe the development of an injectable hydrogel that cross-links by native chemical ligation (NCL), a convenient and chemospecific reaction that has historically been used for building large peptides from small fragments. We adapted NCL for in-situ polymer hydrogel formation and used it as an injectable delivery system for a small molecule inhibitor of prolyl hydroxylases (PHDs). Subcutaneous injection of drug-hydrogel resulted in enhanced regenerative wound healing in non-regenerative mice, in a manner that emulates the basic elements of amphibian regeneration. Follow-up studies will also be described, in which drug and hydrophilic polymer are conjugated through a biologically labile linker to form a polymer prodrug that self-assembles into nanofibers and other structures. These approaches offer new opportunities for delivery of tissue regenerative therapeutic drugs.

In vivo, natural tissues are rarely physically or mechanically homogenous, but rather exhibit a range of elasticities and textures/topographies. Additionally, cells in native tissues reside in a dynamic environment in which the physical environment may change spatially and/or temporally (i.e., during tissue remodeling). Hydrogels are extensively investigated as two- and three-dimensional scaffolds for cells because of their high water content, tunable mechanical and physicochemical properties and their ability to be formed in the presence of biological materials such as cells, proteins and DNA. Therefore, hydrogels in which both topography and elasticity are dynamically tunable undoubtedly represent an important tool for investigating the synergistic effects of inhomogeneities in the physical environment on cell behavior, and better mimic the native microenvironment for tissue engineering applications.
Many strategies and materials to produce hydrogels with controllable and patternable elastic moduli and topographic features have been explored, including techniques such as photolithography, micromolding and microcontact printing. Although physically and mechanically patterned materials are obtained, these techniques generally allow either topographic patterning or elastic patterning, but no hydrogel with controlled variations in both topography and elasticity has been reported. Additionally, most of the demonstrated hydrogel patterns are static or predefined, and cannot be altered dynamically, especially once cells have been introduced into the scaffold. Finally, topography control is typically exerted in a “one-way” manner.
In our laboratory, we utilize a photodegradable poly(ethylene glycol) (PEG) macromers to fabricate hydrogels. Upon exposure to spatially controlled doses of light, topographically and mechanically patterned surfaces can be achieved. Uniquely, our photodegradable hydrogels can act as both positive and pseudo-negative photoresists, depending on exposure time and wavelength. This allows the construction of positive features formed via swelling, and negative features formed via erosion.
Here we report anisotropically patterned hydrogels with programmable gradient stiffness control and sub-micron resolution. Patterning can be done on a surface onto which cells are seeded (2D culture), as well as within the 3D volume of the hydrogel (3D culture). Cell response to these patterned materials is a result of both absolute and relative degrees of degradation as well specific details of the pattern (i.e. pattern dimensions). These hydrogels therefore provide great tunability in both topography and elasticity, which may be useful in elucidating the role of the physical microenvironment in cell behavior.

Current mechanotransduction studies focus on the role stiffness on cell behavior, but tissues are typically viscoelastic, exhibiting relaxation over a characteristic time-scale. We have been studying the impact of biomaterial stiffness and stress-relaxation on both cancerous and stem cells, and developed materials that allow changes in mechanical properties to be decoupled from changes in gel architecture and adhesion ligand presentation. To address the impact of these properties at the single cell level, a gentle microfluidic-based method for encapsulating single cells in an ~5 micron thick layer of hydrogel has been developed.

In the last two decades, significant efforts have been made to develop soft materials exploiting the self-assembly of short peptides for biomedical applications. So-called β-sheet forming peptides are very attractive for the design of biomaterials, in particular hydrogels [Saiani et al., Soft Matter, 5, 193, 2009]. Due to the “simplicity” of the structure formed at the molecular level, the relative robustness of the β-sheet assembly and the ease of functionalization very stable functional hydrogels can be designed with potential application in a range of fields from tissue engineering [Mujeeb et al. Acta Biomat 2013, 9, 4609 & C Diaz et al. J Tissue Eng 2014, 5, 1-12] and cell culture [Szkolar et al. J Pept Sci 2014, 20, 578] to drug delivery [Roberts at al. Langmuir 2012, 28, 16196 & Tang et al. Int J Pharm 2014, 465, 427]. One particular aspect which has attracted significant interest is the ability to use these systems to design 3D injectable scaffolds for in vivo cell delivery.
Here we present our recent work done on the design of an injectable peptide based hydrogel for the in vivo delivery of cell in the heart. First we have investigated the gelation properties, including injectability, of a b-sheet forming peptide based on the alternation of hydrophobic and hydrophilic residues: FEFEFKFK (F: phenylalanine; K: Lysine; E: Glutamic acid). Subsequently the suitability of the hydrogel to sustain rat Cardiac Progenitor Cells (CPCs) biological properties was tested in vitro. Finally a small animal model was used to explore the potential of this hydrogel as a cell delivery vehicle.
Our work clearly showed that these materials can be used both as 2D and 3D scaffolds for the in vitro culture of CPCs. More interestingly, our in vivo work clearly showed the hydrogel good injectability and the potential of this material as a topical delivery vehicle. Using rhodamine nanoparticles we documented that the hydrogels is retained at the site of injection for at least 7-days. In order to investigate the suitability of the injected hydrogel as a vehicle for CPCs delivery in the heart we used a fluorescent peptide (10mg/ml) loaded with Quantum Dot® labelled cells (10⁵ cell/ml). We documented that the peptide gel was able to retain the “cargo” at the site of injection. Moreover, Quantum Dot® labelled CPCs were detected within the myocardium suggesting the ability of the peptide gel to also support cell homing.
Our work clearly shows the potential of these materials as a 2D and 3D scaffold for the in vitro culture of cardiac progenitor cells. In addition, this peptide gel displays proper injectability as well as good properties for the topical delivery of cells, or even drugs, in the host myocardium. These characteristics suggest a possible future application of this biomaterial as a scaffold for cardiac regeneration.
Acknowledgements: The authors are grateful to the EU commission (Grant n0: NMP3-LA-2009-21539 BIOSCENT) for financial support.

BACKGROUND & OBJECTIVES: Angiogenesis, the formation of new vessels, is crucial for tumour progression and metastasis. Typical monolayer cell culture (2D) assays have offered tremendous insight in fundamental cancer biology and drug development. However, the microenvironment within which cells reside and interact with the tumour extracellular matrix is more complex. The objective of this study is to develop 3D vascularized tumour spheroids (Tmu;Sph) that recapitulate the tumour microenvironment and improve the in vitro assessment of cancer therapeutics.
METHODS: For Tmu;Sph formation, HUVECs, MSCs and human breast cancer and hepatoma cell line (MDA, MCF-7, HepG2 and Huh-7) transduced with lentiviruses, were mixed in a fibronectin containing collagen gel or a self-assembled peptide hydrogel. Confocal microscopy and histological immunostaining will be used to assess the formation of a 3D vascular network. Diffusion of the antiangiogenesis and chemotherapy drugs sorafenib and tamoxifen, respectively, will be investigated and their effect on the Tmu;Sph cell metabolic activity, migration as well as angiogenesis and tumour cell metastasis will be assessed in vitro.
RESULTS: Confocal microscopy demonstrated the formation of capillary-like network within the Tmu;Sph with lumen like structures. Tmu;Sph treated with sorafenib and tamoxifen demonstrated that higher drug concentrations are necessary for break down of vascular network, hampering of cell migration and toxicity compared to 2D cell cultures. Tumour and endothelial cell interactions were altered within the 3D Tmu;Sph environment compared to 2D co-cultures. In addition, evidence of the impact of the collagen and self-assembled hydrogels on tumour cells and endothelial cell migration and formation of invadopodia will be presented. Our results demonstrate that both hydrogels provide the appropriate cues and support the formation of 3D vascularised tumour microspheroids; a promising in vitro drug-screening model compared to 2D standard cell culture assays.

Ischemic therapy is based on the stable delivery of angiogenic factors for long-term neovascularization. Here, we developed a new ischemic therapy using engineered mesenchymal stem cells (eMSCs) to express human hepatocyte growth factor (hHGF). The eMSCs were encapsulated for long-term therapeutic effect within self-assembling supramolecular hyaluronic acid (HA) hydrogels. The supramolecular HA hydrogels were prepared by simple mixing in the presence of eMSCs for host-guest interaction between mono-functionalized cucurbit[6]uril - HA (mCB[6]-HA) and diaminohexane conjugated HA (DAH-HA). The angiogenic efficacy was assessed in hind-limb ischemia model mice by laser Doppler imaging, photoacoustic microscopy (PAM), in vivo angiogenic assay, and histological analysis. Especially, as an emerging microangiographic technique, the PAM enabled non-invasive imaging of vascular repair process in deep tissue without any contrast agents. Taken together, we could confirm the feasibility of eMSC angiogenic therapy from the effective vascular repair and enhanced blood perfusion.

Tissue engineering is one of the recent theraputic approaches for both soft and hard tissue repair. Healthy cells taken from the host own tissues or from other sources are used together with scaffolds. Target specific (eg., osteoblasts, chodrocytes, etc.) or preferentially stem cells are isolated, differentiated (in the case of stem cells), or even genetically modified (for instance to express growth factors, eg., BMPs) and are used in two diffetrent approaches. In the first approach they are just loaded into the scaffolds (as seeds) and apply as biohybrid implants. Alternatively, cells are propagated within the pores of scaffolds within bioreactors (in vitro) to form tissue-like structures and then they are implanted for tissue replacement. Scaffolds have large and interconnected pores which allows 3D-cell ingrowth are used. They have to be degradable in vivo, means that they should degrade such a rate that the new forming tissues to replace them properly. Of course both they and their degradation products must be biocompatible. They are produced several techniques, such as moulding/salt extraction, electrospinning, cryogelation, etc. They are made of several natural polymers (e.g., collagen and its denaturated form gelatin) and synthetic polymers (e.g., lactides, glycolide and ε-caprolactone). Several bioactive agents (e.g., growth factors, etc.) may be also incorporated (usually as controlled release formulations) to trigger the regeneration rate and proper new tissue formation. After careful in vitro biocompatibility test, tissue engineering scaffolds (loaded with cells) or biohybrid implants are applied in vivo in proper animal models. Critical size defects (means that the defects do not recover by themselves) are created in animals. In the maxillofacial applications, cranium, cleft palate, zgyoma, mandibula, etc. models have been used for bone tissue engineering. Ear defects are created to study cartilage repair. Several macro-, histological, molecular techniques are used to investigate tissue regeneration. This talk briefly reviews the topics mentioned above by using the experience of the author&’s group in this field.

Due to a lack of proper vascularization methods, the current tissue engineering have limitations to construct large tissue. To solve this problem, endothelial seeded microchannel in scaffold was widely used as vascular network¹. Although these techniques allow immediate perfusion in tissue from very beginning, it is difficult to apply to tissue morphogenesis because the location of vascular network was almost fixed when firstly constructed. On the other hand, angiogenesis-based techniques² that utilize spontaneous network formation of endothelial cells has a potential to induce and control vascularization even after reconstruction of tissues in vitro. Recently, we reported the microsystem that could induce the migration of the angiogenesis-based vascular network from microchannel to lung fibroblast (LF) spheroid used as tissue model³. Here, we cultivated the tissue model containing the pre-vascular network in the microfluidic device and evaluated the anastomosis between the spheroid and network from the microchannel.
In this study, LF and human umbilical vein endothelial cell (HUVEC) co-cultured spheroid was utilized as a vascularized tissue model. To generate co-culture spheroids, LF and HUVE cells were mixed at various number of cells while keeping the ratio at 4:1, and seeded in non-adherent round-bottom 96-well plates. Microfluidic devices were fabricated out of polydimethylsiloxane (PDMS) using soft lithography³. Briefly, the device was consisted of three parallel channels partitioned by microposts to allow the surface tension-assisted patterning of fibrin gel. A central channel (2 mm width) containing the micro well (phi; = 1 mm) was used for cultivation of spheroid embedded in fibrin gel. HUVECs (5×10⁶ cells/mL) were introduced to two microchannel (700 mu;m width) contralateral to the central channel. For HUVECs adhering on the both side of fibrin gel, the microfluidic chip was tilted by 90 degrees and incubated for 30 min respectively. The microdevices were incubated at 37 0C and 5 % CO₂ to sprout newly formed vessel from microchannel.
First, we evaluated the vascular network in spheroid by fluorescent labelling of HUVEC (Lectin conjugated with FITC). The vascular network was confirmed in spheroid after 3 to 7 days in non-adherent 96 well plate. These network was also maintained in fibrin gel and the expanded to outer area. Next, this co-culture spheroid after 4 days culture was introduced to the device and evaluated the anastomosis between spheroid and vascular network from the microchannel. By time-lapse observation, the sprouting cells from microchannel contacted the spheroid at 2 days culture and at day 9, the continuous lumen (Ave. 16.6 mu;m diameter) could be observed in newly formed network from spheroid to microchannel. By developing this vascular navigation system, the various tissue could be cultivated in vitro in the future.
(1) Biomaterials, 35, 7308, 2015. (2) Lab Chip, 13, 1489, 2013. (3) MEMS 2015, 476, 15/01/18-22, 2015.

For tissue regeneration, there have been various efforts using micro-/nano- structures for decades. Many researches have shown micro and nano topographical cues for the interactions between cells and micro-/nano- structures. It is demonstrated that cell shape and spreading on surfaces have played important roles in stem cell fates. Also, it is widely accepted that the composition and distribution of cytoskeletons, and cytoskeletal tensions affect stem cell fates. However, the regulatory mechanism of topographical effects on stem cells fate is not yet clearly understood. In this study, we examine the responses of adipose-derived stem cells (ASCs) on polyurethane acrylate (PUA) films with nanostructures on the surface. We have selected ASCs because they can potentially differentiate into various cell types and they are abundant in adipose tissue. ASCs are cultured on complementary pillar and pit arrays at the nanoscale for investigating the topographical cues of stem cell fates.
For the nanostructures, we first utilize colloidal lithography and plasma etching to fabricate nanopillar arrays on a quartz glass surface. Then, the nanopillar arrays are transferred to UV-curable PUA resin. The replicated nanopit array in PUA film is used as a mold to produce a nanopillar array in PUA film so that we can have complementary nanostructured films. ASCs are then seeded and cultured onto fibronectin coated PUA films. ASCs responses such as adhesion, proliferation and differentiation are periodically observed with time.
This research was supported by the MSIP(Ministry of Science, ICT and Future Planning), Korea, under the “IT Consilience Creative Program” (IITP-2015-R0346-15-1008) supervised by the IITP(Institute for Information & Communications Technology Promotion)

Tissue engineering using whole, intact cell sheets has shown promise in many cell-based therapies. In this work, we describe a new type of surface for the growth and release of cell sheets. Mouse mesenchymal stem cells were grown on slippery, liquid-infused polymeric substrates (SLIPS). Cell sheets transferred from such surfaces showed high viability and similar morphologies to controls grown on standard tissue culture surfaces. Growth and proliferation after transfer proceeded normally. This method of cell sheet growth and detachment may be useful for low-cost, flexible, and customizable production of cellular layers for tissue engineering.

Introduction: Successful repair for bone defects larger than the critical size depends on the regeneration rate of new bone tissue. Bone cells are mechanical sensitive, researches and clinical practices have shown that a proper physical stimulation may play positive roles in osteogenesis and enhance fracture healing, as osteoblasts and osteocytes are given constant and weak mechnical stimulations in the physiological environment. We consider that cells recruited into the implanted scaffold are not able to obtain the proper mechanical stimulation from the physiological environemtn at the beginning stage of implantation, because the scaffold and the defect end of bone have not form close junction each other. Aiming to provide weak but constant micro-deforming stimulations to bone-relating cells that grow in the scaffold, we developed super-paramagnetic responsive scaffolds.
Methods: Scaffolds were fabricated with super-paramagnetic γ-Fe₂O₃ nanoparticles, hydroxyapatite nanoparticles and poly lactide acid using electrospinning technique and implanted to the defect of rabbit lumbar transverse. Pathological observation, radiological analysis of CT and micro CT were performed within 110 days after the implantation surgery. The permanent magnets were fixed in the rabbit cages to provide the static magnetic field. In vitro studies were conducted with osteoblasts and immune cells. Cytokines related with inflammatory responses and wound healing were detected using RT-PCR, ELISA and western blot.
Results: The scaffolds present controllable super-paramagnetic performance as well as fibrous structures with pore diameter of 5~20 µm and diameter of 500 nm in average. The implanted scaffolds recruited monocyte-macrophages, fibroblasts and osteoblast, and were absorbed along with new matrix deposition and vessel-like structures formation. It was demonstrated that the new bone formation and remodelling in situ was faster at each testing time point for rabbits under the magnetic field than that for those in the normal control cages, due to the activation of osteoblasts and the variation of immunological microenvironments induced by the super-paramagnetic responsive scaffold under the applied static magnetic field. Results from In vitro studies supported the in vivo observation.
Conclusions: Super-paramagnetic scaffolds can accelerate the repair rate of bone defects under the applied static magnetic field, displaying promising potentials of application in the scaffold-guided bone regeneration.

All implantable biomedical systems face several risks once in contact with the host tissue. The main problems are i) excessive immune response to the implant; ii) development of bacterial biofilms and iii) yeast and fungi infections. A multifunctional surface coating which can address all these issues concomitantly would significantly improve clinical outcomes. We develop here for the first time a multifunctional coating that allows addressing these three issues simultaneously. We hypothesized that polyarginine (PAR), a synthetic highly cationic polypeptide, can act on macrophages to control innate immune response because arginine is an important component of macrophage metabolism. Moreover, PAR is susceptible to act as an antimicrobial agent due to its positive charges. In order to exploit these properties we developed a new polyelectrolyte multilayer (PEM) films based on PAR and hyaluronic acid (HA), which is a compound also known as an immunomodulator. It is first shown that this multilayer presents a thickness that increases exponentially as a function of the number of deposition steps. The PAR/HA films have a strong inhibitory effect on the production of inflammatory cytokines released by human primary macrophages subpopulations. This could reduce potential chronic inflammatory reaction following implantation. Next, we show that PAR has an antimicrobial activity both in solution and in film format. PAR/HA films were very effective against S. aureus for 24 h. In order to have a long-term antimicrobial activity, we tested deposition of a precursor nanoscale silver coating on the surface before adding the PAR/HA films. A synergistic effect due to the simultaneous presence of PAR/HA and silver coating was observed, enhancing both the short-term antimicrobial activity and conferring a long-term effect. The PAR/HA films can be easily further functionalized by embedding antimicrobial peptides, like catestatin (CAT), a natural host defense peptide. This PAR/HA+CAT film proved to be effective as an antimicrobial coating against yeast and fungi. The cytocompatibility of the PAR/HA films was assessed with human umbilical vein endothelial cells (HUVECs). This all-in-one system that limit strong inflammation and prevent bacteria, yeast and fungi infections constitutes an original strategy to coat implants in an active way, especially the implants located in non-sterile environments such as dental, pharyngeal or tracheal implants.

The nervous system plays an important role in the body to control and regulate various body organ activities. Since the axons or dendrites of continuous cell lines from central nervous system (CNS) are not well-defined, primary hippocampal culture is commonly used instead, as one of the most direct and effective ways of investigating the differentiation, nutritional requirements, and synapse formation of the neuron system. In specific, for the observation of highly mature and extended axons and dendrites of the primary neurons, long-term (generally greater than 1 month) culture of the primary hippocampal neuron cells is of critical importance, which had been shown to be extremely challenging.
One the most conventional ways for a stable culture of the primary neuron cells is using culture plates coated with a positively charged polymer film, poly-L-lysine (PLL), to provide an increased cell-adhesion and growth. However, the culture of the neuron cells on PLL-coated glass is limited only to a couple of weeks due to the low stability of water-soluble PLL, which is readily solubilized out by the neuronal medium during the culture. Therefore, a new surface modification method is highly desired to achieve a stable, long-term culture of the cells.
Here, we introduce a stabilized polymer with acetylcholine-like functionalities conformally coated on cover glass. The surface-modified glass demonstrated strong adhesive characteristics to the hippocampal neuron cells. In addition, the developed polymer coating was not soluble in culture medium, enabling its long-term use. The polymer film was coated via initiated chemical vapor deposition (iCVD), ensuring its high purity, and thus the biocompatibility to primary neuron culture. The neuron cells cultured on glass plates with iCVD coating showed rapidly and extensively extended axon branches and dendrites, resulting in 2.7 and 1.6-fold increase of axon length and dendritic length, respectively than the cells cultured on PLL-coated glass after a 8 day culture. The expression level of microtubule-associated proteins also greatly increased in the neuron cells cultured on the iCVD polymer-coated plates. Furthermore, the uniformly-distributed neuron cells with extended axons and dendrites survived over 50-day culture on the polymer-coated glass, confirming the long-term culture capability of the polymer-modified glass plates.
Considering the aforementioned findings, the surface modification by iCVD polymer can be adopted as a long-term neuronal culture platform, facilitating the research for investigating matured neuronal cells and their further applications to various fields, such as neuron regeneration and neural therapy.

Engineered thick living tissue constructs could enable new in vitro applications in 3D cell studies, drug screening, disease modeling, and, ultimately, therapeutic applications in regenerative medicine. We will highlight our recent efforts on concurrent patterning of cells and vasculature, along with new strategies to achieve active perfusion, long-term stability of thick living tissues (> 1cm thick), all of which are essential for creating a physiologically and therapeutically relevant tissue manufacturing method. As a demonstration of complex architecture and function at a physiologically relevant size scales, we directly differentiate patterned hBM-MSCs towards the osteogenic lineage via the in situ delivery of various factors through a pervasive vascular network, illustrating control over the long-term growth and development of our printed tissue. With control over multicellular architecture, the chemo-mechanical microenvironment, and the ability to support thick, developing tissue for long time points, this method could serve as a platform for studying emergent biological functions in complex engineered microenvironments, and, may ultimately, find applications in vivo.

This study reports the application of radio-frequency (RF) magnetron sputtering to prepare thin CaP-based coatings on the surface of permanent (Ti, NiTi) or biodegradable (Mg-based) metallic substrates.
Pure hydroxyapatite (HA) and silicon-containing HA coatings with the content of silicon 0.5 and 1.72 mol were deposited via a conventional RF magnetron sputtering set-up. Details on the coating deposition regimes can be found elsewhere. Different thickness of the coatings up to 1500 nm was obtained. The physico-chemical and biological properties of the biocomposites were studied via TEM, SEM, EDS, XRD. Wettability, contact angle hysteresis, nanohardness and adhesion strength, corrosion resistance; biological in vitro tests were done.
CaP coating homogeneously covered the entire surface of the substrates. In all cases a single phase coating of HA was prepared. Coating deposition process resulted in a slight increase of the surface roughness and nano-scale grains, generating an amorphous layer at the substrate/coating interface and inducing the growth of a columnar grain structure perpendicular to the substrate surface. A microstructural analysis of the film confirmed that the grain size and crystallinity increased when increasing the deposition time. The potentiodynamic polarization test demonstrated that a 1500-nm thick nanocrystalline HA coating significantly improved the corrosion resistance of the bare AZ31 magnesium alloy in a 3.5 wt.% NaCl solution. In the case NiTi substrates a significant decrease of the Ni release rate compare to the bare substrate was observed. Nanoindentation tests demonstrated that all HA coatings increased surface hardness on both the micro- and nanoscale. The films can significantly enhance the wear resistance of this resorbable alloy. The nanostructured HA-based coatings were not cytotoxic, as proven by in vitro assays using primary dental pulp stem cells and mouse fibroblast NCTC clone L929 cells. HA coatings with different thicknesses stimulated cells to attach, proliferate and form mineralized nodules on the surface better than uncoated substrates. The obtained results revealed that RF magnetron sputtering is a versatile technique to prepare nanostructured CaP-based coatings on the surface of biodegradable and permanent implants. The corrosion resistance of the magnesium-based alloys could be effectively improved via RF magnetron sputter deposited nanocrystalline CaP coatings. The experiments revealed that the bias applied during the deposition of HA coating resulted in a decrease of the grain and crystallite size of the film having a crucial role in providing the enhancement of the mechanical properties of the biocomposites.
ACKNOWLEDGEMENTS: The authors are thankful to the Russian President's Stipend SP-6664.2013.4 and #1052;#1050;-485.2014, NAUKA #11.1359.2014/K.

Over one million meniscal surgeries are performed in the United States every year as a consequence of knee trauma; 89% of the patients receiving these surgeries will develop osteoarthritis [1,2]. The menisci are two semilunar discs that sit on top of the tibial plateau in the knee joint and act to redistribute axial loads generated by walking, jumping, etc. Each meniscus attaches into the tibial plateau at two points through structures called entheses. The enthesis anchors the meniscus into the tibia and allows the meniscus to function. Entheses have a complex tissue structure that mediates the drastic change in modulus from the compliant cartilaginous tissue of the meniscus to the stiff subchondral bone in the tibia. This tissue gradient consists of four tissue types: ligamentous tissue, uncalcified fibrocartilage, calcified fibrocartilage, subchondral bone. This study is aimed at understanding the role biomacromolecules and mineral components play in mitigating the mechanical transition from compliant to stiff tissue by developing a structure-function relationship using correlative Raman spectroscopy and microstrain mapping. The medial caudal entheses of neonatal bovine joints were removed for analysis. Maps of collagen, hydroxyapatite, glycosaminoglycans, and cells were developed across the enthesis using Raman microspectroscopy. The same samples were subjected to tensile loads using a custom-built micro-loadframe. Tensile loading was recorded using confocal fluorescence microscopy, and microstrain maps were developed from a custom MATLAB code. These data were correlated to relate microscopic composition changes with microstrain values over the tissue gradient structure. These results will be used to inform the synthesis of an artificial enthesis construct based on partially mineralized collagen gels for the purpose of producing a fully functional tissue engineered meniscus.
[1] E. A. Khetia and B. P. McKeon, “Meniscal allografts: biomechanics and techniques.,” Sports Med. Arthrosc., vol. 15, no. 3, pp. 114-20, 2007.
[2] C. Rangger, A. Kathrein, T. Klestil, and W. Glötzer, “Partial Meniscectomy and Osteoarthritis Implications for Treatment of Athletes,” Sport. Med., vol. 23, no. 1, pp. 61-68, 1997.

Diseases affecting the competency of the semilunar heart valves, aortic and pulmonary valves, affect millions of patients every year and are a leading cause of cardiovascular mortality worldwide. Diseased semilunar valves can be replaced with mechanical prosthetics or fixed xenograft tissue valves to restore function in patients. However, neither of these solutions is permanent and allow for the patient to remodel and regenerate new, healthy valve tissue. Transcatheter implantation of replacement heart valves is being developed to minimize the risk of this invasive surgery and we ask if this new delivery technique can be merged with permanent tissue engineering approaches to engineer a multiscale semilunar heart valve replacement strategy. Here we show that by using our Rotary Jet Spinning (RJS) nanofiber fabrication technique, we are able to recapitulate semilunar valve structure from the extracellular matrix to the global organ scale. Scaffolds composed of poly-4-hydroxybutyrate (P4HB, polyester), polyglycolide (PGA, polyester), and gelatin (protein) are seamlessly spun into the fibrous scaffold to provide structure, illicit healthy inflammation, and provide a platform for native cell attachment respectively thus potentially encouraging the endogenous regeneration of new and healthy tissue. The polymer:protein hybrid scaffold is tuned to mimic the mechanical properties of the native valve leaflets and ensure proper hemodynamics. Scaffolds are spun three dimensionally on semilunar valve shaped mandrels to produce the organ-level structure and have been functionally testing in vitro with the Pulse Duplicator flow system and acutely in a ovine pulmonary valve in vivo model via transapical catheter delivery. Successful in vitro flow testing under pulmonary conditions, <20% total regurgitation, corresponds with successful, functional valve testing performed in the adult ovine in vivo model (acute, 15hr). Minimal closing volume during diastole, low transvalvular pressures during systole (0-2mmHg), full neutrophil penetration into the valve, and the absence of clotting on the scaffold during these acute in vivo studies (15h) have inspired the next round of chronic animal studies which are currently in progress. Our results suggest that we have engineered a competent, functional semilunar valve replacement by combining a simplified tissue engineering approach with a transcatheter delivery technique for minimally invasive implantation that has the potential to grow and adapt with the patient. This is of particular importance in children born with congenital valve defects who require multiple, invasive surgeries throughout their lifetime using the current replacement strategies.

Prosthetic joint replacement is an important and increasingly common medical procedure. Infections associated with whole joint replacements, however, cause increased morbidity and significant healthcare cost. A prolonged and expensive two-stage procedure is currently the only viable option for revision arthroplasty for an infected prosthesis. Here we report an approach for one-stage revision using implants coated with a self-assembled, hydrolytically degradable polymer-based bioactive coating carrying the antibiotic gentamicin sulfate (GS), and the osteoinductive factor, bone morphogenetic protein-2 (BMP-2). The multilayered coating was assembled using a water-based layer-by-layer approach, in which each element was deposited on the surface in nanoscale layers. The controlled, localized release of GS was essential to eliminate infection and to protect the implant against re-infection for multiple weeks. The subsequent release of BMP-2 induced the rapid and complete integration of new well-developed trabecular bone around the implant. In a rat model the ‘Two-in-One&’ multilayer coatings eliminated the existing infection and bacterial biofilm, and protected the implant against re-infection, facilitated early deposition of bone on the implant, and resulted in at least 10-fold higher bone-implant interfacial shear strength and long-term mechanical stability when compared with the uncoated control implants, demonstrating the potential of this strategy for single-stage treatment of prosthesis-related infections for revision arthroplasty.

The dermal layer of natural skin is mainly composed of densely-packed, random collagen nanofibril. To mimic the structure, we have developed an electrochemical process to align collagen macromolecules two dimensionally (2D) into densely-packed, nano fibrous sheets. The electrochemically aligned collagen sheets were prepared with various degree of crosslinking, porosity,and thickness. The structure of these sheets was characterized by polarized optical microscope and scanning electron microscope (SEM). The biocompatibility of the collagen sheets were tested using adipose-derived stem cells. Furthermore, the moisture vapor transmission rate (MVTR) of collagen sheet was also tested in vitro with or without an temporary epidermis polymer. It appeared that electrochemically aligned collagen has good biocompatibility, degradability, and porosity. Our next step is to evaluate its suitability as a skin substitute in a porcine skin burn wound model.

Since adult cardiomyocytes are not readily available for clinical use, numerous efforts have been made to derive functional cardiomyocytes from pluripotent stem cells in order to develop engineered cardiac tissues using various types of scaffolds. While graphene, a single layer carbon crystal, has recently become a material of interest for tissue engineering applications including osteogenic, neural and stem cell differentiation, its potential for cardiac tissue engineering is yet unknown. The inherent electro-activity of the myocardium makes graphene an especially attractive option for cardiac tissue engineering due to its high electrical conductivity. Thus, a novel hybrid 3D scaffold with graphene has been developed to examine the function of stem cell derived cardiomyocytes. These nanostructured scaffolds have been fabricated using electrospinning process. Scaffolds with random orientation as well as axial orientation have been thoroughly investigated for their mechanical and electrical properties. Improvement in the fiber orientation and overall consistent fiber diameter across its length was observed in scaffolds with graphene. Graphene creates a localized conductive spot which helps electrical charges to concentrate on the polymer beads that act as electrical stress centers which possibly led to continuous stretching of the fibers during electrospinning process. The overall conductivity of the graphene containing PCL scaffold has been investigated using electrical impedance spectroscopy by applying 100 mV AC field at the frequency of 100 Hz across two electrodes. A consistent decrease in the impedance was observed with corresponding increase in the graphene concentrations, suggesting improved conductivity of the nanostructured scaffolds with higher graphene concentration. Our study with mouse embryonic stem cell derived cardiomyocytes (mES-CM) demonstrate the biocompatibility of PCL-graphene scaffolds and functional characterization of cardiomyocytes are currently being investigated. The scaffolds with mES-CM were seeded at 8x10⁶ cells/cm² on PCL and PCL+G scaffold and cultured for 6 days. The study reveals well registered sacromeres present in mES-CM on scaffolds resulting in synchronously contracting tissue that is detected macroscopically. Average spontaneous beating frequency of mES-CM on PCL and PCL+G scaffolds is approximately 1.28±0.20 Hz (n=7±SEM) and 1.21±0.22 Hz (n=8±SEM) respectively. 3D scaffolds express higher MHC (myosin heavy chain) compared to that of 2D culture. mES-CM adheres well and remain highly viable on both PCL and PCL+G scaffolds. This study demonstrates the biocompatibility and feasibility of PCL+G scaffolds for cardiac tissue engineering applications.

Carbonate apatite (CO₃Ap) has attracted great attention due to its higher osteoconductivity and replacement new bone. Interconnected porous. Since replacement of CO₃Ap to a new bone is carried out by osteoclasts and osteoblasts similar to bone remodeling process, interconnected porous structure accelerate bone remodeling process.
In this study, polyurethane foam (PUF) was used as a porogen. Some PUF were coated with polyurethane to increase the thickness of the strut. Ca(OH)₂ slurry was introduced to PUF, followed by sintering at 700°C under O₂-CO₂ mixture to burn out PUF and carbonate Ca(OH)₂. Heat treatment below 650°C result in incomplete burning out of PUF whereas heat treatment above 750°C result in the decomposition of CaCO3 to form CaO. The heat treatment fabricated interconnected porous CaCO3. Then the CaCO₃ was immersed in Na₂HPO₄ solution. No macroscopic change was observed even when the CaCO₃ was immersed in Na₂HPO₄ solution. However, the composition of the porous body became CO₃Ap upon exposure to Na₂HPO₄ solution. Compressive strength of the porous CaCO₃ became lower after its compositional transformation to CO₃Ap.
The cylindrical shaped porous CO₃Ap and hydroxyapatite (HAp) foams made similar to CO₃Ap foam were implanted in the tibia of 17wks rabbits, and evaluated by means of m-CT and histologically. One month post-implantation, new bone and blood capillary were found in the case of CO₃Ap foam. The amount of bone and number of blood capillary were larger when larger pore was made using strut thickened PUF. At this stage, no bone penetration nor blood capillary were found in the case of HAp foam. Three months post-implantation, the amount of bone and number of blood capillary were larger when compared to 1 month results. Formed bone became matured. In the case of HAp foam, bone was formed only at lager pore. No blood capillary was found even at this stage. These tendency were the same even 12 months post-implantation. As a result, HAp foam maintained its shape and was not replaced to bone. In contrast, CO₃Ap foam was replaced to new bone.
It is concluded that CO₃Ap foam has good potential to be used as artificial bone replacement due to its high osteoconductivity and its replacement to new bone.

The use of polymeric scaffolds is an alternative to traditional autografts and allografts to repair non-healing bone defects. Natural polymers, due to their chemical and structural similarity with native tissue components often present several qualities beneficial to applications of tissue regeneration. In this study, we present the characterization of mechanically stable cellulose acetate and collagen based micro-nano structured scaffolds in terms of their ability to promote hMSC&’s progression into osteoblastic lineage in vitro and the biocompatibility of the scaffolds in vivo. Cellulose acetate (CA), poly (lactic-co-glycolic acid) (PLGA) and their collagen-functionalized scaffolds (CAc, PLGAc) were formulated as discs. Each scaffold was seed with 500,000 human bone marrow derived mesenchymal stem cells (hMSCs) and cultured using inductive media for 21 days. hMSC viability, proliferation and differentiation were evaluated temporally by Live Dead, Pico green (DNA) assays, alkaline phosphate activity (ALP), mineralization, changes in gene osteogenic expression (RUNX2, Coll1, ON, and BSP) and presentation of proteins (coll1 and BSP). Further, CA, CAc and PLGA scaffolds were subcutaneously implanted into Sprague- Dawley rats. At set time points, the samples were recovered and evaluated histologically for immune responses and vascularization. Both the test CA and CAc scaffolds maintained higher levels of osteogenic gene and protein expression levels indicating greater long-term osteoinduction on these scaffolds. The test scaffolds also showed greater cellular infiltration and vascularization in the subcutaneous implants. These results indicate the osteogenic potential of CA and CAc scaffolds in vitro and their biocompatibility in vivo.

Elastic hydrogels carry the potential to serve as scaffolds for various biomedical applications. Though some elastic hydrogels are biocompatible, most of the synthetic polymer-based elastic scaffolds lack bioactive sequences to promote cell adhesion or migration, which is important for their applications in tissue engineering. Alternatively, recombinant protein-based polymers such as elastin-like polypeptides (ELPs) are biocompatible and have been widely investigated for biomedical applications. Photocrosslinkable, protein-based biomaterials provide controllable and biocompatible crosslinking, permitting their use as tissue scaffolds and surgical materials.
In this study, engineered ELPs were designed by recombinant expression in Escherichia coli (E. coli), followed by purification by inverse transition cycling. To fabricate the hydrogels, lyophilized ELP was dissolved in phosphate-buffered saline (PBS) and photocrosslinked with UV light (360-480 nm wavelength, 850 mW) for 3 min. The swelling ratios of 10, 15 and 20 %(w/v) ELP hydrogels were evaluated in PBS at 4 °C and 37 °C. Tensile and compressive cyclic testing of ELP hydrogels were performed using a mechanical tester (Instron model 5542) with a 10 N load cell. The gels were extensible up to 420% strain and exhibited fatigue resistance in compression. The inclusion of thiol groups in expressed ELPs allowed for rapid photocrosslinking of hydrogels that maintained the elasticity and biocompatibility inherent in ELPs. The large extensibility of ELPs is important for the engineering of elastic tissues. The recombinant design of these ELPs allow for exceptional control over the presentation of bioactive peptide sequences, which can be used to improve cell viability, proliferation or promote specific cellular interactions in vitro or in vivo. In vivo examination revealed excellent biocompatibility, and minimal degradation over two months. Furthermore, hemostatic functionalization with nanoparticles (NPs) allowed for effective treatment of a lethal bleeding liver wound. With these qualities, such a photocrosslinkable system has potential to be used in biomedical applications as a sealant for soft, flexible tissue injuries like in blood vessels, skin, lung, or cardiac tissue.

Approximately 114 million surgical and procedure-based wounds occur annually worldwide, including 36 million from surgeries in the US. Reconnection of tissues is crucial for restoring proper function and structure. Sutures, wires, and staples are widely used for this purpose. Despite their common use in the clinic, these methods exhibit limitations when applied to fragile and elastic tissues, especially if the intention is to inhibit fluid or air leakage against high pressure, e.g. vascular and lung surgeries. Additionally, the application of these methods can be challenging in minimally invasive procedures with restricted access. To address these limitations, various types of surgical materials have been used for sealing, reconnecting tissues, or attaching devices to tissues. However, existing surgical sealant materials often feature limited adhesion strength, high toxicity, absence of appropriate mechanics, and do not function in wet environments. We aim to overcome these limitations by engineering a highly elastic, biocompatible and biodegradable sealant through photocrosslinking of a human protein. This light-activated elastic sealant proves to be suitable when sealing air and body fluid leakages during surgical procedures.
To fabricate the hydrogels, methacrylated tropoelastin (MeTro) was dissolved in distilled water and photocrosslinked through UV light within 3 min. The pore characteristics and swelling ratios of MeTro hydrogels with different MeTro concentrations and methacrylation degrees were evaluated. MeTro exhibited tunable mechanical properties at varying MeTro concentrations and methacrylation degrees. The adhesion strength of the MeTro sealant, tested by wound closure and lap shear tests, showed superior properties when compared to commercially available sealants such as Evicel and Coseal. Due to the nature of recombinant human protein, improved cell viability and proliferation were achieved by using MeTro materials during in vitro studies. Subcutaneous implantation of the material in rats showed excellent biocompatibility of MeTro and tissue/material integration. In addition, MeTro sealed an incision created on rat lung tissue and promoted lung tissue regeneration as confirmed by our in vivo survival studies using a rat lung incision model. We envision that this sealant will be easily commercialized as it is fabricated from a recombinant human protein, and its degradation and physical properties are controllable. Our in vitro and in vivo data suggest that this material is superior to the existing products on the market and may generate a paradigm-shifting surgical sealant that may not require supportive sutures due to its superior mechanical and adhesive properties.

Severe peripheral nerve injury may result in large gaps and lead to paralysis. Although nerve guidance conduits (NGCs) have been widely investigated to treat peripheral nerve injuries, they are usually tubular guidance channels that lack sufficient surface support for successful peripheral nerve regeneration (PNR). The incorporation of growth factors into NGCs is a general strategy for PNR, but many available approaches are not ideal due to the burst release and early loss of growth factors. To overcome some of these drawbacks, a novel spiral structured nanofiber based NGC is developed for PNR in this study. The NGC is fabricated with a biodegradable and biocompatible polymer, polycaprolactone (PCL), using a combination of solvent casting, salt leaching, and electrospinning to create a conduit that has an inner spiral-structured core with multi-channels, aligned nanofibers and an outer tubular shell composed of random nanofibers. The incorporation of micro-structures in longitudinally oriented channels and aligned nanofibers may provide the directional guiding cues to promote PNR. The novel spiral structured configuration of the NGC can significantly increase surface areas for PNR, provide the mechanical stability, and allow sufficient nutrient supply and metabolic waste removal. The NGC was characterized for morphology, porosity, degradation rate, mechanical properties, and cellular responses of Schwann cells and PC-12 cells. The scanning electron microscope showed the average pore size was around 100 µm, the porosity of the NGC was between 70-80%, and the NGC degraded 20% in mass in 180 days. The mechanical properties of the NGC were around 10.8 MPa which is similar to that of the native nerve. The NGC supported Schwann cell attachment, proliferation and migration and significantly improved PC-12 cells neurite extension.
Additionally, the inner aligned nanofibers were further functionalized by incorporating nerve growth factor (NGF) to enhance PNR. While researchers observed burst release in previous attempts to achieve controlled release of NGF, we overcame this drawback by blending bovine serum albumin (BSA) and polyethylene glycol bis(amine) (PEG-amine) with NGF into the inner aligned PCL nanofibers. The BSA was incorporated into the solution for better retention and release of NGF. PEG-amine provides the functional groups that allows for surface modification. We assessed the release kinetics of NGF from the NGC using enzyme-linked immunosorbent assay and evaluated neurite extension of PC12 cells. The NGF modified NGC had a stable controlled release profile for 21 days and significantly improved PC-12 cell neurite extension compared to the controlled NGC without NGF. This study will provide further insight into the impact of incorporating micro-channels, highly aligned nanofibers, spiral structures and NGF into NGCs for PNR.
Acknowledgement: The work was supported by the DoD (W81XWH-13-1-0320) and NIH-R15 NS074404.

Three-dimensional cell culture formats are desirable in applications ranging from mechanistic analysis of tissue behavior to modeling human responses to new drugs. There is a growing interest in analyzing paracrine interactions between stroma, epithelia, and immune cells in 3D physiological cultures, however the commonly-employed proteolytic methods used to recover cells from 3D gels are often inefficient and can impair fidelity of measurements such as flow cytometry for cell surface protein expression, and they also are not well-suited to allow recovery of locally-released growth factors and cytokines. Here, we present a method for rapidly (<5 mins) dissolving synthetic polyethylene glycol (PEG)-based extracellular matrices (ECMs) using a bacterial transpeptidase with almost no known substrates in native mammalian proteins, in a manner that preserves cell viability and phenotype, and allows 3D passaging of cells. In brief, the approach relies on inclusion of a substrate for the enzyme in the gel crosslinks, allowing the gel to be dissolved by addition of the enzyme and a second simple substrate for the transpeptidase; hence it is translatable to any synthetic gel system. The approach is robust to a range of gel properties and conditions. Specifically, we showed that the method is robust to cross-linking chemistry, degree of cross-linking, polymer architecture (different macromer MW), presence of serum, and that degradation time is tunable through sortase concentration. We used sortase chemistry to recover cells from a 3D PEG hydrogel in <5 minutes without significantly decreasing their viability. Additionally, we passaged epithelial endometrial cells in 3D (from one gel to another) and showed that the cells continue to proliferate and maintained their spheroid structure. We developed a powerful, easily transferable system for recovering cells from 3D matrices in a bio-orthogonal matter. This method does not affect cell viability and could be extremely useful in cell signaling studies and morphogenesis assays.

Arginine-glycine-aspartic acid (RGD) peptide receptors have been used for cell adhesion between cells and extracellular matrix. These features of the RGD peptides were used with Poly(methacrylic acid) (PMAA) to synthesize PMAA-RGD-PMAA block copolymer, which had charged long side chains on the both ends of middle short chain tethering peptides that can interact with cells. We tested the cell adhesion and migration effect of this RGD modified polymer^1,2. Whiteside stamp method was used to pattern a set of gold stripes on the silicon surface³. Self-assembled monolayers (SAMs) of alkanethiols were printed on gold as etch resist while the bare regions of the gold were patterned by potassium iodide/iodine solutions⁴. The characteristics of the striped surface were examined with atomic forced microscopy (AFM). By applying electric field on the surface, the charged synthesized polymer chains stretched and exposed the RGD domain. Cell mobility tests employing fibroblasts were then performed on the surface under confocal microscopy.
References
1.Petersen, Eric, et al. "DNA migration and separation on surfaces with a microscale dielectrophoretic trap array." Physical review letters 98.8 (2007): 088102.
2.Seo, Young-Soo, et al. "DNA separation at a liquid-solid interface."Electrophoresis 23.16 (2002): 2618-2625.
3.Kumar, Amit, et al. "The use of self-assembled monolayers and a selective etch to generate patterned gold features." Journal of the American Chemical Society 114.23 (1992): 9188-9189.
4.Benor, Amare, et al. "Organic transistors realized by an environmental friendly microcontact printing approach." Organic Electronics 11.5 (2010): 831-835.

Hydroxyapatite is a very promising bioceramic for the fabrication of a new generation of bone tissue implants. This material has superior biocompatible properties, due mostly because it composed a big fraction of the inorganic matrix in bone tissue. However, because of its specific crystal phase, hydroxyapatite contains a small elastic modulus which make it a very brittle material. This property has stop, at the moment, the development of new highly biocompatible orthopaedic implants and now a days the work of providing mechanical support to bones is hardly done by low biocompatible, health dangerous metallic implants.
Graphene, a carbon allotrope, provides an alternative to improve the elastic modulus of hydroxyapatite because of their superior Young's modulus (~1 TPa). Furthermore, this 2D material has a large specific surface area that can be used for its functionalization with others nanomaterials such as hydroxyapatite. By using this two materials to synthetized a new hybrid nanomaterial we can obtain the properties needed for the fabrication of highly biocompatible and mechanical resistant bone tissue implants.
In this project we used a hydrothermal method to in situ nucleate crystalline hydroxyapatite nanoparticles on the surface of graphene oxide layers. Through modifications on the synthesis route we have successfully controlled hydroxyapatite morphology producing three different types of this nanomaterial on the surface on graphene, maintaining the hexagonal crystal phase proper of this ceramic. Moreover, we performed a full characterization of the new hybrid nanomaterial using spectroscopy techniques and electron microscopy visualization, verifying the presence of both materials and their three different morphologies and some of their thermal properties.
Finally we performed a cytotoxic assessment employing a MTT assay. Using murine fibroblast cells cultures we measured the cell viability and biological interaction of the new composite graphene/hydroxyapatite. The results shows that this material has a high biocompatibility which makes it a strong material for the fabrication of biological and mechanical improved orthopaedic implants.

Liver transplantation is an exclusive treatment for severe liver failures. Because of the shortage of donor livers, development of bioartificial liver (BAL) is expected. The substrata for BAL require the following criteria; (1) blood compatibility, (2) the capability of hepatocyte attachment, (3) the capability to maintain hepatocyte-specific functions. We have reported that poly (2-methoxyethyl acrylate) (PMEA) and poly (tetrahydrofurfuryl acrylate) (PTHFA) exhibit excellent blood compatibility. More recently, we have shown that non-blood cells can attach on PMEA and PTHFA substrata (T. Hoshiba, Adv Healthcare Mater, 2014). On these substrata, the cell shape can be regulated from spreading shape to round shape through the control of integrin contribution to cell attachment. It has been reported that hepatocyte-specific functions increased when hepatocyte forms round shape. Therefore, it is expected that PMEA or PTHFA can be utilized as the substrata for BAL substrata. Here, we examined the possibility of blood-compatible PMEA and PTHFA to use them as the substrata for BAL.
HepG2, a human hepatocyte model, could adhere on PMEA and PTHFA substrata. HepG2 cells can attach on blood compatible PMEA and PTHFA substrata. The spreading of HepG2 cells was suppressed on PMEA substratum because integrin contribution to cell attachment on PMEA substratum was low and integrin signaling was not sufficiently activated. Hepatocyte-specific gene expression in HepG2 cells increased on PMEA substratum whereas the expression decreased on PTHFA substratum. To address why hepatocyte-specific functions increased on PMEA substratum, we focused on the localization of Yes-associated protein (YAP) whose nuclear localization suppressed hepatocyte-specific functions. On PMEA substratum, YAP is dispersed in the cytosol whereas YAP is localized into cell nucleus on PTHFA substratum to decrease the expression of hepatocyte-specific functions.
These results indicate that blood-compatible PMEA is suitable for BAL substrate. Also, PMEA is expected to be used to regulate of cell functions for blood-contacting tissue engineering.

Bioglass is known to stimulate bone regeneration and have been considered for the fabrication of porous scaffolds for bone reconstruction. Thermal treatments used for the manufacturing of 45S5 Bioglass®-based scaffolds cause the formation of crystalline phases in the material¹. While the effect of the modification of the microstructure during these thermal treatments was previously investigated by different investigators^2-4,the dissolution of Bioglass®-based glass-ceramic scaffold has not been extensively documented. This work presents the in vitro and in vivo microstructure evolution of Bioglass®-based scaffolds produced with a powder metallurgy technology⁶.
Microstructure evolution was studied after immersion in deionized (DI) H₂O, pH 4.5 at 20, 40 and 80 °C, Tris-buffered SBF, pH 7.4 37 °C, and subcutaneous implantation in rats for 3 and 6 weeks. SBF and DI H₂O were changed every 3 and 1 days respectively, in order to maintain a steady environment. In a parallel test, DI H₂O was kept stirring at 20°C without any further change. As sintered microstructures revealed a interdigitated network of crystalline (Na_4.2Ca_2.8(Si₆O₁₈) and CaNaPO₄) and amorphous phases. During immersion in SBF an amorphous hump was visible on the XRD pattern after 7 days, while, in water, amorphization was observed only after 48 days for the specimens immersed at 80 °C and 20 °C (samples in stirred solution). Amorphization of explanted specimen was observed after 6 weeks. The formation of calcium phosphate phase after soaking in SBF and in vivo implantation appeared on XRD pattern after 28 days and 21 days respectively; crystalline phases disappeared completely in SBF while they were still visible in explanted specimens.
Resin mounted samples revealed the formation of an amorphous Si-rich conversion layer in explanted samples, which grew toward the core of the scaffold struts while the external surface was enriched in Ca and P. Similar Si-rich layer formed in stirring H₂O but without the CaP top layer, which could not form in DI H₂O. The resorption of 45S5-based glass ceramic scaffolds, produced with powder metallurgy technology, seems to be associated with the amorphization of the structure. No apparent crystal-glass interface instability was observed. Mechanical consistency was maintained at the end of each soaking test and in ex-vivo samples. Microscopic observation on histologies reveal the increase of the number of vessels from 3 to 6 week experimental point associated to a progressive resorption of the material and enlargement of the pores.
References
1. Bellucci, D. Science of Sintering 2010,42 (3), 307-20.
2. Chen, Q. Z. Biomaterials 2006,27 (11), 2414-25.
3. Jones, J. R. Acta Biomaterialia 2013,9 (1), 4457-86.
4. Comesaña, R. Scientific Reports 2015, 5.
5. Hallab, N. J. Bulletin of the NYU hospital for joint diseases 2009,67 (2), 182-8.
6. Aguilar-Reyes, E.A. Materials Science & Technology 2010 Conference and Exhibition, 2010; 70-7.

The generation of various types of reactive oxygen species (ROS), such as the superoxide anion (O₂^-.), the hydroxyl radical (bull;OH), and hydrogen peroxide (H₂O₂) are very common in aerobic species since they are produced as byproducts during mitochondrial electron transport. Recently, many research groups have started to investigate the potential of nanoscaled materials with antioxidant properties like rare earth oxide nanoparticles, fullerenes and carbon nanotubes. As one of the most novel nanoparticle chemistries, nanoscaled cerium oxide has proven to be a promising candidate for numerous biological applications. Recently it has been reported that nanoceria can scavenge reactive oxygen/nitrogen species, including the superoxide radical, hydrogen peroxide and hydroxyl radicals due to its unique ability to switch its oxidation state from Ce⁺³ to Ce⁺⁴. For these reasons, the objective of the present study was to use a novel chemistry, cerium oxide as an antioxidant at different pH values.
Ceria nanoparticles were synthesized from 1 mL aqueous solutions of 1 M cerium nitrate (Sigma Aldrich, St Louis, MO) and 2 mL of 0.1 M dextran T-10 (Pharmacosmos, Holback, Denmark). These solutions were added dropwise to 6 mL of a 30% ammonium hydroxide (Sigma Aldrich, St. Louis, MO) solution while stirring for 24 hours at 25 ^omicron;C. The synthesized nanoparticles were characterized in terms of their size via Transmission Electron Microscopy (TEM). Cytotoxicity (MTS) assays were carried out with human dermal fibroblast (HDF) cells for 1 day in culture using DMEM, 10% FBS and a 1% penicillin-streptomycin solution. Cells were seeded at a density of 5,000 cells/well, allowed to adhere for 24 hours and the following day, in order to determine the cyto-protective function of ceria nanoparticles, cells were preincubated with ceria at a 250, 500, 1000 µg/mL concentrations for 2, 4, 16, and 24 hours at different pH values pH 6 and pH 7. Following pre-incubation, cells were treated with different (1 mM, 1.5 mM, and 1.75 mM) H₂O₂ concentrations for another 24 hours. After 24 hours of incubation, the MTS assay for cell viability and the carboxy-H₂DCFDA assay was used to quantify ROS generation. 0.1 M dextran coated, sub 5 nm cerium oxide nanoparticles were synthesized, and utilized to rescue HDF cells. Cells were rescued when they were pre-incubated with 0.1 M dextan coated ceria before being exposed to the non-specific ROS source H₂O₂ in a time, concentration and pH dependent manner. Results suggested that as nanoparticle concentration and pre-incubation time increased, its capacity to scavenge higher molarity H₂O₂ was enhanced. Also at physiological pH values ceria showed better antioxidant properties. ROS assay results also suggested that the viability of the cells were directly correlated with the ROS amount that the cells produced. 0.1 M dextran coated cerium oxide particles caused a decrease in ROS generation in the presence of non-specific ROS.

Robust synthetic biolubricants have been searched for the coating of implants and the treatment of diseases, such as osteoarthritis, without real success. A mucin known as lubricin, found in the synovial fluid, is believed to be the glycoprotein responsible for the low friction and wear protection of cartilage. However, lubricin cannot yet be obtained recombinantly and is very expensive to extract, hence a suitable lubricin-mimetic molecule is still to be discovered. In this study, we combined Atomic Force Microscopy (AFM) and Surface Forces Apparatus (SFA) spectroscopy to characterize surface coverage as well as normal and friction forces of a lubricin-mimetic pAA-PEG copolymer (pAA-62kDa, PEG-2kDa) interacting with fibronectin (FN), a protein of the extracellular matrix found in the superficial zone of cartilage.
The pAA-PEG polymer coverage, distribution, and roughness were quantified by AFM for three different polymer concentrations, 0.3mg/ml, 1mg/ml, and 3mg/ml. In parallel, both the normal and the lateral (friction) forces of pAA-PEG were recorded using the SFA, in presence and absence of FN. Our AFM data indicated that the pAA-PEG polymer (i) had an average contour length and a diameter of 72nm and 10nm, respectively, and (ii) could self-associate to form a highly interconnected network onto surfaces, at all three concentrations. Our SFA data showed that the pAA-PEG polymer was only weakly adsorbed onto (negatively charged) bare mica surfaces but became firmly attached when FN was added as a polymer linker. FN also appeared to act as an efficient protector against mica surface damage when shear was applied. All our friction data exhibited (i) low friction coefficients (m asymp; 0.25) up to applied pressures of circa 3MPa and (ii) Amonton&’s like behavior, although poor wear protection was observed in the absence of FN. Friction coefficients also showed a weak shear velocity dependency. Together, AFM and SFA have allowed us for molecular and microscopic characterization of the pAA-PEG, helping us gaining insight into both the lubrication mechanisms and the interactions of the biomimetic polymer with tissue (FN). Collectively, these results indicate that our proposed lubricin-mimetic lubricant might be a promising cheap alternative to lubricin, as it improves lubrication and protects shearing surfaces against wear, in presence of the extracellular matrix proteins present in cartilage.

Silk fibroin scaffolds for bone tissue engineering can be produced by porogen-leaching, lyophilisation or gas foaming^1,2,3. For the first approach, individually tunable pore and interconnection size can be achieved for scaffolds templated by porogens produced by microfluidics, known as inverse opals⁴. The homogeneous architecture of inverse opals has been shown to impact not only cell distribution and diffusion of macromolecules throughout the construct but also differentiation of preosteoblastic cells⁵. In this work, silk fibroin scaffolds exhibiting an inverse-opal structure were developed and compared to others fabricated by the conventional salt-leaching method in terms of performance in bone tissue engineering. Monodisperse polycaprolactone particles were produced in a microfluidic device by creating droplets of a polycaprolactone solution in water and subsequently slowly evaporating the solvent. After washing and assembling them into a close-packed lattice, a heat treatment was conducted to induce necking between the particles. The particle pack was then infiltrated with an aqueous silk fibroin solution. Upon complete evaporation of the water, the porogen particles were dissolved. Salt-leached scaffolds were produced using table salt particles as porogens, as reported elsewhere¹. The scaffolds were seeded with human mesenchymal stem cells and cultured in osteogenic and control medium as described elsewhere¹. The amount of DNA, alkaline phosphatase (ALP) activity and the degree of mineralization were measured using a biochemical assays after 2, 4 and 6 weeks in both osteogenic and control culture. Statistical analysis was done by ANOVA with a posthoc Bonferroni correction. Increased calcium deposition per scaffold (p < 0.01) was observed for the inverse-opal scaffolds, even though no statistical difference in cell number or ALP activity was observed. This increase might have arisen from differences in pore curvature, pore monodispersity and/or pore wall stiffness. In a next step, potentially osteoinductive submicrometric topographical features of a known size were introduced to the pore walls by decorating the pore templates with latex particles.
1. Hofmann S. et al., Biomater. 28:1152, 2007
2. Hofmann S. et al., J. Pharma Biopharma, 85:119, 2013
3. Nazarov R. et al., Biomacromol, 5:718, 2004
4. Zhang YS et al., Macromol. Rapid Commun, 34 :485, 2013
5. Choi SW et al., Langmuir, 26(24) :19001, 2010

Spider silk-based bioengineered biomaterials offer a range of utility for biomedical applications due to the unique material properties, tailorability of sequence, degradability and biocompatibility. The features of silk proteins and ability to provide control over silk protein structural properties through self-assembly were combined with biosilica to develop new hybrid biomaterial systems to promote bone regeneration. To achieve this outcome, genetic consensus domains from the major ampullate spidroin 1 (MaSp1) protein of Nephila clavipes spider dragline were fused to oligonucleotides encoding the R5 peptide derived from the silaffin protein of Cylindrotheca fusiformis. Subsequently, modified dragline silk (15mer, MW 45kDa), (SGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGT)₁₅ domain was directly fused with the R5 peptide (SSKKSGSYSGSKGSKRRIL). The design of these chimeras resulted from the optimization of previously generated and analyzed chimeras. A series of variants were designed to analyze the influence of the position of the R5 peptide (i.e., N-terminal, C-terminal) on material properties and osteogenic differentiation of cells grown on the materials. Additionally, the length of the sequence between silk and the His-tag at chimera termini was decreased. The ability of the 15mer-R5 and R5-15mer proteins to control silicification was compared to the 15mer alone (silk control). No significant differences in biosilicification were observed regarding the position of R5, while protein β-sheet content impacted biosilicification. A higher content of positively charged amino acids analyzed by replacement of R5 sequence with silaffin in the chimera design resulted in increased average silica particle size. The induction of osteogenic differentiation of human mesenchymal stem cells (hMSCs) grown on these chimeric proteins was demonstrated with production of bone sialoprotein, calcium deposition and tracking osteogenic cell signaling pathways. These results suggest that silk-silica chimeric protein designs provide new options for control over biosilicification and silica particle size to generate biomaterial scaffolds for bone regeneration.

Total body irradiation is necessary for bone marrow or hematopoietic stem cell transplantation as a treatment for diseases such as lymphomas, leukemias, and sarcomas. Recovery of the patient is highly dependent upon engraftment of the transplant and subsequent regeneration of the bone marrow compartment post-irradiation. We have shown recently that an enriched subpopulation of osteoprogenitors isolated from human mesenchymal stem cells (hMSCs) can promote engraftment and regeneration of the bone marrow compartment in vivo through the cell secretome. However, these osteoprogenitors constitute a small fraction of the heterogeneous hMSC population. Here we leverage cell-material interactions to induce a heterogenous population of hMSCs to secrete more factors that are beneficial to regeneration. We created polydimethylsiloxane (PDMS) substrata that are cell-culture compatible with varying viscoelastic properties, and demonstrate the effect of the material composition and stiffness on ostepontin expression, a secreted phosphoprotein implicated in pro-angiogenic tissue regeneration and a marker for the osteoprogenitors isolated in our previous studies of bone marrow regeneration. This change in osteopontin expression was inconsistent among hMSC donors, and correlated with the biophysical properties of each donor&’s cell population. This underscores the finding that cytokine and protein expression by hMSCs can be moduated via cell-material interactions, aiding in regenerative clinical applications such as bone marrow recovery, but are also dependent upon intrinsic characteristics of the starting population that are correlative with cell biophysical properties.

‘Regenerative Engineering&’ is the integration of advances in materials science, stem cell technology and developmental biology with tissue engineering to regenerate complex tissues and organ systems. Advanced biomaterial and stem cell science is converging as the mechanisms to guide regeneration and the development of prescribed cell lineages from undifferentiated stem cell populations. Studies in somite development and tissue specification have provided significant insight into the pathways of biological regulation responsible for tissue determination, especially morphogen gradients, and paracrine and juxtacrine signaling. The understanding of developmental biology mechanisms are shifting the biomaterial design paradigm by the incorporation of molecules into scaffold design and biomaterial development that are specifically targeted to promote the regeneration of soft tissues. Our understanding allows the selective control of cell sensitivity, and a temporal and spatial arrangement to modulate the wound healing mechanism, and the development of cell phenotype leading to the patterning of distinct and multi-scale tissue systems.
Building on the development of mechanically compliant novel biomaterials, the integration of spatiotemporal control of biological, chemical and mechanical cues helps to modulate the stem cell niche and direct the differentiation of stem cell lineages. We have developed advanced biomaterials and biomimetic scaffold designs that can recapitulate the native tissue structure and mechanical compliance of soft musculoskeletal tissues, such as woven scaffold systems for ACL regeneration, non-woven scaffolds for rotator cuff tendon augmentation, and porous elastomers for regeneration of muscle tissue. Studies have clearly demonstrated the modulation of stem cell response to bulk biomaterial properties, such as toughness and elasticity, and scaffold structure, such as nanoscale and microscale dimensions. The integration of biological cues inspired from our understanding of developmental biology, along with chemical, mechanical and electrical stimulation drives our development of novel biomaterials aimed at specifying the stem cell lineage within 3D tissue systems. This talk will cover the development of biological cues, advanced biomaterials, and scaffold designs for the regeneration of complex soft musculoskeletal tissue systems such as ligament, tendon, and muscle.

The major cause of heart failure is the regional loss of myocardium following myocardial infarction. Because the loss of tissue is highly localized, and the endogenous response is not sufficient for repair, recent efforts have focused on replacement of the lost cells using a variety of treatment options. These include, but are not limited to, cell therapy, gene therapy and biomaterial-based grafts. These grafts, while promising, have many shortcomings when combined with cell therapy, including poor cell engraftment, survival and differentiation. In vivo, stem and progenitor cells exist in local niches, be it bone marrow, or tissue. These niches likely provide specific cues that influence cell function. Cardiac progenitor cells (CPCs), a progenitor cell found in the myocardium that may contribute to healing following infarction, are one such cell type. While many stem cell trials show early improvements, these are lost over time as the cells die and the paracrine effects are lost. Interestingly, these cells express important proteins such as Notch and beta-integrin on their surface, which promote cardiac development and blood vessel formation. We have developed several hydrogels that not only retain cells at the site of injury, but provide vital cues specific to these progenitor. Using self-assembling peptide nanofibers, we have tethered a peptide-mimetic of the Notch ligand Jagged1 (JAG) and shown that this can improve differentiation in a scaffold concentration-dependent mechanism. When grown under lower concentrations, the hydrogel promoted vascular differentiation and phenotypes. Under higher concentrations, the hydrogel was stiffer and promoted cardiac differentiation under similar JAG concentrations. The self-assembling peptide hydrogels were able to improve cardiac function in a model of ischemia-reperfusion injury in both cell-dependent and hydrogel-dependent manners. In addition to self-assembling peptide hydrogels, we also functionalized PEG hydrogels with VEGF, JAG, or the a11b1 integrin activator GFOGER. Cells cultured on these various hydrogels exhibited different phenotypes based on the peptide sequence and the concentration of the hydrogel. Moreover, human CPCs that had diminished regenerative capacity were rescued by the incorporation of these signals and were able to improve healing in a model of ischemic injury. In summary, our data demonstrate that hydrogels can be tailored to tune the response in human CPCs and either guide the function of the cells for regenerative purposed, or promote healing in a cell-free environment by independent mechanisms.

This talk will focus on heterogeneous cell-biomaterial constructs intended as implantable, physiologically functional tissue replacements for injured tissues with low intrinsic repair capacities including cartilage and heart muscle. We have a great interest in fabricating synthetic elastomeric polymers into 3D scaffolds capable of directing cultured cells to recreate the anatomical weaving, layering and anisotropy of native tissues over hierarchical scales (nm to cm). We have designed multimaterial scaffolds with multiscale pore architectures that can provide nascent engineered tissues growing in vitro with some of the structural and mechanical cues experienced by native tissues developing in vivo. A 3D-woven scaffold infiltrated with a cell-laden hydrogel was shown to produce engineered cartilage with viscoelastic mechanical properties approaching native articular cartilage. A scaffold with accordion-like honeycomb pore design and cardiomimetic mechanical anisotropy was shown to organize the alignment and contractile responses of cultured heart cells. A multilayered scaffold with 3D anisotropic pore structures created by off-set stacking and bonding of polymer grids with rectangular through-pores was shown to direct isolated heart cells into muscle bundles. Scaffolds with permeable microfluidic networks capable providing gas exchange to adjacent compartments were shown enable heart cell survival in vitro and host angiogenic responses in vivo. While previously developed cardiac grafts intended for the repair of heart muscle damaged by myocardial infarction (MI) were typically thin, sparsely cellular and poorly vascularized, our efforts have focused on an integrated scaffold comprising distinct vascular and parenchymal compartments in order to engineer grafts that are thicker, densely cellular and pre-vascularized. Data will be presented to demonstrate a polymer-based platform capable of providing vascular-like transport, a protected niche for exogenous heart muscle cells, template-directed organization of contractile muscle bundles and blood vessels, anisotropic mechanical properties mimicking those of native myocardium, long term mechanical support over the 4-to-12 week time frame needed for MI healing, and slow polymer biodegradation. This work demonstrates new scaffold design paradigms wherein 3D anisotropic pore architectures, microfluidic networks, and tunable material elasticity and modulus can enable the regenerative repair and revascularization of damaged tissues.

Previous human skin models have used either in vitro liquid-air interface cell culture systems or small animal in vivo models to examine the biological properties of human skin. However, the skin of small animals does not exactly replicate that of the human, and current in vitro models lack supporting vasculature making systemic studies impossible. Comprehensive next-generation models of human skin would ideally integrate components lacking in these models thus replicating full thickness, vascularized human skin. Here, blood-vessel-like microvessels composed of poly(ethylene glycol) (PEG) and gelatin methacrylamide (Gel-MA) provide for the necessary structural support, cellular attachment and proliferation required to construct a synthetic blood vessel. Using mild (<10mW/cm2/sec) UV-polymerizable materials these hollow microvessels replicate human venule- and arteriole-like structures and are capable of perfusing cellular media or heparinzed-blood products to deliver nutrients and oxygen to tissues while at the same time removing waste. Sheath-flow based microfluidics incorporate the structural components of the microvessel (PEG and Gel-MA), while cells comprising human blood vessels are simultaneously integrated. Venule-like microvessels require human umbilical vein endothelial cells (HUVEC); while arteriole-like microvessels place HUVEC and human aortic vascular smooth muscle cells (VSMC) into separate, yet adjacent vessel compartments. The resulting microvessels range in size from 100-500um OD; 50-100um ID; here size variance are the result of a fully tunable system whereby appropriate adjustments to flow-rates result in modification to the overall dimensions. Also, to construct viable tissue models, microvessels are then embedded into an addressable manifold device containing an inlet/outlet for fluid transport and an extracellular matrix to support tissue growth. The extracellular matrix material contains dermal fibroblasts to replicate the lower dermis of human skin, while human keratinocytes generate the upper epidermis, thus assembling the major components of human skin into a single vascularized system. By employing the manifold device, these microvessels remain perfusable, supplying cellular growth media to support tissue survival, while at the same time removing unwanted waste derived from cellular respiration and other biochemical processes.

The dynamic modulation of gene expression to illuminate and manipulate cell growth and signaling is an essential aspect in tissue engineering technologies, yet presents a particular challenge in complex tissues that must control phenotypes of multiple types of cells in a region-specific fashion. Specifically, methods to spatiotemporally control gene expression by cells growing within complex tissue scaffolds would provide an essential complement to current biomaterial strategies employed to manipulate cell behavior via extracellular signals. Multiple strategies have been explored for artificial regulation of gene expression in eukaryotic cells, including inducible promoter systems and optogenetic regulation strategies; these systems offer tremendous potential, yet the approaches used do not address the significant difficulties in delivering regulatory components to cells. As an alternative, approaches employing stimuli-responsive nanomaterials have shown promise in generating cellular responses with some degree of spatiotemporal control; however, off-target effects and limitations in efficacy continue to plague these systems. To address these challenges, we have developed novel and tailorable mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) [mPEG-b-P(APNBMA)]-based block copolymers (BCP)s with proven biocompatibility, stable and tunable nucleic acid binding, and light-triggered side chain cleavage leading to rapid and on-demand nucleic acid release. Herein, we report the ability to exploit these materials for spatiotemporally controlled deployment of siRNA cargoes, resulting in the ability to locally “dial-in” precise patterns of gene silencing in NIH/3T3 and adventitial fibroblast cultures. Gene silencing efficiency could be tuned over a range of approximately 0 - 85%, on the basis of varied light irradiation and varied polyplex composition, and spatially controlled gene expression exhibited cell-to-cell accuracy and no observable off-target effects. Using fluorescence correlation spectroscopy and fluorescence resonance energy transfer, we demonstrated that the variations in silencing efficiency were directly controlled by light-induced changes in polyplex structure leading to siRNA release within the cytoplasm, and we show that the polyplexes remain entirely dormant in the absence of illumination. Through development and application of mass action kinetic modeling, we show that the maximal silencing efficiency is defined by a pseudo-steady state balancing the rates of mRNA production and siRNA/RISC-mediated mRNA cleavage. This work establishes the framework for addressing a key challenge in regenerative medicine while also exploring the fundamental mechanisms of nanomaterial intracellular stability and delivery barriers.

The movement of water molecules in and around biological macromolecules is critical to their function, an effect that is now well established for a wide range of important processes including protein folding, cell signaling, and enzyme activity. Similar to macromolecules in biological systems, water dynamics are also likely to mediate the function of synthetic biomaterials. I present a supramolecular peptide-based nanofiber system that has far-reaching applications in the field of regenerative medicine, and demonstrate novel water dynamics measurements through the nanofiber cross-section. These measurements are obtained with sub-nanometer resolution by implementing an advanced magnetic resonance technique termed Overhauser Dynamic Nuclear Polarization spectroscopy. With this new insight into the water dynamics in supramolecular nano-objects, design of molecular subunits for self-assembled biomaterials can be finely tuned to optimize function.

As we approach 15 years of intense nanotechnology funding in the U.S., many are wondering what real medical products have resulted from nanomedicine. This presentation will summarize several FDA approved medical devices that have resulted from nanomedicine research and the impact they are having on improving disease prevention, detection, and treatment. Cancer, orthopedics, antibacterial, and many other diseases and organ systems will be covered. Key current areas of concern in the application of nanomedicine in regenerative engineering will also be covered, such as toxicity and nanomanufacturing. Lastly, emerging future nanomedicine areas will also be discussed, such as the use of nanomaterials to create in-the-body sensors for approaching real personalized medicine.

Local, controlled and sustained delivery of an anti-inflammatory drug, salicylic acid (SA), via a SA-based polymer (SAP) powder has been found to significantly enhance diabetic bone regeneration in vivo through the long-term mitigation of local inflammation. This study formulated SAP into porous scaffolds using heat sintering techniques and obtained products that released SA in a sustained manner (SA continually released in 30-50 days), possessed the porosity (~60%) that favors bone formation, and had the suitable modulus (10-30 MPa) for bone regeneration. Interestingly, the SAP scaffolds had up to 45% SA loading, which is much higher than all the other existing drug-eluding bone regeneration scaffolds. In addition, the physicochemical properties of the scaffold can be easily tuned to meet specific application requirements.
With the unique combination of the chemical advantages of local, controlled and sustained SA release on diabetic bone healing, the physical advantages of the interconnected porosity, and the highest drug loading ever achieaved in bone regeneration scaffolds, the SAP scaffold is a promising candidate to enhance diabetic bone regeneration.

Multidomain peptides (MDP) self-assemble to form nanofibrous scaffolds well suited to tissue engineering and regeneration strategies. MDPs can present bioactive cues that promote vital biological responses. Orthogonal self-assembly of MDP and growth factor-loaded liposomes generate supramolecular composite hydrogels. These biocompatible delivery vehicles allow time-controlled release. MDP-Liposome Composites (MLCs), consisting of peptide nanofibers and liposomes, retain their structural integrity and can be injected in vivo for targeted, localized delivery of growth factors. Release studies from MLCs loaded with the growth factors EGF, MCP-1 and PlGF-1 demonstrated bimodal release in which growth factors loaded in liposomes inside the MDP hydrogel were released more slowly than those encapsulated by the nanofibrous hydrogel alone. In vivo studies have indicated that SDF-1 loaded MDP hydrogels facilitate mesenchymal stem cell recruitment. PlGF-1 can temporally stimulate VEGF-receptor activation in vitro in endothelial cells, and robust vessel formation in vivo. MLCs provide a novel method for the time controlled delivery of growth factors from within highly biocompatible and injectable hydrogels. Time controlled release guided by MLCs induces an unprecedented level of growth factor-mediated neovascular maturity. MLCs offer a unique protein delivery platform for tissue regeneration mediated by angiogenesis.

Natural processes, including tissue development and regeneration, are exquisitely well controlled both spatially and temporally. This motivates the development of bioagent delivery devices that can be precisely controlled in real-time to optimize tissue regeneration and to explore hypotheses related to bioagent delivery timing. The majority of systems that respond to non-invasive or environmental cues, however, typically exhibit a baseline release rate thereby continuously leaking drug. Herein, it is hypothesized that a more complete On/Off switch could be achieved by conjugating growth factors to nanoparticles that would remain physically entrapped within alginate hydrogels, thereby dramatically reducing baseline release; ultrasound is then used to reversibly instruct their release from the self-healing hydrogels.
To examine this hypothesis, gold-nanoparticles (hydrodynamic radii ~ 30, 60, 100 nm) were PEGylated and incorporated into ionically crosslinked alginate gels and microbeads and treated with ultrasound on day 1 or day 5. A dramatic increase in the release rate of PEGylated AuNPs was observed when compared with baseline release rates (day 1 ~ 200-fold; day 5 ~ 5000-fold). Next, bioactive nanoparticles were developed by covalently conjugating recombinant human bone morphogenetic protein-2 (BMP-2) to the gold-nanoparticles through thiol bonds found in cysteine residues of the protein. The bioactivity of these particles was examined by incubating them with mouse mesenchymal stem cells and analyzing their osteogenic differentiation using alkaline phosphatase. The BMP-2-AuNPs at 100ng BMP-2/ml enhanced osteogenic differentiation vs. all controls (DMEM, osteogenic media, unconjugated BMP-2 at 10ng/ml; p < 0.01) and was equally as effective as the maximum dose of unconjugated BMP-2 (500 ng/ml; p = 1.0). Fishy;shy;nally, we confirmed the ability of these osteogenic nanoparticles to be entrapped in the on-demand alginate release system, to remain entrapped in the absence of ultrasound, to be released using ultrasound and to maintain their bioactivity (released BMP-2-AuNPs vs controls, p < 0.015).
This approach to precisely controlled delivery of bioactive factors should allow researchers and clinicians to more precisely control delivery of bioagents; to optimize the temporal delivery of factors; to more efficiently deliver factors at the correct time; and to exploit the favorable physico-chemical properties of nanoparticle-based bioagents. In addition, to our knowledge this is the first example of a system for local on-demand delivery of bioactive nanoparticles; this approach to nanoparticle delivery may help overcome the challenges associated with systemic nanoparticle delivery.

Clinical translation of protein therapies faces multiples challenges; the most significant is how to maintain bioactivity so as to lower dosage and reduce off-target effects. My lab uses heparin and biocompatible polycations to form an injectable coacervate that preserves the bioactivities of heparin-binding proteins. This includes many growth factors and morphogens. I designed the delivery vehicle to mimic the interaction among fibroblast growth factor-2 (FGF2), heparin and FGF receptor. The effectiveness of the coacervate delivery system is demonstrated using multiple proteins (FGF2, VEGF, HGF, HB-EGF, IL10, Shh, PDGF, NGF among others) in multiple animal models (mice, rat, pig). The coacervate provides spatial and temporal control of the release of heparin-binding proteins and promotes angiogenesis, skin wound healing, and cardiac repair post- infarction. In addition, the ease of preparation and administration of the coacervate reduces costs and increases the likelihood of translation and adoption.

Diabetes is a metabolic disorder caused by insulin resistance and/or deficiency, where the complications of impaired bone quality and bone healing are due to altered gene expression, reduced vascularization, and prolonged inflammation. No effective treatments for diabetic bone healing are currently available, and most existing treatments do not directly address the diabetic complications that impair bone healing. Sustained and localized delivery of salicylic acid (SA) via a SA-based polymer has been shown to significantly enhance diabetic bone regeneration in rats.
This work describes the local pharmacokinetics of SA-releasing polymers and the physiological mechanisms induced by the SA-polymer that are needed to optimize and advance diabetic bone regeneration. Specifically, we investigated the local pharmacokinetics of SAPAE using a novel agar-based system and evaluated the cellular and molecular mechanisms of localized, sustained SA release on bone regeneration. With the agar system, a continuous and slow localized release of SA was noted for about 70 days; this data correlates the prolonged local anti-inflammatory effect observed in vivo, as demonstrated by histological analysis. In vivo, low SA concentrations were locally maintained at the bone defect site for more than 2 months. As a result of the sustained SA release, a significantly reduced inflammation was observed in diabetic animals, which in turn, yielded reduced osteoclast density and activity as well as increased osteoblastogenesis. The long-term inflammation reduction down-regulated osteoclast density and activity, and increased osteoblastogenesis, particularly in diabetic animals with elevated chronic inflammation. As a result, higher bone formation was observed in the defect site, particularly in diabetic animals compared to normal animals.
Based upon these results, localized and sustained SA delivery from the SA-based effectively improve bone regeneration in diabetic animals by affecting both osteoclasts and osteoblasts and provide a basis for clinical treatment. This study proves that SAPAE treatment enhance diabetic bone formation through prolonged inflammation mitigation. The SA release profile presented herein serves as a reference for future designs of anti-inflammatory treatment for diabetic bone healing; specifically, an initial lag period of ~ 2 days of low SA concentrations followed by a ~ 2 month release. Future studies will incorporate other osteogenic formulations to further improve system performance, such as utilizing porous scaffold and/or the addition of insulin for diabetic bone regeneration.

Drug-eluting stents (DES) for cardiovascular diseases has reduced the rate of restenosis after stenting. However, there still remains the risk of the late-stage thrombosis and restenosis after the complete emission of the drug. In our previous research, a newly designed drug-release platform with both anti-thrombogenicity and endothelialization was developed. The platform was consisted of an anti-thrombogenic 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer and a micro-patterned cell-compatible diamond-like carbon (DLC) film. The DLC/MPC platform with a model drug presented an effective controlled drug-release capability depending on the covered area of the micro-patterned DLC. Furthermore, the DLC of the platform effectively supported the proliferation of the endothelial cells (ECs) while preserving the anti-thrombogenicity of the MPC.
In this research, we introduced a basic fibroblast growth factor (bFGF) to the DLC/MPC platform and evaluated the bFGF release profile and its effects on the ECs proliferation and the blood compatibility. The MPC films containing bFGF were prepared from the mixture of MPC/ethanol and bFGF/water. A micro-patterned DLC was deposited on the films through a metal grid with 60×60 µm² micro-pores during the DLC deposition. The bFGF release profile from the DLC/MPC film with the micro-patterned DLC was measured using an enzyme-linked immune sorbent assay (ELISA).
It was found that the bFGF-released amount was well suppressed by increasing the DLC-coated surface area. The platelet-adhesion property of the DLC/MPC platform containing bFGF was tested by immersing the substrates in the platelet rich plasma. The anti-thrombogenicity of the DLC/MPC with bFGF showed no significant difference from that of DLC/MPC without bFGF, but a substantial improvement was observed when compared with that of SUS316L, a material used for a bare metal stent. Thus it was confirmed that the introduction of bFGF to DLC/MPC would not affect the anti-thrombogenicity and the platform could be a candidate for a new DES material. The ECs adhesion and its proliferation were finally analyzed by immersing substrates in a cell culture medium. The proliferation of the ECs on DLC/MPC with bFGF was estimated in terms of the cell adhesion area by the fluorescence microscopy. There was no significant difference in the cell adhesion area between the DLC/MPC with bFGF and the DLC/MPC without bFGF up to 12 hours. However, in 24 hours, the cell adhesive area of the DLC/MPC with bFGF became about 1.5 times larger than that of the DLC/MPC without bFGF. Therefore, it was confirmed that the proliferation of the ECs was effectively enhanced by the released bFGF. In summary, the DLC/MPC platform with bFGF was found to have the potential capability to be used as an alternative device of conventional DES. The in vivo test on the anti-thrombogenicity and the promotion of endothelialization would be necessary for the practical medical evaluation.

Controlled release devices have exciting potential for a variety of biomedical applications including tissue engineering. Potent growth factors can be encapsulated into natural or synthetic biomaterials and then released from a scaffold over time to stimulate cell proliferation and differentiation. Because the timing and duration of release are often critical for proper tissue formation, it is important to study the release kinetics from these scaffolds. Assessing in vitro kinetics is generally rather simple, yet these observations may be very different from the release rate in the body due to the complexity of the in vivo microenvironment. As a result, studying in vivo release kinetics is critical for designing a system that achieves temporally appropriate growth factor presentation. Unfortunately, methods for assessing in vivo release are generally costly, low-throughput, and/or lack the time resolution needed for studying protein release in many applications. As a result, we have aimed to develop a method for studying protein release in vivo that is simple, quantitative, and relatively inexpensive. Our system uses the In Vivo Imaging System (IVIS) in combination with commercially-available fluorescent tags including Alexa Fluor® 647 and 680 to observe the release of proteins from polymeric microparticles. Briefly, poly(lactic-co-glycolic acid) (PLGA) particles containing fluorescently labeled ovalbumin or bovine serum albumin were injected into hairless SKH-1E mice to generate a subcutaneous protein depot. As expected, PLGA particles displayed sustained release of protein over the course of weeks as measured by fluorescence using the IVIS. Poly(methyl methacrylate) particles were also used as a non-degradable control. These particles demonstrated an initial release of protein (associated with the release of protein at the surface), but then maintained a stable fluorescent signal for months suggesting that photobleaching is not a major concern. Taken together, these data indicate that this method may be well-suited for studying in vivo release kinetics without the need for expensive or time-consuming methods.

Regeneration of complex orthopedic tissues (such as ligaments, bones, and the tendon-to-bone insertion site) is problematic due to a lack of suitable biomaterials with the appropriate chemical and mechanical properties to elicit formation of tissues with similar structure, organization, and functionality to natural tissues. Additionally, a non-trivial fraction of implanted biomaterials contract bacterial infections, which can lead to implant failure, secondary surgeries, and the spread of infection to other tissues throughout the body. To address these issues, the current study investigated magnesium oxide (MgO) nanoparticles as novel materials to improve orthopedic tissue regeneration and reduce bacterial infection.
Here, MgO nanoparticles and hydroxyapatite (HA) nanoparticles were dispersed within poly-L-lactic acid (PLLA), and the resulting nanocomposites were tested for their mechanical properties, surface roughness, degradation characteristics, antibacterial properties, and their ability to support the adhesion and proliferation of fibroblasts and osteoblasts.
Results showed for the first time that nanocomposites containing both HA and MgO nanoparticles performed best with respect to osteoblast proliferation and mechanical properties. Increases in alkaline phosphatase expression and vinculin focal adhesions within osteoblasts indicated that MgO nanoparticles enhanced the osteogenic properties of HA composites. Further, varying MgO concentrations offered tunable composite degradation kinetics, and the supernatant from degraded MgO-containing composites supported greater osteoblast proliferation compared to non-MgO composites, confirming that the degradation products from MgO nanocomposites were non-cytotoxic. Bacterial experiments with Staphylococcus aureus showed that MgO nanoparticles exhibit powerful bactericidal efficacy, suggesting that MgO nanoparticles should be incorporated into scaffolds for orthopedic tissue engineering to improve cell functions and reduce the risk of bacterial infection with limited antibiotics usage.

Collagen is the most abundant protein in tissue such as tendon and it is a natural resource for healing damaged skin tissues. In this study, under specific micro-environment conditions, mimetic collagen gels have been successfully formed from bovine type I collagen. By controlling ionic strength, temperature and pH, fibrils with native D-banding structure could be created in collagen gels in vitro. For more applicable applications in tissue engineering, collagen fibers were produced through gel spinning process. These fibers are similar in size and structure to that of tendon. By changing fiber spinning and post-spinning processes as well as gelation environments, collagen fibers with natural D-banding pattern were well fabricated. Structural development of gel samples and fibers were recorded through scanning electron microscopy (SEM), atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS). The mechanical performance of these fibers using immersion tensile testing has been investigated, and demonstrates similar property to native tendon. For this reason they can be applicable for supporting cell development. These potential results and applications may show a breakthrough for manufacturing synthetic biopolymers for tissue engineered scaffolds.

Extracellular matrix (ECM) surrounding Schwann cells and neurons provides critical determinants of cellular phenotype during development and essential cues in guiding regrowth. ECM is capable of exerting influence on the rates of transcription, mRNA stability, rates of translation, and post-translational modifications via trans-membrane proteins and cytoskeletal components. In this way, Schwann cells are responsible for guiding nerve regeneration by producing the regeneration-promoting matrix as well as other growth factors. By altering hydrophobicity of thermo-responsive poly(N-isopropylacrylamide) coatings, we generated cell sheets from rat Schwann cells and subsequently layered them onto polycaprolactone fibers for mechanical support. With the use of mild detergents, we were able to decellularize these constructs and isolate ECM produced by Schwann cells with few traces of nucleic acids. Decellularization rendered an acellular allograft that is less immunogenic and could potentially preserve basal lamina tubes and the three-dimensional collagen scaffolding present within native nerve, yet commonly absent within artificial conduits. Thus, we developed a novel scaffold enriched with native ECM to support glial and neuronal growth. Decellularized cell sheets added a larger variety of biological material to the scaffold in more native condition than chemically bound protein, such as EDC/NHS coupling of laminin. This method of deposition of ECM allowed for generation of clinically relevant constructs by providing more proteins than traditional seeding method after decellularization. Additionally, the isolated matrix supported proliferation of Schwann cells better than covalently bound laminin. The proliferation and differentiation of Schwann cells grown on decellularized sheets were complemented by up-regulation of Erbb2 and myelin protein zero. Laminin expression of β1 and γ1 chains was also elevated. Neurite extensions were significantly longer on constructs with aligned fibers and decellularized sheets with an average length of 696.2±30.1mu;m. In comparison, neurite extensions on random fibers with decellularized sheets and aligned fibers alone had length of 317.7±37.4 and 569.3±11.3mu;m. While current fabrication techniques allow for only few molecular cues to be incorporated into the scaffold, this method uses cells as “natural factories” for deposition of various cell-secreted peptides as well as their transcript variants. Our novel approach addresses this important limitation and focuses on genetic expression profile of Schwann cells grown on these regenerative matrices, proving the potential of these scaffolds to be used in enhancing peripheral nerve regeneration.
Acknowledgements: The work is partly supported by the National Institute of Health (NIH-R15 NS074404), and the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Orthopaedic Research Program under Award No. (W81XWH-13-1-0320).

Tissue engineering may provide an alternative approach to treatment of osteochondral defects (OCDs) - loss of articular cartilage and underlying subchondral bone. Given the gradual transition from bone to cartilage in osteochondral tissues, and ideal scaffold should likewise promote tissue regeneration in a spatially controlled fashion. Therefore, using a “materials-guided approach”, the scaffold must contain a gradient of chemical and physical properties to direct the behavior of associated cells. Previously, we have shown that methacrylated star polydimethylsiloxane (PDMS_star-MA) can induce osteoinductivity and bioactivity when incorporated with diacrylate (PEG-DA) in a hydrogel. In this study, a technique combining solvent-induced phase separation and salt leaching was use to provide tunable pore size and interconnectivity throughout the PDMS_star- PEG scaffolds. Scaffolds were fabricated using different salt sizes (<75 - 460 mu;m) in order to produce porosities typically associated with superior generation of bone and cartilage tissues. The ratio of PDMS_star-MA/PEG-DA was also varied to control the formation of hydroxyapatite and, ultimately, the differentiation of mesenchymal stem cells (MSCs) into osteoblast-like phenotypes. The morphology, hydration, and modulus of the scaffold zones were evaluated as well as the formation of scaffolds containing multiple zones.

Calcium phosphate cements (CPC) are composed of a mixture of particles, such as tricalcium phosphate (TCP), tetracalcium phosphate, or dicalcium phosphate dihydrate (DCPD) etc., and they show self-hardening reaction upon mixing with liquids to form hydroxyapatite or mainly DCPD. There are many publications about how additives to CPC improve its setting time and the mechanical properties. In this study, we investigated crystalline phases and mechanical properties of alpha TCP cements mixed with citric acid (CA), into which tilapia scale collagen (Col) or hyaluronic acid (HyA) as a dispersant was mixed. Alpha TCP particles heated at 13000C were prepared with a conventional wet method using Ca(OH)₂ and H₃PO₄. The precipitate was adjusted with three methods to obtain different particle sizes and densities; first one was spray-dried (SD), second freeze-dried (FD), and third sintered after molding with cold isostatic press (CIP) and crushed. The 14 mu;m in size SD particles were mixed with FD particles at 45mu;m or CIP particles at 134mu;m in different ratios. The CA liquid was added to the particles and kneaded under different liquid/solid (L/P) ratios. After chelating with calcium ions dissolved from the particles for 1 day (hardening), the mixtures containing CIP particles (23 MPa) showed lower compressive strength than those including FD particles (28MPa) even though the packing density was high. This indicates that the size of the particles is an important factor for a chelating reaction. The SD/FD cements mixed and kneaded with the CA including Col exhibited no other phase of alpha TCP but showed a decrease of XRD peak for alpha TCP. However, the cement containing HyA showed XRD peaks for the DCPD phase. The compressive strength of Col (33MPa) cement was higher than that HyA (23MPa) containing cement. This can possibly be explained by the inhibition of chelating reaction of HyA.

45S5 Bioglass® has generated a lot of interest in the last decades and different products were introduced in the market in the form of powders, granules or putty for bone reconstruction. Different technologies have been, also, developed to produce 3D porous 45S5 Bioglass® but these materials are not yet available commercially. The porogen and the freeze casting methods allowed the production of scaffolds with high mechanical strength but such scaffolds exhibit low pore interconnectivity and evident anysotropy^{1, 2}. On the other hand, scaffolds prepared through foam replication technique were the first Bioglass® based scaffolds which showed a 3D glass-ceramic network with good pore interconnectivity, pore size and porosity adequate for new bone regeneration and vascularization³. However they exhibit low mechanical strength⁴. The bioactivity of these structures has been demonstrated³ and the process has been adapted to other organic templates in order to increase the mechanical properties⁵. Another approach to increase the mechanical stability of Bioglass® 3D porous construct is to build “shell scaffold”, providing outer walls while maintaining fluid permeability⁶. A powder metallurgy approach has been recently adapted to produce Bioglass® foams with interconnected porosity and good mechanical properties⁷.
This work compares the microstructure and bioactivity in SBF of different 45S5 Bioglass®-based scaffolds. Three of them were produced via replication of different templates: synthetic polyurethane foam (45 ppi), natural foams “spongia agaricina” and “spongia lamella”⁵. The other samples were a shell scaffold⁶, replicating a polyurethane foam, and a 3D porous scaffold produced via a powder metallurgy-inspired technology⁷. Microstructures of resin-mounted specimens have been studied using SEM-EDX in the as-sintered condition and after SBF soaking for 7, 14 and 28 days at 37°C with 1.5 g/L immersion ratio. XRD has been applied to show the different crystalline phases while cross section SEM imaging allowed distinguishing different pore and strut morphologies, contingent defects and distribution of the different phases inside the struts. The production process has an impact on the structure and microstructure of the materials providing unique phases and phases&’ distribution. It is likely that Bioglass® based of scaffolds, produced with different fabrication methods, will have their specific strengths and weaknesses and the final selection of the scaffold type will strongly depend on the intended application.
References
1. Jones, J. R. Acta Biomater 2013,9 (1), 4457-86.
2. Bellucci, C. Ceramic International 2011, 37(5), 1575-85.
3. Chen, Z. Q. Biomaterials, 2006, 27, 2414-25.
4. Chen, Z. Q. J Am Ceram Soc, 2011, 94(12), 4184-90.
5. Boccardi, E. Adv Appl Ceramics, 2015, (accepted for publication)
6. Bellucci, D. J Mater Sci: Mater Med, 2012, 23, 1397-1409.
7. Aguilar-Reyes, E.A. Materials Science & Technology 2010 Conference and Exhibition, 2010; 70-7.

There has been a lot of effort to engineer biocompatible and biodegradable scaffolds to study biological, chemical, and mechanical features of cells in vitro. Extracellular matrix (ECM) proteins and synthetic polymers have been mainly investigated to make in vitro scaffolds. ECM proteins can mimic natural tissues by providing biological cues for cell attachment and growth. On the other hand, synthetic polymers are cheap and easy to handle. However, ECM proteins are too expensive to be suitable for large-scale manufacturing and synthetic polymers are lack key ECM biological and structural cues. Here, we suggest that soy protein/polycaprolactone (PCL) blend nanofiber scaffold can be used as a cost effective platform for large-scale tissue engineering. We built soy protein/PCL blend nanofibers by using rotary jet spinner, which generates high centrifugal force to polymers in solution and makes continuous nanofibers of polymers. In an effort to optimize structural cues of these scaffolds - fiber diameter, alignment, and porosity were respectively controlled by tuning the molecular weight of the polymer solution, adjusting a mandrel to improve fiber alignment and co-spinning sacrificial gelatin nanofibers. In addition, after neonatal rat ventricular myocytes (NRVMs) culture, NRVMs on soy protein/PCL nanofibers showed plausible cell contractions, proving good biocompatibility owing to variety of bioactive amino acids in soy protein. Our results demonstrate how structural and biological cues of soy protein/PCL blend nanofiber scaffold can be engineered for tissue engineering applications. We anticipate our scaffold to be one of alternatives to replace expensive ECM protein-based scaffolds for commercial scale uses because of low cost and good biocompatibility. For instance, soy protein/PCL blend nanofiber scaffold could be used as not only a platform for various drug testing, but also a wound healing patch.

Stimuli responsive hydrogels exhibit a sol-gel transition in response to small changes in temperature, pH, light, electric field, and magnetic fields. These hydrogels have potential applications in biology and medicine fields, including drug delivery system, biochemical sensor and bio-engineering. Among these hydrogels, thermo-responsive hydrogels have been extensively investigated due to their simple application and low adverse effects on tissues, compared to other stimuli. In particular, the thermo-responsive properties make them potential candidates for drug delivery and tissue engineering. The common feature of thermo-responsive hydrogels is that they exhibit a sol-gel phase transition at certain temperature, and two types of behavior are observed. One is lower critical solution temperature (LCST) and the other is upper critical solution temperature (UCST). Below the LCST, the polymers are soluble, and above the LCST, they form the gel mainly due to hydrophobic interaction. In contrast, polymer solutions with LCST form the gel upon cooling below the UCST. In general, the hydrogel with LCST than the UCST is often useful in biomedical fields.
Hydroxypropyl methylcellulose (HPMC) is one of cellulose derivatives with a certain degree of substitution of hydroxyl propyl and methyl groups, and is widely used in personal care products, foods and pharmaceutical applications. HPMC is well known to have LCST behavior due to their substituents. The thermal transition of HPMC solution was closely associated with concentration, heating rate and degree of substitution. Furthermore, thermo-responsive hydrogelation is also affected by salt. A salt, such as NaCl, has a greater affinity for water than HPMC, resulting in the removal of water from the hydrated polymer and thus a lowering of its sol-gel transition temperature due to an enhancement of the hydrophobic association of HPMC chains. The gelation temperature of HPMC solution significantly decreases with increasing of the salt concentration. According to the Hofmeister, the effect of salt in reducing the gelation temperature becomes significant with increase in magnitude of anion. Then, the ability of salts to “salt out” or “salt in” effect is well classified in Hofmeister series.
In this study, we investigate the effect of HPMC concentration and inorganic nanoparticle precursor salt concentration on the gelation temperature of HPMC solution. Also, we synthesize nanoparticles in HPMC hydrogel using two kinds of inorganic nanoparticle precursor salts and also examined the influence of nanoparticle and residue ion on HPMC hydrogel.

Electrospun nanofibers from natural and synthetic polymers have been widely used in regenerative medicine, including tissue engineering applications. The physiological performance of these nanofibers in a particular situation is determined by the fibers&’ composition, diameter, shape, surface morphology, porosity, and by their arrangement and interaction in a scaffold. Most common capillary needle high-voltage DC-electrospinning has been proven to be efficient in controlling these fiber parameters. However, it takes a long time to fabricate extended fibrous structures by this method. On the contrary, the less common centrifugal spinning technique allows for a high rate of production of polymer fibers, yet the fiber sizes and geometries do not often reach the desired parameters. Initial experiments in the present study have demonstrated, for example, that while cells can attach and proliferate on electrospun polycaprolactone (PCL) nanofiber meshes prepared by electrospinning, there was little success with centrifugally spun PCL material. A combined electrocentrifugal method has been realized to address the issues with the biomedical nanofibers' quality, performance and scalable manufacturing. Using dextran and PCL as exemplary biomedical polymers, the simultaneous effects of the electrocentrifugal process parameters, including DC-voltage (5kV - 15kV) and angular velocity of the spinneret (4000 rpm - 15000 rpm), on the production and geometry of the fibers were investigated with the polymer solution concentrations in the range from 14% w/w to 20% w/w for PCL. Significant improvements in the fiber size control fiber diameter (diameter range 300 nm - 4 mu;m), and shape uniformity have been observed in the electrocentrifugal method when compared with only centrifugal spinning technique. Initial results of in vitro testing of PCL fibers with the selected cell lines are discussed. This work was supported in part by the National Science Foundation award OISE-1261154.

Currently there is a growing research for synthetic solutions for anterior cruciate ligament (ACL) ruptures repair or replacement, to replace the autografts, the most chosen by physicians, but with limited availability. It is a challenge to choose appropriate materials that could fit as load support during ACL recovery while simultaneous promotes their regeneration requires above all biocompatibility and mechanical functionality. Biodegradable polymers such as polylactic acid (PLA) have been studied for being used on a temporary approach of the natural ligament replacement, combined with Tissue Engineering techniques, instead of the permanent approach. This approach aims to recover damaged natural ligament remaining from a rupture, by using a biodegradable scaffold that allows the growing of the new tissue, promoting regeneration. Biodegradable scaffold should degrade during ligament recovery period conserving essential mechanical properties until scaffold is no longer required.
Despite being already approved by FDA, PLA presents faint mechanical properties that may be improved with mechanical reinforcements, namely its resistance to fatigue and creep permanent deformation, promoting laxity or rupture of the device. To improve PLA mechanical properties carbon nanostructures as graphene nanoplatelets (GNP) and functionalized carbon nanotubes (CNT-COOH) were assessed as mechanical reinforcements for PLA matrix. Small weight percentages of GNP (up to 2 wt.%) as well as CNT-COOH (up to 1 wt.%), separately, were added to PLA by melt mixing followed by compression moulding, for the production of PLA/CNT-COOH and PLA/GNP nanocomposites. Mechanical study of the nanocomposites was developed, proving considerable improved creep and fatigue results.
Biocompatibility and cytotoxicity were also assessed both in vitro and in vivo testing. In vitro assays were performed in triplicate using neonatal human dermal foreskin fibroblasts-1 (HFF-1, ATCC SCRC-1041), resorting to MTS cytotoxicity and BrdU incorporation assays. Taking PLA as the control specimen, for the three time points studied (24, 48 and 72 h), values of cell proliferation are quite acceptable with minimum limit of 70% observed for 48 hours, predicting a good biocompatibility of the material with human tissue, namely fibroblasts. Cell viability for all nanocomposites is substantial being slightly reduced for time point of 72 h, but above 70% viability of fibroblasts, being a good indicator that the cytotoxicity of nanocomposites is insignificant in comparison with the PLA. For in vivo, biocompatibility tests were developed, by inserting subcutaneously nanocomposites circular specimen into C57 black 6 mice. Two time points were evaluated in particular 2 and 4 human weeks. These times were chosen considering hydrolytic degradation results of the nanocomposites on PBS medium at 370C during 16 weeks.

Introduction: Autologous chondrocyte implantation (ACI) has become a general therapy for damaged cartilage. To obtain enough chondrocytes for ACI, chondrocytes need to be grown. However, chondrocytes lose their specific functions after the subculture. Recently, it has been cleared that round chondrocytes keep their functions. The cells adhere on polymer substrate through the interaction between integrin and proteins adsorbed on the substrate, which leads cell spreading. Therefore, cells are expected to form round on the substrates on which small amount of proteins adsorbed. We have previously reported that poly (2-methoxyethyl acrylate) (PMEA) suppressed protein adsorption due to the existence of intermediate water, which is loosely bound water in hydrate polymers. Moreover, PMEA has cellular adhesiveness. Here, we tried to regulate bovine articular chondrocyte (BAc) morphology on the substrates coated with PMEA analogous polymers containing intermediate water to keep their specific functions during subculture.
Methods: PMEA, its analogous polymers (PMe2A and PTHFA), and poly (2-methacryloyloxyethyl phosphorylcoline-co-n-butyl methacrylate) (PMPC) were used after the coating on tissue culture polystyrene (TCPS) and polyethylene terephthalate (PET). Intermediate water contents in these polymers are in order of PMPC> PMe2A> PMEA> PTHFA > PET, TCPS. The amounts of adsorbed proteins were measured with mu;BCA assay after the immersing in serum containing medium for 1 h. Primary BAc (BAc-P0) or 3-times-subcultured BAc (BAc-P3) were incubated on the substrate for 1 h . The adherent cells were counted after crystal violet staining. DAPI-labeled cell nuclei were counted with image-based cytometry for cell growth assay. Cell morphology was observed under a confocal laser microscope after the visualization of actin fiber and vinculin as an indicator of focal adhesion.
Results and discussion: The amounts of adsorbed FBS proteins were in order of PMPC>> PMe2A, PMEA> PTHFA, PET, suggesting that protein adsorption decreased with the increase of intermediate water contents. Approximately 10% of seeded BAc-P0 adhered on the substrates except PMPC. Notably, the numbers of BAc-P0 adhered on PMEA and PMe2A were approximately twice as high as those on PET and PTHFA. In contrast, approximately 60% of seeded BAc-P3 adhered on all substrates except PMPC. BAc-P0 and BAc-P3 could grow on all examined substrates but not on PMPC. The cell growth decreased with the increase of intermediate water contents. BAc-P0 cultured on PET and PTHFA substrate for 5 days formed many focal adhesions and were spreading, but the cells cultured on PMEA and PMe2A maintained round shape. Similar results were obtained in the case of BAc-P3.
Conclusions: PMEA and PMe2A suppressed protein adsorption and have high cellular adhesiveness. Furthermore, BAc on PMEA and PMe2A kept round. It is expected that chondrocyte can keep their functions on PMEA or PMe2A during the subculture.

Recreating the microenvironment of native tissues faces significant challenges in cartilage tissue engineering due to their complex and dynamic biochemical and biomechanical nature [1]. Glycosaminoglycans (GAGs) such as hyaluronic acid (HA) and chondroitin sulfate (CS) are abundantly present in the extracellular matrix (ECM) and play significant roles in a variety of cell-ECM-protein interactions [2]. Their presence and specific organization in cartilage is particularly important for tissue mechanics and biological function. Recently, our laboratory has exploited peptides that bind HA and CS to specifically and non-covalently bind HA or CS to mimic the dynamic nature of ECM [2]. Here, we incorporated these GAG-binding peptides into collagen-mimetic protein hydrogels to recapitulate the cartilage microenvironment. Recombinant bacterial proteins such as Streptoccocal collagen-like 2 (Scl2) offer a versatile ‘blank slate&’ platform that can be modified with multiple bioactive and biodegradable epitopes [3]. We modified the terminal ε-amines of lysines on Scl2 with a heterobifunctional linker using NHS conjugation chemistry to generate acrylate-functionalized Scl2 proteins. The HA or CS-binding peptides were tethered to these proteins via thiol-acrylate reactions, and the functionalized proteins were cross-linked into hydrogels using a matrix metalloproteinase 7 (MMP7)-degradable peptide sequence for cell-mediated degradation. Interestingly, the HA-binding and CS-binding peptides had different but complementary effects on human mesenchymal stem cells encapsulated within the hydrogels. The presence of the HA-binding peptide resulted in significantly greater chondrogenic differentiation with enhanced collagen type II, aggrecan, and SOX9 gene expression, leading to the highest matrix accumulation. Hydrogels functionalized with the CS-binding peptide induced significantly higher MMP7 gene expression and activity, which is important for the cleavage of ECM components during tissue remodeling. These results introduce the possibility to tune degradation kinetics and differentiation as desired by incorporating multiple peptides within a single hydrogel system. Our novel hydrogel platform demonstrates a high degree of modularity, highlighting the potential to tailor cell-mediated processes and create functional engineered tissues for regenerative medicine and tissue engineering applications.
[1] Place ES, Evans ND, Stevens MM. Complexity in biomaterials for tissue engineering. Nature Materials 2009;8:457-70.
[2] Chow LW, Armgarth A, St-Pierre J-P, Bertazzo S, Gentilini C, Aurisicchio C, McCullen SD, Steele JAM, Stevens MM. Peptide-directed spatial organization of biomolecules in dynamic gradient scaffolds. Advanced Healthcare Materials 2014;3:1381-6.
[3] Parmar PA, Chow LW, St-Pierre J-P, Horejs C-M, Peng YY, Werkmeister JA, Ramshaw JAM, Stevens MM. Collagen-mimetic peptide-modifiable hydrogels for articular cartilage regeneration. Biomaterials 2015;54:213-25.

Introduction: Recent years, stem cells have attracted great interests for regenerative medicine. For its realization, technologies controlling stem cell differentiation are strongly required. We have previously reported that poly (2-methoxyethyl acrylate) (PMEA) and its analogous polymers showed excellent biocompatibility owing to the existence of intermediate water which is one of the water structures in hydrated polymers and whose contents are determined by primary structures of polymers.^{1, 2)} We have also reported that intermediate water in hydrated PMEA analogous polymers influenced protein adsorption to the substrates. ^{1, 3)} It is thus expected that stem cell differentiation can be controlled on the substrates coated with PMEA analogous polymers with different intermediate water contents through the regulation of protein adsorption. In this study, we examined the differentiation of a mouse mesenchymal stem cell model, 3T3-L1, on PMEA and its analogous polymer substrates.
Materials and methods: PMEA and three types of PMEA analogous polymers (PMEA, PMe2A, PMe3A, and PTHFA) and poly (2-methacryloyloxyethyl phosphorylcoline (MPC)-co-n-butyl methacrylate) (PMPC) were used for this study. The amounts of adsorbed proteins on the PMEA analogous polymers-coated substrates were evaluated with mBCA assay after the immersing in 10% serum containing medium for 1 h. The adipogenic gene expression levels were measured by real-time PCR analysis after 4 days of culture under adipogenic condition. For cell adhesion inhibition assay, the cells were seeded on each polymer substrate in the medium with or without EDTA. After 1 h of incubation, the attached cells were counted under optical microscope observation after crystal violet staining.
Results: We found that the total amount of adsorbed FBS proteins was suppressed with the increase of intermediate water contents. On the other hand, adipogenesis of the cells were facilitated with the increase of intermediate water contents. To clarify the mechanisms why adipogenesis was facilitated on the polymers with higher intermediate water contents, we focused on integrin-mediated cell attachment mechanism using a cell attachment assay with EDTA, which is an inhibitor of integrin-dependent attachment. As a result, we found that integrin-dependent cell adhesion was weakened on polymers with higher intermediate water content. These results demonstrates that stem cell differentiation can be controlled by utilizing PMEA analogous polymers that possess different intermediate water contents through the tuning of protein adsorption and the degree of dependence on integrin-mediated cell attachment.
Conclusion: PMEA analogous polymers with different intermediate water contents have the potential of controlling MSC differentiation through the regulation of protein adsorption.
References: 1) M Tanaka et al., Biomaterials, 2000, 21, 1471-1481. 2) K. Sato et al., Macromol Biosci, in press. 4) T. Hoshiba, E. Nemoto et al., submitted

Cranio-maxillofacial (CMF) bone defects can result from trauma, infection, congenital deformities or tumor removal and represent a significant clinical need. Currently considered the gold standard of CMF bone defect treatments, transplantation of autologous grafts is often hindered by donor site morbidity and limited availability, among other limitations. Additionally, a particular difficulty is shaping and fixing the rigid autograft tightly into the defect in order to obtain osseointegration and ultimately prevent graft resorption. Tissue engineering has been explored as an alternative strategy for the treatment of CMF bone defects. Essential to the success of a tissue engineering approach is a scaffold that is able to conformally fit within an irregular defect - to overcome the limitations of autografting - while also having the fundamental biodegradability, pore interconnectivity and bioactivity. Thus, we have prepared a “self-fitting” shape memory polymer (SMP) tissue engineering scaffold that could conformally fit into an irregular CMF defect though the application of warm saline (~60 #8304;C). In this work, SMP scaffolds were prepared from diacrylated macromers comprised of inorganic polydimethylsiloxane (PDMS) and organic poly(ε-caprolactone) (PCL) segments, AcO-PCL_n-block-PDMS_m-block-PCL_n-OAc. High pore interconnectivity was achieved via a revised solvent-casting/particulate-leaching (SCPL) technique. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. By varying macromer composition and fabrication variables, the impact on pore interconnectivity, self-fitting and shape memory behaviors, degradation and in vitro bioactivity was systematically studied. The impact of ethylene oxide sterilization on scaffold properties is also evaluated. Overall, this approach represents a promising alternative to autologous grafting and conventional bone substitutes for CMF bone defect repair.

Nanosized hydroxyapatite (NHA) is the main component of mineral bone, which possesses exceptional biocompatibility and bioactivity properties with respect to bone cells and tissues. Living bone constantly undergoes a coupled resorptive-formative process known as bone remodeling. The process involves simultaneous bone removal and replacement through the respective activities of Osteoblasts and Osteoclasts, with the accompanying vascular supply and a network of canaliculi and lacunae. Compared to conventional ceramic formulations, NHA properties such as surface grain size, pore size, wettability, could control protein adsorption, configuration, and bioactivity. To this end, we have synthesized biomimetic hydroxyapatite nanoconstruct (nanosized hydroxyapatite, NHA) using a chemical gradient across lipid bilayer at room temperature for the surface modification of a Titanium implant. Thus synthesized NHA was characterized by DLS, XRD, TEM, FTIR, and in-vitro Osteoblast proliferation. As synthesized NHA was found to be highly stable in aqueous condition and exhibit the hydrodynamic diameter of ~150 nm. Powder X-ray crystallographic diffraction shows the formation of NHA with characteristic reflections corresponding to 2theta; degree (211), (130), and (213). The chemistry of the NHA was confirmed by Fourier Transferred Infra-Red (FTIR) spectroscopy analysis. Typical bands corresponding to the hydroxyapatite spectrum were observed at 560- 640, 963, and 1028 to 1110 cm^-1 correspond to the phosphate group (PO₄^3-), and the band at 3572 cm^-1 corresponds to structural -OH group. With these confirmation we moved forward to study the Normal Human Osteoblast (NHost) cell proliferation in metal surface. NHost when cultured on to the surface of NHA coated Titanium, a distinct proliferation pattern and cell-communication via cytokines secretion on the substrate surface was observed. Whereas in bare titanium surface NHost cells were found to have a diminished size with a minimal adherence on the surface. NHA along with phospholipid bilayer mimics the environment required for proper cell attachment on the surface as it occurs in live animals. Hence, this in-vitro studies indicated that inclusion of NHA to a fractured bone along with Titanium solid support during the treatment process would be an effective in fast healing of fractured bone.

Functional properties of tissue in the human body are heavily dependent upon the structure of its primary macromolecules. In particular for soft tissues such as articular cartilage and the lubricating synovial fluid surrounding them, these primary macromolecules are biopolymers. We are interested in developing biomaterials to address the suboptimal mechanical properties that typically accompany the disease osteoarthritis, a progressive degeneration of hyaline cartilage in joints. In a healthy state, the fluid surrounding the articulating cartilage surfaces contains the biopolymer hyaluronic acid, which imparts to the fluid rheological and tribological properties that prevent cartilage wear over decades of use. As the disease progresses, hyaluronic acid and other lubricating polysaccharides are structurally compromised and decrease in concentration; the fluid experiences a decrease in lubricating ability, and cartilage wear occurs. As there is a large and increasing population of individuals affected by osteoarthritis, there is a significant need to develop biomaterials that can remedy the material deficiencies, namely the propensity for wear, in articulating joints.
To address this need, we report the development of polymeric lubricants derived from poly(2-methacryloyloxyethyl phosphorylcholine) (polyMPC), a polymer known for its biocompatibility and ultra-low friction when grafted to two-dimensional surfaces. Macromolecular polyMPC networks of various network architecture, network concentration, crosslinking density, and crosslinker composition were synthesized via free-radical polymerization initiated by ammonium persulfate. Aqueous polymer solutions were characterized by rheometry, and their lubricating properties were evaluated with ex vivo bovine cartilage specimens. Cylindrical osteochondral samples were subjected to a simultaneous torsion/creep cartilage-on-cartilage regimen consisting of 10080 rotations under a constant stress of 0.78 MPa. Coefficient of friction and compressive strain were computed, and cartilage was graded semi-quantitatively for wear. The polyMPC lubricants were found to lower coefficients of friction by up to 40% compared to native synovial fluid, and to determine mechanism of friction attenuation, a Stribeck curve of coefficients of friction at various articulating speeds and viscosities was generated, indicating effective boundary-mode lubrication. The key polymer design criteria for minimizing cartilage friction and wear will be presented, along with preliminary rodent in vivo efficacy data.

Hydrogels have emerged as promising platforms for culturing cells in vitro because their mechanical and biochemical properties can be readily tuned to achieve functional, defined ECM mimics. One method to impart biological activity into hydrogels is to covalently tether biomolecules (e.g., the adhesive peptide RGDS) to the hydrogel backbone. While this approach is commonplace for peptides, proteins (such as growth factors and cytokines) are still traditionally added to culture media as soluble factors. Increasing evidence suggests, however, that the context in which proteins are presented to cells (e.g, as soluble verses tethered signals) can influence their bioactivity. Indeed, many growth factors exist bound to the ECM in vivo suggesting cells frequently interact with immobilized signals. Moreover, not all cells within a given niche are exposed to the same concentrations of growth factors at a given time. Here, we present an approach to reversibly tether proteins to hydrogels with spatiotemporal control to recapitulate protein-ECM interactions in vitro.
We drew inspiration from chain-transfer agents employed in reversible addition-fragmentation chain-transfer polymerization to identify an allyl sulfide functionality that allows for reversible, sequential thiol-ene reactions (addition of a biomolecule regenerates the 'ene' functionality and releases the previously tethered biomolecule). Previously, we have employed this methodology to achieve reversible thiol-ene-mediated immobilization of peptides in hydrogels. The focus of this work has involved expanding this approach to achieve reversible protein immobilization. Specifically, we have employed strain-promoted azide-alkyne cycloaddition polymerization to generate poly(ethylene glycol) hydrogels that contain pendant allyl sulfide functionality. Fluorescently-labeled, thiolated ovalbumin (Ova) was swelled into the hydrogel and immobilized with spatial control using photolithographic techniques. Protein release is enabled by a subsequent thiol-ene reaction of a thiolated small-molecule to regenerate the allyl sulfide functionality and release Ova. Current studies are ongoing to optimize the kinetics of protein release and subsequent protein additions. We shall leverage this methodology to study how the contextual presentation of two growth factors, transforming growth factor-β and fibroblast growth factor-2, influence fibrosis of aortic valvular interstitial cells as a model of valve disease.

How to fabricate a microvascular network to support the tissue growth is crucial to tissue engineering. The major challenge in bottom-up synthesis of large-scale neotissues for transplantation is revascularization. Herein, we report on a generic route to generate endothelialized microvessel networks in a fibrin gel system with the aid of senescent fibroblasts. The microvessels rapidly develop in the vicinity of senescent fibroblasts within 6 days and potentially can be retrieved as a free-standing bio-compatible transplant towards regenerative medicine. Senescent fibroblasts (FB), induced by bleomycin treatment, secrete significantly higher levels of VEGF, IL 6, and IL8, compared to normal human lung fibroblasts. The sustained elevated level of VEGF promotes proliferation of HUVECs towards vasculogenesis in fibrin gel (2 mg/ml fibrinogen). Premature endothelial sprouts after a 24-hour co-culture with senescent fibroblasts. High quality interconnected capillary-like (20~80 mu;m in diameter) microvascular structures develop in 4-6 days. We process the 2-D images to compute the total length and the number of branches, and find that HUVECs and HUVEC+FB groups do not give rise to a lot of capillary-like micro vessels. Instead, most of them are cell aggregates or sheets. But the HUVEC+senescent FBs group does give capillary like micro-vessels. Further investigation would confirm whether 3-D lumen-like EC sprouts are present in the HUVECs only and HUVEC+FB group at early stages (day 2-4). Compared to HUVECs cultured alone in fibrin gel, senescent fibroblasts significantly accelerate the vasculogenesis process and improve the quality of the microvessels with increased length, branches, and more uniform capillary diameters. In addition, senescent fibroblasts do not proliferate, so they don#700;t take much space as other mural cells such as non-senescent fibroblasts and mesenchymal stem cells, and thus maintain the high degree of vascular integrity.

Orthopedic tissue engineering would help thousands of patients every year suffering from severe bone defects, e.g. following bone tumor resections. For such a therapy a suitable scaffold is needed to support cell growth and the build-up of bone tissue. As bone is a complex and highly dynamic tissue, autologous bone has the best clinical outcome but suffers from donor site morbidity and its limited availability [1]. Therefore, there is the need for synthetic materials that enhance bone cell growth and promote bone formation as demonstrated already successfully for porous polymeric or ceramic materials. Nevertheless, the ideal scaffold still needs to be created.
It has long been known that Si enhances bone cell activity and improves bone formation [2]. Therefore, next to the biomimetic ceramic hydroxyapatite (HA) Si releasing materials are promising candidates for biomaterials in bone tissue engineering. As silicon nanomaterials, such as silicon nanowires (SiNWs), have recently been found to dissolve in physiological environment [3] and thus release silicate into the surrounding, they may be used as biomaterial to enhance bone cell activity [4], either in combination with e. g. HA or on their own.
Here, SiNWs with a diameter distribution around 100 nm and a length towards the millimeter range were grown bottom-up on Si substrates in high density by vapor-liquid-solid growth. The use of SiNWs is a fast and scalable route to resemble collagen fibrils structurally. Grown in high density, they build up highly porous layers similar to electrospun fibers. As a step towards a 3D scaffold material, the SiNW layers were also rolled up mechanically, producing SiNW fleeces.
The dissolution behavior of the SiNW scaffold was studied in PBS and cell culture medium as simulated physiological environments by measuring Si release using ICP-AES. A clear increase of Si release in the protein containing cell culture medium was measured compared to the solely ion containing PBS. Further, MC3T3-E1 osteoblastic cells were cultured on the SiNW substrates to assess cell proliferation by MTT assay. Preliminary data indicate that short-term cultivation (1 week) had a stimulating effect on cellular activity compared to cell cultivated on control substrate, whereas the cultivation for 3 weeks resulted in diminished cell proliferation. Further studies are performed to investigate cell differentiation behavior.
[1] Stevens 2008, Mater. Today 11, 18-25
[2] Carlisle 1981, Calcif. Tissue Int. 33, 27-34; Kim et al. 2013, Biol. Trace Elem. Res. 152, 105-112
[3] Zhou et al. 2014, Nano Lett. 14, 1614-1619
[4] Popat et al. 2006, J. Orthop. Res. 24, 619-627

Poly(L-lactic acid) (PLLA) has received much attention due to its bioresorbable and biocompatible properties in tissue engineering. Recently, the incorporation of biodegradable hydrophilic polymers into PLLA has been applied for biomedical materials. These copolymers are expected to improve poor hydrophilicity of PLLA and to encourage the cell growth on scaffolds. In this study, we selected methyl poly(ethylene glycol) (MPEG) as a hydrophilic polymer and synthesized methyl poly(ethylene glycol)-block-poly(L-lactic acid)s (MPEG-b-PLLAs) and investigated the effect of incorporating MPEG segments on cell adhesion and proliferation.
MPEG-b-PLLAs were synthesized by the ring-opening polymerization of L-lactide using hydroxyl-terminated MPEG as an initiator and stannous octoate (Sn(Oct)₂) as a catalyst. The block length of PLLA could be adjusted by changing the proportion of the monomer and initiator. The synthesized polymers were characterized by using ¹H NMR, FT-IR, and GPC. Molecular weights (M_n) of MPEG-b-PLLAs increased linearly with the molar ratio of MPEG and L-lactide. The polydispersity index (PDI) remained as low as 1.2 with high monomer conversions up to 100%. M_ns of MPEG-b-PLLAs were in the range of 13,000 to 68,800 which were determined by GPC, and the unit molar ratios (MPEG : PLLA) calculated by ¹H NMR were in the range of 1 : 0.6 to 1 : 7.19. The diblock copolymer thin films were prepared by spin-coating 1% (w/v) chloroform solution onto glass slides. The influence of the surface morphologies on physicochemical properties was investigated by contact angle measurements and cell adhesion rate measurements using MC3T3-E1 cells. The results of contact angle measurements showed that the hydrophilicity of the copolymers increased with the incorporation of MPEG segment into the polymer. In order to create porous scaffolds for soft tissue engineering, MPEG-b-PLLA fiber membranes with various MPEG block ratios were fabricated by electrospinning. MPEG-b-PLLA was dissolved in 2,2,2-trifluoroethanol (HFIP) to obtain viscous solution. The obtained highly viscous solutions were electrospun at a high input voltage of 25-30 kV to create nanofibers with fiber diameters of 1.18 µm to 1.48 µm. The morphology of fiber membranes were analyzed by SEM, and it was found that the obtained nanofiber membranes have a porous structure suitable for cell adhesion and growth. The porosities of the fiber membranes were about 87-93%. The cell adhesion ratio and proliferation were examined by culturing MC3T3-E1 cells on these substrates. The results concluded that the cell adhesion and proliferation were significantly affected by the fiber diameters and shapes rather than the compositions of copolymers and spinning solvents.

The paper describes a method to fabricate three dimensional (3D) scaffolds from synthesized glycopolymer photocurable resin. The 3D structure of disc (1 mm height x 6 mm diameter ) from polymer system compromising of polyethylene glycol diacrylate (PEGDA) /hexanediol pentaacrylate (HDPA) with additional of 2.5 and 5.0% of synthesized glycopolymer (Glc) is successfully fabricated using envisionTec Desktop Digital Shell Printer (Desktop). They are then subjected to swelling ratio and sol fraction to determination the degradability of the structure. Dumbbell shape test piece are also fabricated and subjected to micro-tensile measurement. The swelling ratio has increased 20 and 33% after 2.5 and 5.0 % of Glc are added to the PEGDA/HDPA whereas the degradation increased 35% for 5.0% of Glc addition. The tensile strength of the structure has shown slight increment from control to 5.0% of Glc. In conclusion, microstereolithography technique is feasible to develop glycopolymer for biomedical application of hydrogels.

Titanium and its alloys are widely used in dental implants due to their combination of properties closely related to a surface oxide layer formed, such as adequate biocompatibility and high corrosion resistance [1]. However, the studies show that it has very poor bone integration, after the implantation. Researchers reported that the conjugation of biomolecules onto metal oxide surface can improve the biocompatibility and bioactivity of the metallic implants and accelerate the osteointegration process [2].
The objective of this study is to improve the osteointegration of Ti-surfaces by adding biomolecules and study the remineralization process. Our hypothesis is that peptides derived from DMP1 will enhance cell adhesion on the titanium surface and can lead to remineralization.
In this study TiO₂ and ZnO were synthesized by sol-gel method. Discs of titanium cp were coated with the metal oxides by spin coating technique and then annealed in order to form nanocrystalline particles. The peptides are derived from DMP1, an acidic phosphorylated non-collagenous protein that plays a critical role in biomineralization [3]. Peptide pA (ESQES) and peptide pB (QESQSEQDS). These have been identified as hydroxyapatite binding peptides. Use of these peptides can lead to remineralization. The peptides pA and pB were immobilized by covalent bonds onto metal oxides surface by coupling reactions on self-assembled monolayers (SAMs), using thiol-, silane-, hydroxyl- and amino-based SAMs.
XRD measurements indicated that rutile and wurtzitte polymorphic crystalline phase were obtained for TiO₂ and ZnO, respectively. SEM images showed that the particles sizes are around 100 nm for titania and 30 nm for ZnO. XPS data indicates that the SAMs were attached upon the oxide surface by hydroxyl groups. As the ratio of 1:4 of pA to pB was determined to have superior dentin remineralization, based on hydroxyapatite morphology and calcium/phosphorus ratios, we have decided to use a similar ratio of the peptides for coating the functionalized titanium surface. Tests with stem cells are being conducted in order to determine their adhesion and osteogenic differentiation. Such method of functionalization of inorganic substrates by anchoring biomolecules to the surface is a very powerful way to tailor surface properties. This methodology can overcome material deficiencies while maintaining its bulk material properties and improve osteointegration.
1. Alves, A. C. et al. Tribocorrosion behaviour of anodic treated titanium surfaces intended for dental implants. J. Phys. Appl. Phys. 46, 404001 (2013).
2. Care, A., Bergquist, P. L. & Sunna, A. Solid-binding peptides: smart tools for nanobiotechnology. Trends Biotechnol. 33, 259-268 (2015).
3. Padovano, J. D. et al. DMP1-derived Peptides Promote Remineralization of Human Dentin. J. Dent. Res. 94, 608-614 (2015).

Introduction: One of the most predominant symptoms of skin aging is wrinkle formation, which results from both intrinsic aging and environmental damage. Current anti-aging and anti-wrinkle materials often induce a toxic response, which results in inflammation, to increase tissue growth under the skin. Although effective, a much safer and more effective way to alleviate the effect of aging and thereby winkle formation needs to be developed. In this study, fluorescein isothiocyanate (FITC) peptide amphiphiles that consist of both cell penetrating peptides and biomimetic sequences were designed, synthesized, characterized, and tested for their ability to promote skin growth in a healthy manner. The ability of these peptide amphiphiles to penetrate the skin and to promote fibroblast functions (specifically, increasing adhesion, proliferation, collagen synthesis, and decreasing elastase and collagenase synthesis) was determined.
Materials and Methods:Material characterization. Self-assembled amphiphiles were characterized by zeta potential to determine their charges, by dynamic light scattering to examine their size, and by TEM to observe their morphology. Cell adhesion and proliferation. To determine cellular adhesion/proliferation, keratinocytes and human dermal fibroblasts were seeded at a density of 10,000 cells/cm². Both types of cells were incubated under standard cell culture conditions for a certain period of time. The MTS assay was used to determine cell density after incubation. Skin penetration/permeation. Skin penetration and permeation were determined using Static Franz diffusion cells with porcine skin as the membrane between donor and acceptor compartment due to their resemblance to human skin. Peptide amphiphiles were applied in the donor compartment, and PBS was used as the receptor solution. The donor compartment was sealed with aluminum foil and the system was maintained at 37°C in water bath. The skin samples were fixed and sectioned to examine penetration using confocal microscopy. Measurement of total collagens. A sircol soluble collagen assay (Biocolor, UK) was used to quantify total soluble collagens after 7, 14 and 21 days of culturing. All experiments were conducted in triplicate and repeated at least three times. Differences between means were determined by ANOVA and student t-tests.
Results: While peptide amphiphiles showed moderate toxicity at a higher concentration, they provided improved skin penetration across the stratum corneum when compared to using short peptides alone. Also it was found out that these peptide amphiphiles have the abilities to promote cell prolifereation as well as total collagen synthesis in the long term.
Conclusions: Through the above experiments, various peptide amphiphiles were identified that can be added to current skin cream formulations to promote penetration of active ingredients, to increase fibroblast growth, as well as to enhance collagen synthesis.

Hydrogels have been widely studied, especially as injectable materials for applications as biomaterials. These hydrogels may possess thermosensitive features near body temperature (LCST - Lower Critical Solution Temperature - around 370C), rapid gelation, carry drugs, cells or cell growth factors. The current challenge is to make these materials biodegradables and mechanical properties optimized for their use in cardiac tissue engineering. The aim of this study was to synthesize hydrogels of poly(N-iropropylacrylamide) (PNIPAAm) from a new class of macromer consisting of 2-hydroxyethyl methacylate (HEMA), poly(L-co-D,L lactic acid) and trimethylene carbonate (PLDLA-co-TMC) in order to give the hydrogel biodegradation characteristics and mechanical properties suitable for future applications in cardiac tissue engineering. Hydrogels were synthesized by free radical polymerization, the macromer having concentrations ranging from 5%, 10% and 15% (w/w) to evaluate the influence in chemical, mechanical and thermal properties. Macroscopic evaluation showed that the hydrogels exhibit characteristics of injectable and thermosensibility. Swelling tests showed swelling hydrogels with absorption of 20, 29 and 63% of water for compositions. Nuclear Magnetic Resonance (NMR ¹³C e ¹H) confirmed the copolymerization of HEMA with PLDLA-co-TMC as well as the copolymerization of hydrogel PNIPAAm-co-AAc-co-HEMAPLDLA-co-TMC. Infrared spectroscopy (FTIR) corroborated the data found in (NMR ¹³C e ¹H). Differential Scanning Calorimetry (DSC) showed the LCST for samples at 290C, 300C and 240C, temperatures being lower than 370C, indicating these hydrogels may be used in the human body. Mechanical tests showed hydrogels with Young&’s Modulus of 531, 922 and 725 kPa, and over 500% strain in all synthesized compositions. Degradation of materials followed by weight loss demonstrated stability of hydrogel during first 4 weeks and after 10 weeks showed the onset of weight loss. Finally, the biological interactions assays showed the material as cytocompatible and non-toxic to Vero cells. Mouse embryonic stem cells (mESC) were grown in control conditions (polystyrene plate) and hydrogel, and the expression of β-Actin, Brachyury-T, Isl-1, Nkx2.5 and MHC6 transcripts was analyzed via quantitative PCR (qPCR). Because the expression levels presented are similar to the control, and does not exhibit relative significance between conditions of the differentiation time, it can be inferred that there are no changes on cardiomyogenesis pathway between the control and the hydrogel, namely the material allows cell differentiation. Thus, the synthesized hydrogels are promising for future applications in cells carriers for tissue engineering.

Considering the limited physiological relevance of 2D cell culture experiments, significant effort was devoted to the development of materials that could more accurately recreate the in vivo cellular microenvironment, and support 3D cell cultures in vitro. [1] One such class of materials is conducting polymers, which are promising due to their compliant mechanical properties, compatibility with biological systems, mixed electrical and ionic conductivity, and ability to form porous structures. [2,3] In this work, we report the fabrication of a single component, macroporous platform made from poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) via an ice-templating method. [4] PEDOT:PSS scaffolds offer tunable pore size, morphology and shape through facile changes in preparation conditions, and are capable of supporting 3D cell cultures due to their biocompatibility and tissue-like elasticity. Moreover, these scaffolds are functional: they exhibit excellent electrochemical switching behavior and significantly low impedance. The adhesion and pro-angiogenic secretions of mouse fibroblasts cultured inside the porous structure can be controlled by switching the electrochemical state of the polymer prior to cell-seeding. This allows for noninvasive control of spatial localization of cells, an attractive approcah for tissue engineering. The electrochemical activity of the scaffolds enables their use in the active channel of the state of the art diagnostic tool of bioelectronics, i.e., the organic electrochemical transistors. Hence, these smart materials hold promise not only as extracellular matrix-mimicking structures for cell culture, but also as high-performance bioelectronic tools for diagnostic and signaling applications.
[1] M. Holzwarth, P. X. Ma, J. Mater. Chem. 21, 10243#8208;10251 (2011).
[2] L. H. Jimison, J. Rivnay, R. M. Owens, in Organic Electronics, Wiley#8208;VCH Verlag GmbH& Co. KGaA, 27#8208;6 (2013).
[3] A. I. Cooper, Advanced Materials, 21, 1291#8208;1295, (2009).
[4] A. M.-D. Wan, S. Inal, T. Williams et al., J. Mater. Chem. B, DOI: 10.1039/C5TB00390C (2015).

High-density cultures of cells can mimic immature condensates present during many developmental processes. Presenting specific soluble signals, such as growth factors, exogenously in tissue culture media can regulate cell behavior in these cultures and promote new tissue formation. However, shortcomings of this approach include transport issues, limited spatial control over signal presentation, and required repeated dosing in the media. We have engineered technology that overcomes these challenges by incorporating polymer microspheres containing bioactive signals within the high-density cell cultures, which permits localized spatial and temporal control over the presentation of these regulatory signals to the cells. In this talk, I will present our research using this strategy to engineer a variety of tissues, including bone, cartilage and trachea. The capacity to deliver diverse signals, including growth factors and plasmid DNA, for driving new tissue formation will be demonstrated. In addition, the value of this technology for engineering a wide range of tissue shapes, including spheres, sheets, rings and tubes will be shown. Finally, the utility of providing cell-instructive bioactive factors from biomaterials in a controlled manner, for the assembly of modular tissue units to engineer complex constructs comprised of multiple tissue types, will be explored.
Acknowledgements: This work was supported by the National Institutes of Health (R01AR063194), the Department of Defense Congressionally Directed Medical Research Programs (OR110196), the AO Foundation, and a New Scholar in Aging grant from the Ellison Medical Foundation.

With over 2.3 million soft tissue filler procedures performed within the United States in 2014, injectable materials have become an effective way of treating anti-aging, rejuvenating facial defects and correcting volume loss.^1,2 Despite the popularity of current fillers, a need exists for the development of better tissue fillers. These novel, next-generation materials will not only support tissue bulking and volume retention, but should a) initiate tissue regrowth via cell mediated degradation and integrated delivery of growth hormones and cell signaling factors, and b) fill a range of mechanical stiffness without compromising injectability. Towards this need, new silk protein-based shape memory sponges were investigated for minimally invasive soft tissue bulking. Silk fibroin is a naturally derived, fibrous protein which is non-immunogenic, FDA-approved for reconstructive surgery and capable of volume retention up to at least 18 months in vivo. Shape memory polymers (SMP&’s) are a subclass of stimuli-responsive “smart” materials that can recover from a deformed shape back to an original, pre-defined shape in the presence of an external stimulus, such as temperature or hydration. These systems are useful for minimally invasive delivery of bulky devices or filler materials which are expected to undergo high compression during deployment and complete recovery post-implantation. Our data demonstrate silk shape memory sponges can be designed to rapidly recover near their original volume after compression to 80% strain. An open pore morphology supports high cell infiltration and extracellular matrix deposition over 12 weeks in vivo. Silk memory sponges can also be designed to swell beyond their original volume in the presence of aqueous media, in excess of 40X their compressed volume. The high elasticity and tissue regenerative properties of silk memory sponges make them suitable as next generation minimally invasive tissue fillers.
References:
1. American Society of Plastic Surgeons. 2014 Plastic Surgery Statistics Report. Available at: http://www.plasticsurgery.org. Accessed June 1, 2015.
2. Attenello NH, Maas CS. Injectable Fillers: Review of Material and Properties. Facial Plast Surg 2015;31(1):29-34.

Due to the limited supply of tendon/ligament autografts and unsatisfactory performance of allografts or xenografts, there is a strong need to develop synthetic tendon implants that mimic tendon extracellular matrix (ECM) and are able to repair or regenerate the injured tendon (or ligament). Here we used an environmentally-friendly electrochemical process to align soluble collagen macromolecules into a densely-packed, aligned nanofibrous structure in the form of sheets or fibers. These sheets or fibers were highly biocompatible and supported the attachment, migration, and proliferation of both tendon fibroblasts and adipose-derived stem cells. Both the wrapped sheets and braided collagen fiber scaffolds were tested in a critical size tendon gap defect model in Lewis rats. Following 16 weeks' implantation, the tendon implants were retrieved and tested for critical load to failure and examined for histology. Both the wrapped aligned collagen sheets and braided aligned collagen fiber scaffolds demonstrated equivalent critical load to failure to normal, healthy tendon controls. Histological analysis by H&E staining and Von Gieson's staining confirmed the formation of tendon-like collagen tissue and organization of tenocytes. This study strongly suggest the feasibility of tendon/ligament repair and regeneration strategy with novel electrochemically aligned collagen scaffolds.

In the field of regenerative medicine, vascularization of solid organs such as the liver is a considerable challenge. Not only is the liver made of over 100 billion hepatocytes, each of them must be within 50 µm of a blood stream. The body fulfills this demand by placing modular microscale structures in close proximity with the blood vessels. The communication between these cells, in the form of paracrine signals that allow the hepatocytes and endothelial cells to coordinately expand, has been shown to be important in the regulation of liver processes such as development and regeneration.
Previously, we have developed microtechnology tools to control the organization of multiple cell types within a 3D context and shown that architecture impacts heterotypic paracrine signals. Emerging from these model systems appears to be a reciprocal interaction between hepatocytes and endothelial network via paracrine signals. To combine the effects of flow-mediated perfusion with paracrine signaling, we fabricated fibrin and polydimethylsiloxane-based vascularized tissues consisting of: a) a central vascular channel, embedded in a fibrin matrix and lined with endothelial cells, b) liver constructs, consisting of aggregated hepatocytes and fibroblasts embedded inside the fibrin matrix, and c) flow-based setup to modulate the shear-stress experienced by the endothelial cells. When cultured under oscillatory flow, each device produced on the order of ~20 µg albumin/mL into the perfusate for up to a week and stained for both hepatic (Arginase-I) and endothelial (VE-cadherin) markers. Thus, we introduce a new model system of engineered vascularized liver microtissue to develop insights into how vascular architecture and associated perfusion affect the paracrine signaling network that supports liver function and regeneration. In the future, we will attempt to assess the functional role of network architecture and perfusion on graft expansion in vivo

There is no doubt on the importance of fluorophores and fluorescent materials. Fluorescent materials have numerous applications in biological and biomedical fields ranging from protein/cellular labeling and imaging to analyte biosensing. Developing new fluorophores and fluorescent materials has been sought-after in biomedical fields. For examples, conventional theranostic drug delivery nano systems conjugated or encapsulated with organic dyes or quantum dots (QDs) suffer from multiple challenges for clinical translations such as poor photo-bleaching resistance, low dye-to-particle conjugation ratios, high toxicity, increased particle sizes, added complexity, and high risk of adverse biological reactions. In tissue engineering, an in situ real-time method to accurately monitor and quantify scaffold degradation and tissue regeneration without traumatically explanting samples or sacrificing animals is urgently needed. There have been significant efforts in developing new fluorophores and fluorescent materials such as poly(amido amine) (PAMAMs), cabon nanodots (CDs) and our recently developed biodegradable photoluminescent poymers (BPLPs) to address the challenges in the above biomedical applications. However, the fluorescence mechanisms of these new fluorophores remain unclear. Therefore, the rational design of new fluorophores and materials remains a significant challenge due to our limited ablity to correlate fluorescence and molecular structures.
Our recently developed BPLPs could potentially address the above challenges in tissue engineering and theranostic drug delivery. BPLPs are synthesized from three monomers, aliphatic diol, citric acid, and α-amino acid. BPLPs have shown complete degradation, high quantumn yield (as high as 80%), and long fluorescence life time (>12 ns), which can be tuned by varying the choices of amino acids and monomer feeding ratios. The emission wavelengths can be tuned as “red” as 725 nm. BPLPs also possess excellent photostability unlike many other organic dyes. BPLPs have already been used in many biomedical applications that traditional biodegradable polymers or fluorescent molecules appear unsuitable, including non-invasive monitoring of tissue engineering scaffolds and theranostic cancer imaging and drug delivery. In this presentation, a complete understanding on the fluorescence mechanism of BPLPs and a rational design of a series of new citrate-based fluorophores and fluorescent polymers will be discussed. Specific applications to be covered include soft and hard tissue engineering, sweat test for the diagnosis of cystic fibrosis (biosensing), and immune-cell mediated cancer drug delivery.
Acknowledgement
The authors are grateful for the funding support from a National Institutes of Health Award (NIBIB EB012575, NCI CA182670, NHLBI HL118498), and National Science Foundation (NSF) Award DMR 1313553.

The role of polymer-inorganic composite electrospun fibers as tissue engineering scaffolds are well recognized. We report here the fabrication of electrospun nanofibers from Poly(methyl methacrylate) (PLA) with hydroxyapatite synthesized using eggshell. The ability of the composite electronspun nanofibers to support the attachment and growth of mammalian cells was examined by culturing the transformed ATCC CRL 11372 and SW480 cell line (ATCC CCL-228) with the fibers in vitro. Cells were physically deposited on PMMA fibers with and without hydroxyapatite nanoparticles, which were immersed in growth medium (ATCC McCoy&’s medium) and incubated in a humidified tissue culture incubator at 37^oC and 5% CO_2. Cell attachment and growth were followed by light microscopy. The FE-SEM images revealed that the nanofibers were well-oriented and incorporated with the nanoparticles, which were distributed on the nanofibers. The energy dispersive spectrum (EDS) confirmed the inclusion of the nano particles on the surface of the nanofibers. X-Ray Diffraction (XRD), Fourier Transform Infra-red (FTIR) showed the particles used are hydroxyapatite. The light microscopy showed that SW480 cells can attach and grow very well on the fibers. Images from the light microscope also showed that the composite fibers can support the attachment and growth of the bone-osteoblast cells (ATCC CRL-1372) but took a long time than SW480 cell line. Further studies are ongoing to assess the potential of the composite fibers for bone tissue engineering.

Thin and flexible polymer membranes are finding vital applications in regenerative medicine, whether it is to recapitulate critical physiological features that undergo cyclic stress or to study cellular responses to mechanical stresses. However, existing membranes used to impose cyclic stress are limited either by the lack of biocompatibility or the necessary strain profile. Commonly used polymers to fabricate these membranes such as Polydimethylsiloxane (PDMS) have been reported to cause undesired effects such as absorption of small molecules [1] and limited compatibility with large scale production [2].
To address these limitations, we are investigating the application of Polyurethane (PU) to develop thin (<20 mu;m), flexible, and biocompatible membranes. PU is well known for applications in various biomedical devices and has been shown to cause minimal absorption of small molecules compared to PDMS [1]. We are studying different types of PU including GSP 1552-2 (G.S. Polymers, Inc) and Bayhydrol (Bayer Material Science). Optically transparent membranes with thicknesses ranging from 6 mu;m to 20 mu;m were fabricated using spin coating on silicon and polymeric substrates. However, it is difficult to release the thin membranes from the substrates without any treatment. We report a unique technique to reproducibly release the membranes form the substrates and mount them on a frame for device integration. The membranes were characterized for their elastic properties using a microfluidic based bulging test system. The Young&’s modulus of 10 micron films was found to be ~112 kPa. It is possible to apply up to 6 kPa fluidic pressures to execute cyclic stress on 1 mm diameter membranes without failure. Adenocarcinomic human alveolar basal epithelial cells (A549) and Human microvascular endothelial cell (HMVEC) were co-cultured on the apical and basal sides of the PU membrane, respectively. The morphology and the viability of the cells were compared to cells cultured on standard tissue-culture plates.
Our experiments suggest that stretchable PU membrane may be useful for various tissue culture applications including the development of in vitro models of the lung, skin, heart, etc.
References:
[1] K. Domansky, D.C. Leslie, J. McKinney, J.P. Fraser, J.D. Sliz, T. Hamkins-Indik, G.A. Hamilton, A. Bahinski, D.E. Ingber, Lab on a Chip, 13 (2013) 3956-3964.
[2] P. Gu, T. Nishida, Z.H. Fan, ELECTROPHORESIS, 35 (2014) 289-297.
LA-UR-15-24567

Critically sized composite silk scaffolds with aligned pore architecture have been developed for applications in cardiac tissue repair, repair of volumetric muscle loss in the limbs, and generation of patches for repair of craniofacial defects. Our recent work has focused on the generation of aligned silk sponges^{1, 2} using silk fibroin and decellularized muscle tissue from either cardiac (cECM)³ or skeletal muscle (sECM) origin. Addition of the ECM to the silk solution improves cell-based degradation of the sponge and enhances in vivo cell infiltration ².
To create these sponges, we have utilized directional freezing methods followed by lyophilization to achieve sponges with anisotropic pore architecture. Utilization of silk-cECM sponges in vivo demonstrates improved host cell infiltration in a subcutaneous model.² Further results demonstrate an increase in functional recovery following implantation over the scar tissue formed following permanent ligation of the coronary artery. Preliminary data demonstrates that the ejection fraction and fractional area shortening in silk-cECM composite patches with anisotropic pore architecture were statistically similar to the pre-infarct values after 6 weeks post repair when evaluated in a rodent model of myocardial infarction (p>0.05, n=3). In vitro evaluation using both human embryonic stem cell derived cardiomyocytes and an atrial carcinoma cell line showed improvements in protein production and cell attachment compared to isotropic silk and aligned silk only sponges (e.g., myosin heavy chain, connexin 43).²
Similarly, we have utilized silk-sECM sponges to create in vitro models of skeletal muscle behavior, demonstrating that C2C12 murine myoblasts are able to growth and differentiate within these sponges, improving production of mature muscle markers (e.g., desmin, Pax7).
Overall, our work demonstrates that directional freezing and subsequent lyophilization and water annealing generates silk-based biomaterials with adequate integrin binding resulting in materials suitable for both in vitro and in vivo application.
1.Rnjak-Kovacina, J.; Wray, L. S.; Burke, K. A.; Torregrosa, T.; Golinski, J. M.; Huang, W.; Kaplan, D. L., ACS Biomater Sci Eng 2015,1 (4), 260-70.
2.Stoppel, W. L.; Hu, D.; Domian, I. J.; Kaplan, D. L.; Black, L. D., Biomed Mater 2015,10 (3), 034105.
3.Williams, C.; Budina, E.; Stoppel, W. L.; Sullivan, K. E.; Emani, S.; Emani, S. M.; Black, L. D., 3rd, Acta Biomater 2015,14 (0), 84-95.

Carbon based materials have been explored for their application in energy storage and electronics, however, very limited studies have evaluated their potential in tissue engineering. In this study, we investigated potential of carbon-based scaffolds with different geometries and surface functionalization for skeletal muscle tissue engineering. Recreating native microenvironment and providing appropriate guiding cues are instrumental in the design of tissue-engineered scaffolds. For examples, skeletal muscle possesses highly anisotropic tissue architecture. In addition, conductive materials have shown to improve skeletal muscle cell (myoblast) differentiation. Thus, we engineered multiscale hierarchical scaffolds based on carbon materials, highly porous carbon foams and highly anisotropic carbon fibers. Both porous foam scaffolds and anisotropic fibrous scaffolds were nanofunctionalized with carbon nanotube (CNT) and CNT-silica coatings on their surfaces. It was hypothesized that anisotropic fibrous structure and nanoscale CNT coatings will provide synergistic guidance cues to myoblast differentiation.
CNT coating increased contact angle of carbon foams from 65-75° to asymp;160° showing higher hydrophobicity while further coating of silica on CNT-coated foams reduced contact angle to asymp; 66.5°, suggesting hydrophilicity of CNT-foams similar to uncoated carbon foams. Moreover, surface functionalization did not significantly change the porosity of foams and fibers. CNT and Si-CNT coating on carbon foams enhanced myoblast cell adhesion, growth and differentiation, thereby enhancing MHC positive cells. However, continuous and fused myotube structures were not observed. On the other and, highly aligned CNT-coated carbon fibers not only promoted differentiation into MHC-positive cells but also facilitated formation of multinucleated continuous, and fused myotube structures. These results collectively indicate the combined role of nanoscale surface modification (CNT functionalization) and aligned fibrous structure (microstucture) to facilitate cell adhesion, growth and differentiation. Future studies will focus on myogenic gene expression on these scaffolds.

We have developed a customizable assay to observe the differential fate of stem cells in response to multiple parallel and competing cues with single cell resolution and high-throughput. Stem cell fate is regulated by the interplay between bio-chemical and physical microenvironmental cues, much of which still remains unclear. Our model attempts to better understand the influence of individual factors.
We used a 193nm ArF excimer laser (near-Gaussian distribution, ~120mJ/cm² fluence, 100 Hz pulse frequency) to micromachine “sunburst” and “horse-race” patterns into cell adhesive hydrogel substrates (10%w/v gelatin cross-linked with 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-Hydroxysuccinimide). A sunburst pattern consisted of a central circle (diameter 520um, depth 580um) with 8 emanating radially-symmetric channels (depth 630um, length 1000um, widths alternating 10/20um). A horse race pattern consisted of 8 circles with one channel emanating from each, spaced >1mm apart with their centers aligned.
To provide chemical cues, one 10um and one 20um wide channel was coated with a ~50um thick layer of an extracellular matrix protein solution. To provide physical cues, the elastic properties ("stiffness") of the substrate was varied by changing the gelatin concentration and cross-linking density, as well as by using different hydrogels. Embryoid bodies (EB) ~500um in diameter were generated from spontaneous aggregation and differentiation of pluripotent embryonic stem cells (ESD3). The patterns contain one EB within a circle, and allow migration into a channel one or two cells at a time. The EBs were precisely transferred into the circles using the matrix assisted pulse laser evaporation direct write (MAPLE DW) bioprinting technique. We have shown that this technique can successfully deposit large 3-D microspheres in specified locations with high precision, and that embryonic stem cells maintain their pluripotency following deposition. Following culture in standard conditions, induced differentiation and migration was analyzed using immunocytochemistry and time-lapse microscopy.
This versatile assay will allow for high-throughput studies into microenvironmental cues governing stem cell fate. In the future, we could investigate the influence of additional bio-physicochemical cues, including coating channels with ligands, chemo-attractants, drugs and other bioactive compounds, producing chemical gradients in channels, micro-texturing the surface of channels and varying the slopes of channels.

Combining paper-based diagnostics with highly sensitive nucleic acid amplification techniques has the potential to enable rapid molecular diagnostics for point of care use throughout the world. Here, we report results from our extensive investigation into the effects of numerous biomaterials on two different isothermal amplification schemes, helicase dependent amplification (tHDA) and loop-mediated isothermal amplification (LAMP), an on PCR for four separate DNA and RNA targets. Biomaterials selected for testing were chosen based on their use in current rapid diagnostics and upstream sample preparation capabilities. These substrates were cellulose chromatography paper, polyether sulfone filters, track-etched polycarbonate, glass fiber, and nitrocellulose membranes.
The effects of these materials on LAMP and tHDA cannot be predicted by traditional PCR amplification within the materials, nor easily classified by the membranes nominal pore sizes, isoelectric points, or microstructures. The mere presence of glass microfiber and nitrocellulose membranes strongly inhibited all LAMP and tHDA reactions. The presence of cellulose still allowed for isothermal amplification. However, when the reaction occurred entirely within the cellulose paper, with no excess fluid volume, no significant amplification occurred. While the presence of polycarbonate itself did not hinder amplification, tHDA reactions were impeded when performed within this membrane. Only polyether sulfone allowed for both LAMP and tHDA in all reaction conditions.

Fibrous proteins are intriguing polymers for biomaterials-related needs, due to their structural hierarchy, functional properties and similarities to synthetic polymers in terms of repetitive sequence. Silks are unique proteins with respect to these features due to the unusual sequence chemistry and the associated opportunities to modulate structure, morphology and functional features via various process controls. These proteins provide useful attributes from a materials perspective, such as self-assembly, robust and tunable mechanical profiles, enzymatic degradability in vivo, stabilization of bioactive compounds, low biological activation, and related opportunities. Examples of this suite of tunable control features related to biomaterial scaffold designs for regenerative medicine will be discussed. Examples such as shape-memory silk materials, large scaffold designs for tissue defects and novel microfolded or 3D printed designs for tissue-specific needs will be discussed to highlight the versatility and robust biomaterial features achievable via the use of silk proteins.

Introduction: Strategies for the regeneration of tendon tissue include the use of biomimetic scaffolds in combination with physical and/or biochemical stimulation. Physical cues may arise from the topography of the scaffold, whereas biochemical signals can be delivered via exogenous growth factors. Electrospun fiber matrices are attractive tendon scaffolds as they mimic collagen fibers naturally found in the extracellular matrix and are easily fabricated. Growth factors such as GDF-5 have been observed to drive tendon differentiation, however, clinical use of growth factors proves to be a challenge, as high doses are often required for effective use. Peptide insulin has been shown to differentiate bone marrow derived MSCs after 24 hours of treatment on TCP towards tendon lineage with increased expression of tendonogenic markers^[1]. The current study further explores the use of insulin treatment on electrospun fiber matrices.
Materials and Methods: PCL polymer solution was dissolved in methylene chloride and ethanol mixture and electrospun to create randomly aligned nanofibers matrices. Human BMSCs were expanded to passage 5. Prior to seeding onto nanofiber scaffolds, cells were treated with DMEM containing 1pM, 0.1nM, 1nM insulin; 1ng/ml, 10ng/ml, 100ng/ml GDF-5 or control. Cells were seeded onto the scaffolds at a seeding density of 50,000 cells per scaffold. At various time points, changes in proliferation and gene expression (Col I, Col III, TNMD, SCX) were evaluated using picoGreen and SYBR green qPCR analysis, respectively.
Results and Discussion: Increased proliferation levels were found with all treatment groups when compared to control at Day 14. Cells exposed to insulin or GDF-5 for 24 hours showed upregulation of tendonogenic markers. Group treated with 1nM insulin had the highest expression of scleraxis ^[1]. Collagen I staining showed increased production on scaffolds treated with 1nM insulin. This suggests that a single 24-hour treatment of insulin or GDF-5 is sufficient to cue cells on fibrous scaffolds towards tendon differentiation. These results indicate that treatment of stem cells with supplemented media prior to scaffold implantation would negate the need to deliver growth factors or other biological factors into the body.
Conclusion: An increase of tendonogenic markers was observed in cells treated with a single bolus of insulin prior to seeding on nanofiber scaffolds. Ongoing studies will look at protein levels and immunohistochemistry.
Acknowledgements: Authors acknowledge the funding from the National Science Foundation (IIP-1355327, IIP- 1311907 and EFRI-1332329), Raymond and Beverly Sackler Center for Biomedical, Biological, Physical and Engineering Sciences and Department of Defense-OR120140.
References:
[1] A. D. Mazzocca, M. B. R. McCarthy, D. Chowaniec, M. P. Cote, C. H. Judson, J. Apostolakos, O. Solovyova, K. Beitzel and R. A. Arciero, Arthroscopy: The Journal of Arthroscopic & Related Surgery 2011, 27, 1459-1471.

The culture and maintenance of stem cell phenotype is integral to their use in regenerative medicine applications. Current cell culture methods rely on the use of fetal bovine serum (FBS), a relatively undefined cocktail of chemical factors that may have unwanted or unknown influences on stem cell phenotype. The influence of individual serum-borne biomolecules (such as heparin) on cell behavior is difficult to isolate due to the random nature of serum protein adhesion. Chemically defined, functionalizable surfaces provide the necessary control over cell-material interactions that are important to maintaining potency while also providing a vehicle for directing differentiation when desired. Here is presented a crosslinked copolymer coating with the capability to be applied to a wide variety of substrates for stem cell culture. The polymer is synthesized by living free radical polymerization and consists of poly(ethylene glycol), vinyldimethyl azlactone (VDM) and glycidyl methacrylate (GMA). The resultant PEG-ran-VDM-ran-GMA polymer is a thermally crosslinkable, cytophobic surface that can be functionalized post-polymerization via ring-opening reaction with the VDM group with a range of bioactive molecules. Crosslinking through the GMA group can be carried out at low temperatures (90 C), enabling the coating of relatively low-melting-point materials such as tissue culture poly(styrene) (TCPS). The polymer coating shows similar surface characteristics regardless of underlying substrate type as shown by water contact angle. This material allows stem cells to be cultured in the presence of single, specific signals as well as tunable combinations of signals. We report the ability to incorporate Arg-Asp-Gly (RGD) containing and Lys-Arg-Thr (KRT) containing peptides into the polymer coating in a single-step, room temperature reaction via a thiol-containing cysteine side chain. The RGD-containing peptide provides an integrin-based cell adhesion ligand, while the KRT serves to bind heparin, a glycosaminoglycan well known for its ability to sequester growth factors. The combination of the two peptides functions to enhance growth factor signaling in the adhered cells and results in enhanced proliferation even at low FBS concentration. As a result, we observe increased proliferation of hMSCs in culture media as well as enhanced osteogenic differentiation in the presence of bone morphogenic protein and osteogenic induction medium where the heparin-binding KRT peptide is present.

Control of prosthetic limbs that mimic human limbs will require novel advances in brain machine interfaces. To acheive closed loop performance that mimics the human system, signals from the motor cortex must be decoded to understand motor intent followed by movement of the prosthetic limb, and sensory information from the limb must be converted to electrical pulses to the somatosensory cortex. To acheive this grand vision, quality brain-machine interfaces to the peripheral nervous system are vital. In this work, we detail our latest nanoscale device that can interface to the peripheral nervous system and that is capable of delivering high quality signals.

Co-culture of stem cells with a desired cell type is an effective method to promote the differentiation of stem cells into desired cell type. The features of membranes used for co-culture are crucial in order to achieve the best outcome. Not only should the membrane act as a physical barrier that prevents the mixing of co-cultured cell populations, but it should also allow effective interactions between the co-cultured cells. Unfortunately, conventional membranes, such as track-etched PET membranes , used for the co-culture do not sufficiently meet these requirements. In addition, cell harvesting using proteolytic enzymes following the co-culture impairs cell viability and extracellular matrix (ECM) produced by the cultured cells. To overcome these problems, we developed nano-thin and highly porous (NTHP) membranes with thermoresponsive property for the generation of transfer-printable sheets of cells differentiated from stem cells through co-culture.
The ultra-thin and nanoporous structure of NHTP membranes was realized by the vapor-induced phase separation (VIPS) mechanism during spin-coating process. By using this process, the pore size of NTHP membranes can be finely tuned through simple changes in processing parameters. It was found that membranes with ~ 100 nm pores prevented cell-cell direct contacts while membranes with ~ 900 nm pores allowed for such interactions but were ineffective in providing a physical barrier for the separation of co-cultured heterogeneous cells. Optimized pore size at ~ 400 nm not only enhanced the stem cell differentiation compared with the conventional co-culture methods but also facilitated the fast and easy collection of differentiated cells.
Furthermore, we were able to utilize initiated chemical vapor deposition (iCVD) process to graft a thermoresponsive polymer conformally onto the NTHP membrane without altering the porous structure of the membrane. This modification allowed for the generation of ECM-preserved, highly viable, and transfer-printable multilayered sheets of differentiation cells after the co-culture. The membrane developed in this study is expected to facilitate the effective co-culture for differentiating stem cells and producing various types of multilayered cell sheets applicable in regenerative medicine.

Electrospinning has become increasingly favoured in tissue engineering, in particular for soft tissues. This is due to electrospun nanofibres having a very similar structure to the nanofibrous collagen found in the extracellular matrix (ECM) of many tissues. These collagen networks contribute significantly to the mechanical properties of the tissues. Mimicking the morphology of the networks in the natural tissue will improve the effectiveness of an artificial tissue, allowing similar mechanical properties to the natural healthy tissue resulting in a successful replacement.
This work considers the mechanical properties of electrospun meshes in relation to the polymer solutions used, and the morphology of the fibres within the mesh. Eight different solutions of gelatin in acetic acid were electrospun repeatedly, with the resulting meshes analysed by scanning electron microscopy (SEM) and unidirectional tensile tests. As the concentration and bloom strength of the gelatin increases, the average diameter of the fibres was found to increase from 75 nm to 254 nm, as well as an increasing variability in the diameter. However, the concentration and bloom strength were shown to have little effect on the mechanical properties of the meshes. The viscosity and the conductivity of the solutions also had little effect on mechanical properties, although viscosity does strongly influence the fibre diameter. Further analysis showed that the mechanical properties displayed little dependence on fibre diameter. This result is unexpected and contradictory to many other author&’s findings, however indicates that another aspect of the electrospun mesh structure has a greater influence on mechanical properties, such as pore size or the interaction between fibres. Single nanofibre properties are compared to the mesh properties to investigate the effect of the mesh structure.

Star-shaped poly(#671;-lactic acid) polymers with varying arm-numbers and arm-lengths were synthesized, and self-assembled into microspheres, which were either smooth or fibrous on the nano-scale, and either non-hollow, hollow, or spongy on the micro-scale. We demonstrate via both experiments and simulations that the molecular architecture and functional groups determine the structure of the resulting microspheres on both the nanometer and the micrometer scales. This exciting mechanistic discovery guides us to design new polymers, poly(#671;-lactic acid)-block-poly(#671;-lysine) copolymers, to fabricate nanofibrous spongy microspheres (NF-SMS) as advanced injectable stem cell carriers for tissue regeneration. In one example, NF-SMS were employed as human dental pulp stem cell (hDPSCs) carrier for dentin tissue regeneration. NF-SMS with interconnected pores throughout the nanofibrous microspheres could enhance the proliferation and odontogenic differentiation of hDPSCs, compared to nanofibrous microspheres (NF-MS) without pore structure and conventional solid microspheres (S-MS) with neither nanofibers nor pore structure. During the first 9 d in culture, hDPSCs proliferated significantly faster on NF-SMS than on NF-MS and S-MS (p < 0.05). Following in vitro odontogenic induction, all the examined odontogenic markers (ALP content, OCN, BSP, COL1), DSPP gene expression levels, calcium content and DSPP protein content were significantly higher in the NF-SMS group than in the control groups. 6 wk after subcutaneous injection of hDPSCs and microspheres into nude mice, NF-SMS enhanced dentin matrix deposition compared to NF-MS and S-MS. In another example, NF-SMS were used to carry hDPSCs into the pulp cavity to regenerate living dental pulp tissues. In addition to enhanced hDPSCs attachment, proliferation and odontogenic differentiation, NF-SMS also enhanced angiogenesis, as compared to control cell carriers. Additionally, NF-SMS promoted vascular endothelial growth factor (VEGF) expression of hDPSCs in a 3D hypoxic culture. In a subcutaneous tooth implantation model in mice, hypoxia-primed hDPSCs/NF-SMS complexes were injected into the cleaned pulp cavities of rabbit molars for subcutaneous implantation in mice. According to the histological analysis after 4 weeks, the hypoxia group significantly enhanced angiogenesis inside the pulp chamber and promoted the formation of ondontoblast-like cells lining along the dentin-pulp interface, as compared to the control groups (normoxia-primed hDPSCs/NF-SMS group, hDPSCs alone group, and NF-SMS alone group). Furthermore, in an in situ dental pulp repair model in rats, hypoxia-primed hDPSCs/NF-SMS were injected to fully fill the pulp cavity and regenerate pulp-like tissues with a rich vasculature and a histological structure similar to the native pulp. Collectively, NF-SMS were demonstrated to be excellent stem cell carriers for dental tissue engineering and potentially the regeneration of other types of tissues.

A major challenge in the development of in vitro organ-specific models is to reconstitute physiologically relevant microenvironments that are capable of maintaining cell differentiation and tissue-specific functions. Engineering an organ-specific model requires multidisciplinary approaches involving chamber/channel fabrication, scaffold integration, fluid handling, cell/tissue culture, process control, and sterilization. Organs-on-a-chip platforms are predominantly fabricated using Polydimethylsiloxane (PDMS) based soft lithography techniques. However, the use of PDMS and soft lithography offers limited flexibilities to account for the complex microenvironments typically associated human organs. Furthermore, several undesirable features of PDMS such as small molecule absorption, costly and time consuming integration of design iterations, and the difficulty in large-scale production obligate the investigation of alternate fabrication methods. In this work, our objective is to investigate a rapid prototyping technique based on laser patterned polymeric membranes/films/sheets and lamination to develop highly integrated microfluidic platforms for long-term cell/tissue cultures. In particular, we will present the development of a complex bioreactor model for an artificial lung. The lung model includes compartments to represent both the bronchiolar and the alveolar region. It integrates several materials to recapitulate the intricate tissue development environment, sterilization, and long-term stable tissue culture. Polyester membranes are used to develop the bronchiolar tissue, whereas thin and flexible polyurethane membranes are used to develop the alveolar tissue. Microfluidic channels are patterned using laser cut Polycarbonate, Polyimide, and PET sheets. Each layer is laminated using biocompatible silicone based adhesive transfer tapes to form the highly integrated bioreactor. Specific flow management systems were also developed integrating micro-valves and pumps. This allowed cell seeding, tissue growth under static conditions, transition to air-liquid interface, integrate flow, and to execute breathing motions. We have demonstrated co-culturing of human bronchial epithelial cells (BEAS-2B), human alveolar epithelial cells (A549) and human lung microvascular endothelial (HLMVE) cells within these complex bioreactors. Our approach is suitable for developing miniaturized lung organ models to perform systemic absorption, distribution, metabolism, excretion, and toxicology. The versatile combination of available materials for integration, rapid prototyping, and compatibility with commercially available large-scale production tools make our approach suitable to swiftly engineer any in vitro human organ-specific model.

Research on cardiac tissue repair has motivated the creation of tissue equivalents, capable of mimicking extracellular matrix conformation. Modern micro-scale fabrication techniques enable the creation of corresponding scaffolds, imitating the cell-supporting and mechanical function of extracellular matrices and thus allowing for controlled growth and contraction of cells. The combination of passive micro-structures with actively beating myocytes, functioning as biological actuators, facilitates the design of bio-actuated medical and biomimetic devices.
In this work we investigated 2D-structured sheets, suitable for unidirectional deformation, actuated by myocytes. We designed geometries with meso-scale properties that allow for effective adhesion and cell contraction. 2D-periodic structures with feature sizes of 1.8 - 6.0 µm were found to be most promising to facilitate adhesion while retaining structural stability. Strongly directional Young's Moduli in ranges of 20 - 50 kPa and 800 - 850 MPa were achieved and correspond well with the values reported in literature for cell actuated deformation. We used finite element (FE) simulations, homogenization technique and analytical modeling to assess the structures' response to myocardial contraction and their dependences on parametric variations. Comparing the analytical results to the FE simulations and the homogenization data, a maximum deviation of 1.14% was found.
We fabricated selected structures from elastomers, using 3D-lithography based on two-photon absorption. Microcontact printing of proteins and topology modification have been investigated for fostering cell alignment. We are currently seeding C2C12 cell lines as monolayers onto the functionalized substrates and differentiate them into myotubes that can be stimulated to contract simultaneously.

Regenerative medicine is one of the grand challenges for science and medicine given the rising life span and cost of health care worldwide. One of the important strategies is the development of bioactive biomaterials that can interact with endogenous or transplanted cells to promote regenerative processes. Supramolecular structures designed with components found in natural extracellular matrices would be ideal as they could disintegrate into harmless molecules. Biology utilizes one-dimensional fibrous assemblies in the form of protein polymers to mechanically support cells and bind the growth factors and receptors engaged in regenerative signaling. This lecture will describe dynamic supramolecular biomaterials that can serve those functions built with peptides, DNA, and saccharides. These materials signal cells in vitro and in vivo to trigger the growth of tissues such as bone, blood vessels, muscle, and axons of the spinal cord.

Healthy articular joints exhibit highly efficient lubrication and wear protection over the course of a person&’s lifetime. They maintain low coefficients of friction and resist wear over a wide range of pressures and sliding speeds. While extensive research has contributed to our understanding of the role of synovial fluid components in mediating these remarkable tribological properties, the role of the glycoprotein fibronectin (FN) has remained unstudied. FN is present in the outermost superficial zone of cartilage (i.e. the cartilage-synovial fluid interface in articular joints), but absent from other regions of cartilage, which raises the question of what contributions FN makes to the lubrication and wear protection of the underlying cartilage surface. In this work, we have investigated the molecular interactions between FN and various components of the synovial fluid which are believed to contribute to joint lubrication, specifically lubricin (LUB), hyaluronan (HA), and serum albumin (SA). Using a Surface Forces Apparatus, we have measured the normal (adhesion) and lateral (friction) forces between layers of individual synovial fluid components physisorbed onto FN-coated mica substrates. Our chief findings are: (i) FN strongly tethers LUB and HA to the underlying mica substrate, as indicated by sustainable (brush-mediated) long-range repulsive interactions between the surfaces, and (ii) FN and LUB synergistically enhance lubrication and wear protection of the surfaces during shear, as suggested by the structural robustness of FN+LUB films when subjected to a wide range of shearing velocities and pressures up to circa 4 MPa (i.e. above the physiological range). These findings provide new insights into the role of FN in articular joints and our understanding of cartilage damage, and represent a significant step forward in the design and tethering of cartilage replacements following total knee arthroplasty.

Complex structural organization in the human body is a key factor to produce the appropriate tissue functionality. To achieve engineering biomimetic tissues, therefore, a technique for mimicking microstructures in native tissues is important. In skeletal muscle, the muscle fibers are highly oriented to produce its mechanical functions, and vasculature and neurons also have well-organized structures. In this study, we have developed a micropatterned thermoresponsive cell culture substrate to produce a cell sheet composed of aligned skeletal muscle myoblasts. Using this anisotropic tissue unit, we have engineered 3D muscle tissue with well-organized microstructures, required for future tissue engineering.
The original procedures for the preparation of micropatterned thermoresponsive surfaces have been reported previously. Briefly, thermoresponsive polymer poly(N-isopropylacrylamide) was grafted on glass substrates, and then hydrophilic polymer poly(N-acryloylmorpholine) was further polymerized spatio-selectively through photolithographic process.
Human skeletal muscle myoblasts were seeded onto the patterned substrate (stripe patterns: 50 mm / 50 mm). After reached confluence, the cells were cultured at 20 °C for harvesting a single continuous cell sheet. To produce a 3D tissue construct, multiple cell sheets were layered using a gelatin gel-coated manipulator. Furthermore, within the cell sheet construct human neurons and human umbilical vein endothelial cells (HUVECs) were incorporated for creating a complex tissue construct made of various cell types.
Myoblasts were aligned on the surface and were harvested as an anisotropic cell sheet by lowering culture temperature. Using a gelatin gel-coated plunger, multiple anisotropic cell sheets were successfully layered with maintaining the designed cell orientation. As a result, a 3D myotube construct with a single orientation was produced. Next, neurons and endothelial cells were simply sandwiched between multiple cell sheets. After 5 days of layering, both type of cells formed oriented networks in the construct. Neurite outgrowth was guided by the myoblast orientation of the cell sheets, and they finally formed an oriented neural network. HUVECs also formed vascular-like branching networks by the cell sheet layering. Importantly, the networks of neurons and endothelial cells showed anisotropy originated from the orientation of the myoblast sheets. This indicated that the cells recognized the anisotropy and self-organized native-like microstructures within the tissue construct.
In conclusion, we have developed a novel technique to create a muscle tissue construct through a cell sheet layering process. Uniquely, microstructures of the muscle tissue construct can be organized by using anisotropic myoblast sheets. The structural design and sequential functionalization in future work could lead to truly biomimetic tissue generation, and development of in-vitro physiological tissue models.

Enzymatic polymerization of polyphenols has been extensively studied to augment the oxidation potency of natural flavonoids. These polymers have received considerable interest in recent years. Current research focuses on developing methods to enhance the oxidation properties, processability and environmental compatibility of this material. Numerous chemical and electrochemical approaches have been reported for obtaining these polymers in a processable form. Furthermore, the traditional polymerization process involves tiresome chemistry or severe chemical treatment. The difficulty of contemporary procedures in the chemical synthesis of these polymers combined with the harmfulness of the process has led to the growth of biochemical methods for the synthesis of water-soluble and highly antioxidant polymers. Horseradish peroxidase (HRP), activated by hydrogen peroxide, has been widely used for the polymerization of phenols in water. Using green chemistry we report the homopolymerization of rosmarinic acid and its copolymerization with hydroxytyrosol. The resultant product of these reactions is the formation of two large molecular weight polymers. The biological activities of these polymers will be reported for the first time. It is recognized that polyphenols have a potential protective effect on neurodegenerative disorders characterized by microglial activation, due to their anti-inflammatory and antioxidant properties. The activation of microglia is associated with neurodegenerative disease, characterized by increased oxidative stress and neuroinflammation, including Alzheimer and Parkinson diseases and multiple sclerosis. Indeed, microglial cells display a ramified morphology but, when activated in response to various immunological stimuli and neuronal injuries, turn into an amoeboid type and release neurotoxic factors, including nitric oxide (NO), prostaglandin E2, inflammatory cytokines, and reactive oxygen species (ROS) that cause lipid peroxidation. Microglial cells were obtained from the cerebral cortex of 1- or 2-day old decapitated rats. Microglia, detached from the astrocyte monolayer, were re-suspended in D-MEM/F12 supplemented with FCS and penicillin/streptomycin. We evaluated the potential neuroprotective effect of the two synthesized polymers on primary rat microglial cultures by using MTT assay and detecting LDH activity released in H₂O₂-activated microglial cells Cells were pretreated with the two polymers before H₂O₂ stimulus. Moreover, it will be presented the effects of the polymers on release of NO, inflammatory cytokines, ROS and lipid peroxidation biomarkers in H₂O₂-activated microglial cells Finally, the structural assessment and property of these polymers will be presented, as well as their applicability as antioxidant drug. This is a novel, direct and inexpensive approach that can open new avenues of synthesis for complex and highly efficient antioxidant substances.

Additive manufacturing technologies are increasingly being used to replace standard extrusion or molding methods in engineering polymeric biomedical implants, which can be further seeded with cells for tissue regeneration. The principal advantage of this new technology is the ability to print directly from a scan and hence produce parts which are an ideal fit for an individual, eliminating much of the sizing and fitting associated with standard manufacturing methods. The question though arises whether devices which may be macroscopically similar, serve identical functions, and are produced from the same material, in fact interact in the same manner with cells and living tissue. The polymer we selected for printing, compression molding and spun casting was poly-lactic acid (PLA) - not only an effective material for cell scaffolds, but also one of the most popular filaments used in commercial 3D printers. Data from an FTIR spectrum, water contact angle, and qualitative SEM analysis were used to characterize physical differences between scaffolds. We found that fundamental differences can exist between 3-D printed, molded and spun cast scaffolds. Different manufacturing methods produce features on multiple length scales. Dental pulp derived cells (DPC) seeded onto different scaffolds differed significantly in their attachment, morphology, growth and differentiation. Substrate heterogeneity in both morphological and mechanical features determine difference in expression of extracellular matrix proteins, determined by RT PCR, and quality of DPC produced calcified tissue. Furthermore, we found that surfaces produced by different manufacturing can be affected differently by various sterilization techniques, which in addition alters cell adhesion and potential lineage selection. These findings are in great importance in perspective of 3D printed scaffolds application for tissue regeneration.

The motivation for this work is to make pre-mineralized scaffolds, ultimately for regeneration of bone. This study describes a paper-based system to culture osteoblasts and evaluate the deposition of calcium phosphate by the cells in these paper scaffolds. The deposition of calcium phosphate was assessed using colorimetric, microscopic, and spectroscopic methods. This work demonstrates that paper can be used as a matrix to induce template-guided bone growth. The paper-based system offers a new way to study mechanisms of biomineralization, and perhaps to repair mineralized tissues.

Human body tissues consist of multiple cells and extracellular matrix with nanofibrous architecture. The construction of nanofibrous cell-laden and ECM-like structures is therefore critical for the successful regeneration of human body tissues. Electrospinning has been widely used for making tissue engineering scaffolds with nanofibrous architectures. However, the dense structure of electrospun scaffolds limits cell infiltration and migration. Our previous work demonstrated a new approach in fabricating nanofibrous and 3-D cell-laden scaffolds by using concurrent electrospinning and cell electrospray. In the current investigation, this new approach was used to construct complex nanofibrous and 3-D cell-laden scaffolds for the regeneration of complex body tissues. Since endothelium and smooth muscle layer are major structures of various tubular tissues, such as blood vessels and gastrointestinal tracts, an endothelial cell, human umbilical vein endothelial cell (HUVEC), and a smooth muscle cell, human aortic smooth muscle cell (HASMC), were used in our new cell-laden scaffolds for reconstituting the endothelium and smooth muscle layer, respectively. Through emulsion electrospinning, growth factors including vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) were incorporated in nanofibers within specific cell-laden layers for promoting the growth of HUVECs and HASMCs in scaffolds. For emulsion electrospinning, emulsions made from PLGA polymer solution (oil phase) and growth factor-containing PBS (water phase) were used. For cell electrospray, coaxial electrospray was conducted where the cell suspension and Na-alginate polymer solution flowed through the inner capillary and the outer capillary of the coaxial nozzle, respectively, followed by crosslinking of Na-alginate by CaCl₂ and cell-encapsulated Ca-alginate microspheres were formed. Concurrent emulsion electrospinning and cell electrospray were performed, forming complex scaffolds. The nanofibrous scaffolds with embedded cell-containing microspheres were dripped by Na-citrate solution to dissolve the Ca-alginate microspheres and hence the encapsulated cells were released in situ in the scaffolds. The bilayer nanofibrous scaffolds contained one VEGF-incorporated and HUVEC-laden layer and one PDGF-incorporated and HASMC-laden layer which were subsequently studied using SEM and confocal microscopy. The in vitro release behaviors of VEGF and PDGF from specific layers of scaffolds were assessed. Cell behaviors such as cell viability, cell proliferation, and cell phenotype for the cells cultured in the nanofibrous scaffolds with the 3-D cell arrangement were evaluated using different techniques (live/dead cell viability assay, MTT assay, and histochemical staining, etc.). This research demonstrated a viable approach for making biomimetic cell-scaffold constructs for the regeneration of complex body tissues.