Organic-inorganic hybrid materials composed of interpenetrating molecular networks of polymer and silica are potential candidates for synthetic bone grafts as they can mimic the complex structure of a natural bone. Combining the properties of an elastic polymer and bioactive silica with molecular level interactions, the hybrids are expected to exhibit congruent degradation rate and tailored mechanical properties. Property control is primarily achieved through covalent bonds between the components, which can be achieved by functionalising the polymer with silane coupling agents that will then bond to the sol-gel silica. In this work we present new organic- inorganic hybrids of alginate and silica and compare the use of two different coupling agents: 3-glycidoxypropyl trimethoxysilane (GPTMS) or 3-aminopropyltriethoxysilane (APTES). The reaction between alginate and GPTMS involved nucleophilic opening of the GPTMS epoxy ring and formation of ester bonds with alginate carboxylate groups while the reaction with APTES was performed using carbodiimide chemistry. 1H solution state NMR analysis was used to monitor in situ the reaction between the alginate and the coupling agents. It revealed that within 72 hours of reaction most of GPTMS epoxy rings were unreacted or partially hydrolysed to diols indicating a slow functionalization process. 13C CP solid state NMR of aged gel samples did show formation of ester bonds between GPTMS molecules and alginate, showing that the aging step is necessary for covalent coupling to occur (confirmed by SIMS). The reaction between APTES and alginate readily occurred yielding elastic APTES - alginate hydrogels containing amide bonds, confirmed by FTIR, 1H NMR, 13C CP NMR and 15N CP NMR. Depending on the coupling molecule used in the synthesis the silica-alginate hybrids showed different degradation profiles and mechanical properties. GPTMS based hybrids showed faster dissolution in Tris buffer solution at physiological pH compared with APTES based hybrids, which had controllable dissolution behaviour relevant to their coupling degree. Scaffolds were produced by freeze drying APTES / alginate hybrids (pore size 150 µm) and compression tests showed that the scaffolds had an elastic behaviour suggesting that they would be appropriate for cartilage regeneration while the GPTMS based hybrids had improved compressive strength (150 MPa) and toughness comparable with the cortical bone and thus suitable for bone tissue application.

Designing artificial instructive coatings at the cell-biomaterial interface have become an appealing strategy to stimulate tissue regeneration and improve implanted material biocompatibility. Guiding cellular activities, such as adhesion, viability, and differentiation, on the biomaterial surface in a robust and cell-selective manner is often essential for both accelerated healing and the long term clinical success of the surgical intervention. In the biological milieu, however, stability of coatings on the material surface is restricted under the abrasive conditions, such as highly hydrated saline environment accompanied with constant mechanical wearing. Various organisms adapted to living in the seashores, or intertidal zones, suffer from the analogously harsh physical and chemical instabilities. To remain sessile, for example, mussels synthesize a highly complex, spatiotemporally evolving glue containing high amount of 3,4-dihydroxy-L-phenylalanine (DOPA). Herein, we imitate this mechanism by incorporating this chemical residue to self-assembling peptide amphiphile building blocks. Self-assembly of these molecules with complementary peptide amphiphiles carrying biofunctional ligands, such as REDV, DGEA, and KRSR, form nanofibrous networks, which closely mimic both structure and biochemical properties of the native extracellular matrix. Being capable of mediating both non-specific surface adhesion and cell type-specific behavioral guidance, we demonstrate general applicability of this supramolecular adhesive concept. We firstly apply this glue as bioactive cardiovascular stent coating, where endothelial cell adhesion and survival is favored over the smooth muscle cells, which is the main cause of re-closure of the arteries (restenosis) in the long term. We further develop an orthopedic/dental implant coating where mineral-depositing cells are selectively promoted over soft tissue forming fibroblasts. We also develop novel mussel-inspired nanofibers with biofunctional ligands that can synergistically deposit bone-like hydroxyapatite in body fluid mimetic conditions. This paves the way to develop a second generation orthopedic/dental implant, which efficiently mediates differentiation of human mesenchymal stem cells into functional osteoblasts. Altogether, we here concentrate on a mussel-mimetic, hybrid supramolecular adhesive interfaces for biofunctionalization of metal implant surfaces in order to direct the cellular behaviors at the molecular level for long-term success of the regenerating tissue.

Biomaterials play a critical role in the design of medical devices and the implementation of new therapies to improve patient care. Our laboratory pioneered the development of citrate-based polyesters, referred to as polydiolcitrates. Polydiolcitrates can be engineered to have properties that improve the function of medical devices and for applications in regenerative medicine. As an example, aberrant oxidative stress and inflammation play a significant role in the development of neointimal hyperplasia, a problem that eventually leads to the failure of small-diameter vascular grafts and stents. By incorporating antioxidant polydiolcitrates into the design of the device, one can control excess reactive oxygen species (ROS) contributing to oxidative stress and inflammation, reduce neointimal hyperplasia, and potentially increase the lifespan of the device. As for applications in regenerative medicine, when polydiolcitrates are combined with mesenchymal stem cells and hematopoietic stem cells one can create vascularized and innervated bladder tissue that may one day benefit patients with bladder cancer or bladder-related complications from spina bifida. In another example, the efficient healing of diabetic skin ulcers can be a significant challenge due to deacreased vascularization, chronic inflammation, high oxidative stress, and biofilm formation. To address this challenge we are investigating the use of thermoresponsive polydiolcitrate oligomers referred to as nanonets. Nanonets self-assemble into an elastic hydrogel with hierarchical nano- and microscale architecture within seconds upon contact with tissue above 30°C. The hydrogel can effectively entrap and slowly release bioactive compounds, proteins, and transgene products that modulate the wound healing response.

Age-related macular degeneration is a major ophthalmic disease that causes visual impairment and blindness. Although transplantation of autologous peripheral cells has been tested by injection of cell suspensions, limited visual improvement resulted due to the low viability of the injected cells in the subretinal tissue. As an alternative approach, there have been several reports on the development of natural and synthetic substrates for local delivery of retinal pigment epithelial (RPE) cells using collagen, poly(ethylene terephthalate) and poly(methyl methacrylate). However, these engineered substrates with micrometers in thickness (6 mu;m at thinnest) and several millimeters in size are not sufficiently flexible to be aspirated and injected through a conventional syringe needle into the narrow subretinal space. Thus, a large incision of the sclera and retinal tissue would be required for the injection of these rigid substrates. Such an incision might result in leakage of vitreous fluid and lead to post-surgical infection. Therefore, miniaturization of the substrates is an important approach to achieve minimally invasive delivery of the engineered tissue. In this study, we developed micropatterned polymeric nanosheets consisted of biodegradable poly(lactic-co-glycolic acid) and magnetic nanoparticles (MNPs, 10 nm in diameter) toward local delivery of the RPE cells. The micropatterned nanosheet encapsulating MNPs was fabricated by microcontact printing techniques. Owing to the magnetic property and flexible structure, the micropatterned nanosheet with 170 nm thick was manipulated remotely and delivered to the subretinal space of a swine eye. The micropatterned nanosheet also directed growth and morphogenesis of the RPE cells, and allowed for the injection of an engineered RPE monolayer through syringe needles flexibly without loss of cell viability. Such an ultra-thin flexible carrier has the promise of a minimally invasive delivery of organized cellular structures into narrow tissue spaces.

Introduction: Success in tissue engineering of the vocal fold lamina propria extracellular matrix (ECM) for treatment of chronic vocal fold scarring demands an understanding of how cells integrate the signals presented from the scar microenvironment, in combination with the signals from the biomaterial scaffold, to alter their response. To date, approaches to biomaterial development for scarred lamina propria treatment have examined the response of “normal” (non-scar) vocal fold fibroblasts (VFFs) to the biomaterial. As a result, they have failed to capture cellular responses, from activated macrophages and from resident scar-tissue VFFs (also known as myofibroblasts), from the in vivo implant environment which critically impact the quality and rate of VFF ECM production. In the present work, we conjugate cytokines, previously identified as anti-fibrotic and/or immunomodulatory, to biocompatible poly(ethylene glycol) [PEGDA] hydrogel formulations shown to have mechanical properties which preserve mucosal wave activity with low average phonation threshold pressures. We demonstrate these biomaterials influence macrophage polarization — shifting activated macrophages from a pro-inflammatory phenotype to a phenotype that is anti-inflammatory and pro-healing — and shift myofibroblasts to a “normal” or anti-fibrotic VFF phenotype.
Results and Discussion: Pro-inflammatory macrophages and/or myofibroblasts were encapsulated in PEG-bFGF gels. Following 3 days of culture in activation(pro-inflammatory, pro-fibrotic) media, expression of the fibrotic marker αSMA by myofibroblasts in PEG-bFGF gels was reduced over 7-fold relative to non-bFGF containing gels. Similarly, the expression of genes associated with matrix turnover (e.g. MMP1) were increased in response to bFGF. Activated macrophages exposed to bFGF gels displayed over a 50% reduction in inflammatory makers Nos2 and IL-12β relative to non-bFGF hydrogel controls. This reduction in fibrotic markers in myofibroblasts and inflammatory markers in macrophages in bFGF-containing gels occurred despite the continued presence of activating media and is consistent with anti-fibrotic and anti-inflammatory properties reported for bFGF. These results suggest that PEG-bFGF gels could be used to transition the activated cells present in chronic vocal fold scar to more phenotypes more conducive to healing.
Conclusions: The design of material environments to transition activated cells that are present in wound environments to more “normal” cell phenotypes would be desirable in the treatment of chronic scar and other chronic wounds. The present results represent an important step toward using tissue engineering principles to modulate cell phenotypes within an active wound environment.

The selection of a scaffold material is both a critical and difficult choice that will determine the success of failure of any tissue engineering (TE) strategy. We believe that natural origin polymers are the best choice for many approaches. In addition, we have been developing an all range of processing methodologies to produce adequate scaffolds for different TE applications. Furthermore an adequate cell source should be selected. In many cases efficient cell isolation, expansion and differentiation methodologies should be developed and optimized. We have been using different human cell sources namely: mesenchymal stem cells from bone marrow, mesenchymal stem cells from human adipose tissue, human cells from amniotic fluids and membranes and cells obtained from human umbilical cords. The potential of each type of cells, to be used to develop novel useful regeneration therapies will be discussed. Their uses and their interactions with different natural origin degradable scaffolds and distinct nano and micro-carriers, and smart release systems, will be described. A great focus will be given to the different sources of stem cells, the isolation of distinct sub-populations, ways of differentiating them, as well as their interactions with different 3D architectures and materials for culturing them. The use of bioreactors to control cell differentiation, as well as the surface modification of the materials in order to control cell adhesion and proliferation will also be illustrated. Several biomimetic and nanotechnology based strategies to engineer mineralized tissues will be described.

Biomedical adhesive is important in clinical medicine and various surgical conditions require new and better polymeric adhesives. The biomedical adhesive should be wet adhesive, nontoxic, physiologically stable, rapidly crosslinkable and physically flexible. A new bio-inspired adhesive for medical applications was synthesized from three monomers, N-methacryloyl-3,4-dihydroxyl-L-phenylalanine, acrylic acid N-hydroxysuccinimide ester and acrylic acid. 3,4-dihydroxy-L-phenylalanine (DOPA) containing polymer segment has a function of strong wet adhesion properties. Poly(acrylic acid) is a water soluble segment and N-hydroxysuccinimide ester rapidly forms covalent bonds with thiols on thiol terminated 3-armed poly(ethylene glycol) cross-linking agents. The adhesive with DOPA incorporated demonstrated increased adhesion strength that is measured by lap shear strength tests on wet porcine skin mimicking human tissues. Crosslinking the polymers significantly enhanced the maximum adhesion strength. The novel adhesive shows viscosity drops at high shear rate, demonstrating its potential to be injected through syringe needles. The enhance moduli of crosslinked adhesive revealed that its robust stability after the adhesive attached on intended sites.
Recent developments in various invasive fetal diagnosis and fetal therapeutic technologies has been contributed to improve fetus&’ health, but the limited healing capability of fetal membrane may cause clinically dangerous Preterm Premature Rupture of Membranes (PPROM) from the medical device punctured site. This problem could be solved by the developed new bio-medical adhesives. The wet adhesive properties of the new terpolymer were evaluated in the context of sealing punctured fetal membranes. In order to perform realistic in vitro test on human fetal membrane, a new test device was designed and manufactured to mimic maternal uterus and amniotic sac. The new adhesive successfully sealed needle punctures on fetal membrane. Also the new terpolymer was directly compared to commercially available medical adhesives and was found to be more efficient in the presealing treatment of fetal membrane.

Microbial infections endanger public health unremittingly and cause a huge economic burden to our society. Numerous efforts have been made to develop molecules which are able to inhibit pathogens. Novel broad-spectrum antimicrobial materials were developed based on natural polysaccharide. Firstly, a group of antimicrobial materials were synthesized by quaternization and alkylation of chitosan.(1) An argon plasma-ultraviolet (UV) induced coating method for hydrogel surface immobilization was developed, which can be applied on diverse soft biomedical surfaces. A novel mechanism of these hydrogels based on “anion sponge” concept was proposed and proven. The optimized coating formulation and conditions show excellent antimicrobial potency. The in vitro and in vivo studies suggest this antimicrobial coating is biocompatible with mammalian cells. Secondly, a peptidopolysaccharide that mimics the bacterial peptidoglycan structure, which is a feature unique to bacterial membrane but absent in mammalian cells, was designed, synthesized and tested.(2) By the ring-opening polymerization of N-carboxyanhydrides (NCA), a polysaccharide backbone was copolymerized with cationic polylysine, and the resulting optimized peptidopolysacchride shows high selectivity to bacteria over mammalian cell. Preliminary results also suggest that the compound stimulates little or no inflammatory response. These two classes of polysaccharide based materials reveal new directions for the design of antimicrobial materials, and they have a promising prospect in further applications.
References:
1. Peng Li, et al. Nature Materials, 2011, 10(2), 149-156.
2. Peng Li, et al. Advanced Materials 2012, 24(30), 4130-4137.

Controlling the spatial organization of cells with well-defined architecture is a vital aspect of tissue engineering. Odontoblasts are the cells that synthesize dentin. They are densely packed and line the pulpal wall while anchored into dentin through their processes. Recreating the odontoblast layer and morphology may facilitate dentin development in-vitro. Objective: To craft an in-vitro construct that is able to position cells in a configuration that mimics the highly organized layer of odontoblasts in teeth using micro porous membranes and promote secretion of a collagenous matrix that mineralizes. Method: A two-step lithographic process was used to generate a master-mold on a silicon wafer. Polycaprolactone (PCL) was spin cast onto the mold to create a membrane with 1-3µm pores, and pillars with 10µm heights surrounding each pore. Commercial cell culture inserts with 1 or 3mu;m pores and homemade porous PCL membrane were used to enable high-density, single-layer cell accumulation and formation of cell protrusion. MC3T3 cells were seeded at confluent densities onto membranes and centrifuged at 700 rpm to position. Cells were allowed to mineralize under a chemical gradient for up to 21 days. Results: MC3T3 cells formed a monolayer of cells on membrane after centrifugation and produced collagenous matrix, which appeared continuous over large areas. Chemical gradient created with serum, led to the formation of cell protrusions extending through pores at day 21. However, it was observed that MC3T3 cells lose its 3D organization on the porous membranes within 24hr with the absence of pillar structures. Presence of pillars on PCL membrane may prolong cellular organization for a longer time and enable organized matrix production. Conclusion: A porous membranes engineered with micropatterning technology can be used to confine one cell to a single pore while inducing cell protrusion successfully, providing us the possibility of recreating the odontoblastic morphology in-vitro.

Collagen is a naturally occurring biopolymer and its&’ architecture is important in tissue development and repair. We have explored the nucleation, assembly and the 3-D architecture in collagen hydrogels prepared under the conditions commonly employed in biomedical, tissue engineering and medical device research. Employing in situ multi-photon optical microscopy that combines nonlinear optical phenomena of second harmonic generation (SHG) and two-photon fluorescence (TPF) signals we showed that drastically different microstructures are prepared by changing collagen solid content, incubation temperature or ions and moreover that the non-zero cross-linkers for example genipin significantly alter the originally assembled 37 C architectures. Specifically, genipin induces formation of aggregated fluorescent fibers concomitantly with a 3.5 fold drop in SHG contrast (after 24-hour reaction at 37 C) suggesting that this modification disaggregates initial collagen microstructure within the materials at the expense of forming the new fluorescent features. The 800 nm excitation wavelength generates complex TPF contrast within genipin-modified materials. It is centered within the blue-green (~ 490 nm center) as well as red (~ 615 nm center) spectral regions and spatially distinct. The ratio of the intensity of 615 nm emission band to the intensity of 490 nm emission band is 5.3 (after 24-hour reaction at 37 C). There is a slight bathochromic (red) shift when the excitation wavelength is varied between 760 nm and 900 nm. The findings will be discussed within the context of our ongoing tissue engineering and wound repair research.

In the recent years, there is an alarming increase in obesity worldwide. Obesity associated health concerns as well as stigma has driven affected people for reconstructive surgeries. With this, the amount of adipose tissue discarded as a clinical waste has increased proportionally. Literature suggests different protocols that decellularize adipose tissue. These protocols rely on combinations of physical, chemical and enzymatic agents to remove cells and lipids. The end-product of decellularization yields extracellular matrix (ECM) that supports the cells in an organ; both structurally and functionally. Although the previously designed protocols have been successful in the decellularization, they span a number of days. Our study involves a purely enzymatic, chemical or physical process of decellularization. Further, we distinguish them based on their ability to remove lipid and cells. We then tested the capability of these processes in preserving the essential biomolecules such as collagen, Glucoseaminoglyxcans (GAG), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Additionally, we tested the ability of ECMs generated by the enzymatic (ECM-E), chemical (ECM-C) and physical (ECM-P) to support various cell types. We report that the physical method of decellularization is advantageous due to its shorter time; efficient lipid and cell removal; and collagen, GAG, VEGF and bFGF conservation. Therefore, we recommend the use of ECM-P for scaffolds and coats to apply in tissue engineering.

Engineered tissues require enhanced organization of cells and the extracellular matrix (ECM) for proper function. To promote cell organization, substrates with controlled micro-and nanopatterns have been developed as supports for cell growth, and to induce cellular elongation and orientation via contact guidance. Micropatterned ultra-thin biodegradable substrates with precisely defined chemistry are desirable for implantation in the host tissue. These substrates, however, need to be mechanically robust to provide substantial support for the generation of new tissues, to be easily retrievable, and to maintain proper handling characteristics. Chitin is an ideal substrate. Chitin is a naturally abundant polysaccharide, which is mechanically stable, biodegradable, nontoxic, and physiologically inert. Here, we introduce ultra-thin (<10 mm), self-assembled chitin nanofiber substrates micropatterned by replica molding for engineering cell sheets. These substrates are biodegradable, mechanically strong, yet flexible, and can be easily manipulated into the desired shape. As a proof-of-concept, fibroblast cell attachment, proliferation, elongation, and alignment were studied on the developed substrates with different pattern dimensions. On the optimized substrates, the majority of the cells aligned (<10 mu;m) along the major axis of micropatterned features. With the ease of fabrication and mechanical robustness, the substrates presented herein can be utilized as a versatile system for the engineering and delivery of ordered tissue in applications such as myocardial repair.

Recent developments in stem cell differentiation methods have enabled the derivation of human heart muscle cells, or cardiomyocytes, from stem cell sources at exceedingly high efficiencies. The resulting cells demonstrate many of the properties of immature human cardiomyocytes, but they do not spontaneously align and form mature, highly-structured myofibrils that are typically observed in vivo. In our studies, we&’ve developed a platform that utilizes soft lithography and micropatterning techniques to control cell geometries in a highly specific manner. The patterned geometries consisted of rectangular features of varying sizes and aspect ratios to investigate how cardiomyocyte aggregates responded to the geometric constraint. Pure populations of immature, human embryonic stem cell-derived cardiomyocytes were seeded onto these fibronectin/matrigel geometries. Myofibril assembly of the cardiomyocytes was assessed by observation of cardiomyocyte sarcomere structure using actin and alpha-actinin stains. Alignment of the cells was assessed using DAPI staining and subsequent analysis of nuclear alignment. Results showed that the human cardiomyocytes aligned much more strongly, and formed much more robust sarcomere structures, on geometries with widths ranging from 20µm to 80µm. Features whose shortest side was more than 80µm failed to produce cardiomyocyte aggregates that properly aligned. We hypothesize that this improved alignment of the cardiomyocytes contributes to the enhanced myofibril formation due to improved cell polarity, as well as a focal adhesion/costamere confinement that forces stress fibers to align more consistently with the cell axis. The cells cultured on optimal geometries demonstrate a much more physiologically-representative size, as well as a mature-like myofibril expression.

Previous investigations have established the utility of porous silicon in electrospun microfibers of biocompatible polymers such as polycaprolactone (PCL) for possible applications such as orthopedic tissue engineering1 and treatment of ocular disease.2 For the latter, the presence of a polymer such as PCL or poly-L lactic acid (PLLA) facilitates processing and provides opportunities for multistage drug delivery dependent on the dissolution kinetics of the porous Si and polymer components, respectively. In this presentation, we outline a process for the spatial control of particle location of nanostructured porous silicon in the polymer network and ideal control of drug release from this platform. Control of the loading amounts is discussed through processing techniques of the composite and the flexibility of the method to easily manipulated variables. We seek to use the porous silicon as a chemical reservoir for the sustained release of either ophthalmological-relevant drugs or cellular growth factors, but begin with proof-of-concept using a fluorescent dye as an easily-tracked proximate. After a discussion of composite microstructure using scanning electron microscopy (SEM) characterization and an evaluation of the stability of such structures in cell-based media, kinetic-based studies of the release of therapeutics from these composites are presented.
1 Dongmei Fan, Giridhar R. Akkaraju, Ernest F. Couch, Leigh T. Canham, and Jeffery L. Coffer, Nanoscale, 2011, 3,354-361.
2 Soheila Kashanian, Frances Harding, Yazad Irani, Sonja Klebe, Kirsty Marshall, Armando Loni, Leigh Canham, Dongmei Fan, Keryn A Williams, Nicolas H Voelcker and Jeffery L Coffer, Acta Biomaterialia , 2010 , 6, 3566-3572.

The leading cause of implant failure is poor implant osseointegration. The use of electrically polarized biomaterials provides one possibility to address poor osseointegration. Electrically polarized hydroxyapatite has been shown to improve osteoblast attachment and proliferation; however, the mechanism by which this occurs is not fully understood. During the processing of hydroxyapatite, for example, there can be differences in a number of factors that would also influence osseointegration, i.e., surface roughness, Ca/P ratio, etc. In this work, we demonstrate the use of a ferroelectric substrate (lithium niobate, LN) with a permanent polarization (in the z-cut direction) as a novel platform to study the effect of surface charge for improving osseointegration while minimizing the factors described above. Each of the transparent LN samples used has the same chemistry and approximately the same surface roughness. Thus, any differences in the performance between the positively and negatively charged substrates can be ascribed to the surface charge. The biocompatibility of LN and its ability to promote osseointegration were characterized using cell proliferation and mineralization assays.
Fluorescence-based assays in cultured cells demonstrated for the first time the biocompatibility of LN. MC3T3 osteoblast cells cultured for up to 11 days attached and proliferated. These studies revealed an enhancement of cell attachment on the charged LN surfaces compared to uncharged LN, i.e., x-cut LN and a glass control. By 9 days there was a 30% proliferation enhancement on charged compared to uncharged surfaces, further demonstrating biocompatibility and enhanced cell metabolism (via MTT assay). We also report the ability of the cells to undergo mineralization in calcium-supplemented media. The cells on charged LN produced 26% more mineralized calcium than the unpoled LN surface after 30 days. We conclude that the surface charge allows for enhanced osseointegration and provides a novel platform for understanding the effect of surface charge on cellular processes.

Electromechanical coupling in collagen type I has been studied extensively since the discovery of piezoelectricity in several biosystems in the mid-1900s. It is known that induced currents (via piezoelectricity) activate the healing process in tissues under compression or tension. The structural similarities between collagen type I and other fibrillar collagens (type II, III, V, etc.) suggest they are piezoelectric as well. Collagen type II comprises 3 identical alpha-1 polypeptide strands twisted together to form a helix; whereas the collagen type I helix comprises two alpha-1 strands and one alpha-2 strand. In this study, piezoresponse force microscopy (PFM) is used to probe the electromechanical properties of reconstituted collagen type II fibrils at the nanoscale. The fibrils are found to exhibit shear piezoelectricity, showing the same cosine dependence of the signal on the angle between the cantilever and fibril axes as has been observed for collagen type I. The piezoelectric coefficient of collagen type II was found to be one order of magnitude lower than that of type I. This reduction could be due to a higher density of cross links in collagen type II, resulting in a lower degree of freedom of movement along the fibril axis and thus, lower shear piezoelectricity. The investigation of piezoelectricity in collagen type II may be relevant for possible regenerative biomaterial devices in the case of damaged cartilage or as piezoelectrically-active cell scaffolds or coatings.

The functional role of piezoelectricity in collagenous materials is a topic which has been under debate since the discovery of piezoelectricity in bone in 1957. Various studies have shown the potential of piezoelectricity in collagen to play a role in bone regeneration. However, in order for collagen piezoelectricity to have biological significance in the case of bone, the property must be observed on the length scale of the piezoelectric component (collagen) in physiological conditions; a topic in the literature with contradictory results. Macroscopically, hydrated bone has been shown to have an insignificant piezoelectric effect compared with dry bone. However, at the nanoscale the piezoelectric effect is observed in both dry and wet bone. Here, using single and multifrequency (band excitation (BE)) piezoresponse force microscopy (PFM), the electromechanical properties of collagen type I have been investigated as a function of humidity, from ambient to 90% relative humidity (RH), which surpasses the hydrated range for collagen in physiological bone (12% moisture content corresponding to ~ 40/50% RH). BE PFM allows for the detection of the cantilever response across a band of frequencies corresponding to the contact resonance at each pixel in an image, enabling contact resonance frequency and quality factor (energy dissipation) mapping in addition to electromechanical property imaging. The role of the water layer between the tip and surface with varying humidity and applied voltage has also been investigated, revealing no change in tip-sample capillary adhesion between on and off voltage states. The results show that collagen remains piezoelectric in a highly humid environment. These results lay to rest the question of whether collagen is piezoelectric in physiologically-relevant moisture-rich environments, and will facilitate future studies investigating the biofunctionality of piezoelectricity in physiological conditions.

Piezoelectricity in biomaterials is a ubiquitous phenomenon of high scientific interest, not only because of a possible biological function, but also in terms of potential applications as cell scaffolds and biocompatible sensors and actuators. Deoxyribonucleic acid (DNA) is one of the most important bionanomaterials that shows potential for biopiezoelectric applications. As DNA strands are stable polymers that are easy to replicate, they are ideal candidates for molecular nanodevices. Thymine is one of the nucleobases in DNA that are held by backbones of deoxyribose and phosphate. The coupling of nucleobases through hydrogen bonds and van der Waals forces might play a fundamental role in electric and possibly electromechanical behavior and are therefore interesting to study separately in the form of crystals.
In this work, thymine crystals were grown from aqueous solutions having different thymine concentrations (0.1 - 4.5 mg/ml). Centrosymmetric anhydrate and monohydrate crystals were prepared at room temperature in a covered beaker containing 40 ml of 4.5 mg/ml solution. X-ray diffraction (XRD) measurements show that both forms of these crystals are monoclinic with space group P21/c, in agreement with literature. The centrosymmetric structure of thymine anhydrates was corroborated using second harmonic microscopy (SHM) and local piezoresponse force microscopy (PFM) measurements, which showed an absence of both second harmonic signal and piezoelectric response. However, evidence of possible non-centrosymmetric crystal structure was observed for micro-sized crystals prepared under certain evaporation conditions. These microcrystals exhibited second harmonic signal and piezoelectricity. In plane (IP) and out of plane (OOP) piezoelectric response was measured with local effective piezoelectric coefficients of around 1 pm/V IP several pm/V OOP. SHM measurements confirmed that the piezoresponse is not a surface effect. However, the exact origin of this non-centrosymmetric behavior could not be determined, as microcrystals are unsuitable for conventional XRD.
In some cases, temporal switching of domains was observed. As cell adhesion and proliferation are known to be influenced by charge (e.g., poled hydroxyapatite) this opens up possible applications for nucleobase crystals as functional biomaterials in regenerative tissue engineering.

Biomaterials that simultaneously present electrical, chemical and topographic cues are ideal substrates for neural tissue engineering. Significant amount of research has been conducted to utilize combinations of these cues on a rainbow of different substrates for increased cell adhesion, proliferation and alignment as well as enhanced neurite outgrowth. The nature of anodized aluminum oxide (AAO) membranes intrinsically provides fine control over topographic and chemical cues for enhanced cell interaction; hence these biomaterials are widely used for connective tissue applications. Neural tissue engineering research with AAO substrates, however, is still very limited and the reported literature mainly focuses on the influence of topography on neural response. Herein, we present the fabrication and characterization of conductive AAO (CAAO) surfaces for the ultimate goal of integrating electrical cues for improved neural tissue behavior on the nanoporous substrate material. Parafilm was used as a protecting polymer film, for the first time, in order to obtain large area (50 cm2) free-standing AAO membranes. Carbon (C) was then deposited on the AAO surface via sputtering. Morphological characterization of the CAAO surfaces revealed that the pores remain open after the deposition process. The presence of C on the material surface and inside the nanopores was confirmed by XPS and EDX studies. Furthermore, I-V curves of the surface were used to extract surface resistance values and conductive AFM demonstrated that current signals can only be achieved where conductive C layer is present. Finally, novel nanoporous C films with controllable pore diameters and one dimensional (1-D) C nanostructures were obtained by the dissolution of the template AAO substrate. Regarding cellular data, naked AAO surfaces showed cell growth stimulation, negligible cytotoxicity and improved cell adhesion for differentiated PC 12 cells. Cellular adherence, cytotoxicity as well as neurite formation are currently being investigated for CAAO surfaces and the details of these studies will be presented. The combination of chemical, topographic and electrical cues will investigated for enhanced nerve regeneration by comparing growth factor doped and/or electrically stimulated CAAO surfaces having different porous topography.
This work was supported by The Scientific and Technological Research Council of Turkey, Grant No. MAG-111M686

Supramolecular photo-responsive hydrogels are prepared from partial methacrylation of branched polyethylenimine (PEI). The properties of the PEI-based hydrogels in terms of swelling and porosity can be controlled during synthesis by the amount of functional methacrylate groups on the polymer backbone. Different reaction conditions were used, and the synthesis was optimized to give superabsorbent hydrogel materials with water retaining properties higher than 95%. The hydrogel microstructure was characterized using several techniques including small-angle x-ray scattering and fluorescence microscopy to visualize the gel porous network. The PEI-based hydrogels are activated by multi-photon laser irradiation and can be patterned on the micron scale when molecular probes with free carboxylic acid or hydroxyl groups are present in solution. This approach offers the possibility for precise immobilization of bioactive signals into three-dimensional matrices without the need of a photoinitiator. Direct patterning of the hydrogel matrix in solutions with several types of biomolecules was demonstrated in multi-photon confocal microscopy experiments. In combination with bioactive molecules, these hydrogels represent a novel cell-instructive platform that can be selectively encoded with active signals, with relevant applications in biotechnology and medicine.

Hydrogel application is limited by its poor mechanical property for structurally demanding tissue engineering. Here, we designed highly stretchable, tough, yet stiff hydrogels based on fiber reinforced hydrogel composites. We used 3D printing technology to fabricate three dimensional patterned fibrous structures. Impregnating the fiber mesh with highly stretchable and tough PAAM-alginate hydrogel constructs the fiber reinforced hydrogel. Synthetic gels can reach fracture energies of around 9,000 J m-2. But modulus of these tough hydrogels is only about 100 kPa. Here, we designed fiber reinforced hydrogels, which can reach fracture energy of about 20,000 J m-2 and modulus of around 3MPa. Stretrability of these fiber reinforced composites can still reach about 10, which is even larger than many pure gels. The enhancement of toughness is due to multi-scale energy dissipation mechanism which spans over multiple length scales ranging from nanometers to millimeters. This design of fiber reinforced hydrogel composites can serve as a model to expand the application of hydrogels for biomedical devices and soft machines.

Damaged articular cartilage (AC) tissue shows poor regenerative capacity due to its avascularity. Current (AC) treatment modalities lack longevity or are primarily palliative in nature. The authors aim to respond to the clinical challenges presented by cartilage cell culture in electrospun scaffolds by exploring the optimal delivery environment or Bilaminar Cell Pellets (BCPs). BCPs are comprised of mesenchymal stem cells and nucleus pulposus cells that undergo chondrogenesis without hypertrophy.^1 BCPs were applied to biodegradable Poly(ε-caprolactone) (PCL) scaffolds. These multi-layer scaffolds were electrospun to provide 3D architecture for defect applications, while FDA-approved fibrin was selected for its adhesive and wound healing properties. The scaffolds provide superior cellular infiltration due to their dual-scale and laser-etched architecture. BCPs were seeded into laser-etched and non-etched scaffolds with fibrin gel. Single layer scaffolds were encased in fibrin gel for immunofluorescence to visualize type 2 collagen and aggrecan. The scaffolds were cultured for 21 days for biochemical and histological analysis, which is currently pending. Cellular attachment and infiltration was assessed using Scanning Electron Microscopy (SEM), Glycol-Methacrylate and Paraffin histology. In addition, quantitative Real Time Polymerase Chain Reaction was performed to quantitatively assess the gene expression of chondrogenic markers. The PCL nanofiber diameter was analyzed by SEM to be approximately 200-600 nm, while the PCL microfiber diameter was around 2-3 um. The laser-etched pore diameter was approximately 467 nm. SEM images indicated that the nanofiber and microfiber scaffold structure was retained after laser etching. This is a novel study showing that dual-scale electrospun meshes can be modified by commercial laser etchers to increase porosity, allow for the application of pelleted BCPs, and can be held together with fibrin gel to provide desired thickness for cartilage tissue engineering techniques.
Citation:
1. M.E. Cooke, A.A. Allon, T. Cheng, A.C. Kuo, H.T. Kim, T.P. Vail, R.S. Marcucio, R.A. Schneider, J.C. Lotz, T. Alliston. Structured three-dimensional co-culture of mesenchymal stem cells with chondrocytes promotes chondrogenic differentiation without hypertrophy. Osteoarthritis and Cartilage. 2011;10:1210-1218

The natural extracellular matrix (ECM) is made up of protein nanofibers, which provide mechanical support as well as topographical and chemical signals to the embedded cells. Bioinspired materials designed as scaffolds for regenerative therapies attempt to replicate this environment by taking into account the chemical, biological and physical cues to the ECM. Electrospinning has emerged as a versatile, inexpensive, and scalable method to create materials that that readily recapitulate the physical environment of the ECM. In order to fully realize the potential of electrospun materials as ECM scaffolds, it is necessary to devise a method of tuning the biological signaling component independently of the electrospinning process. To accomplish this, we have created a scaffold combining the use of electrospinning to control the morphology of the material with multivalent peptide conjugates embedded in the fibers to control their biochemical environment. The material is based on 800 kDa hyaluronic acid that has been conjugated with the cell-binding peptide bspRGD(15) using carbodiimide and maleimide-based chemistry. The final valency of the conjugate averaged 23 peptides bound to each hyaluronic acid molecule as measured by in-line SEC, multi-angle light scattering, differential refractive index and UV absorbance measurements. This conjugate was electrospun from a water-based solution with a high molecular weight 1 MDa poly(ethylene glycol) carrier and 200 Da poly(ethylene glycol) diacrylate, which was crosslinked to render the structure water-stable. The fibers produced had an average diameter of 538 ± 262 nm. Alignment was achieved by using a 76 mm drum rotating at 6000 rpm as the electrospinning target, resulting in 86.4% of the fibers being aligned to within 10 degrees of the main axis. Induced pluripotent stem cell-derived cardiomyocytes were seeded onto the electrospun fibers and attached and maintained their cardiac phenotype. The cells formed beating structures that aligned with the underlying fibers. Long range beating capable of deforming the scaffold was observed after five days and continued for over two months. This study has demonstrated we can independently vary the macroscale mechanical properties, nanoscale topography and alignment of the electrospun fibers, and the chemical and biological signals presented to cells. Possible applications of this material include use as a cardiac patch to reinforce the weakened myocardial wall post infarction and as a wound dressing .

As the formation of hard tissues in human consists of nano-sized hydroxyapatite (nHAp), research on the probable bio implant applications of nHAp has been increasingly gaining importance. In spite of having sufficient biocompatibility, HAp cannot perform the expected mechanical properties of a hard tissue. It is considered that, the production of composites by adding a variety of materials to nHAp would improve mechanical properties. Accordingly, nano-sized hexagonal boron nitride (hBN) having different compositional percentages was added to the nHAp to form novel composites with expected properties. For the advanced characterization of the materials, structural and morphological analysis methods were used. For the structural characterization, XRD, Raman Spectroscopy and EDS were used, and for the identification of morphology and surface features, TEM, SEM and FIB and AFM techniques were utilized. The comprehensive analytical study was helpful to reveal the microstructural and compositional changes information in detail.
Acknowledgements: The authors would like to thank TUBITAK 112M592 and 112M591 projects for financial support, also the support of Prof. Dr. Mehmet Ali Gulgun for TEM investigations is gratefully acknowledged.

The interaction between cells and implant materials is influenced by the composition of the surface structure and / or the surface of the material, subject studied and proven by several authors.
The process of osseointegration is benefited by changes in the surfaces of dental implants which are responsible for the acceleration of adhesion, migration and cell proliferation.
An ideal surface roughness is desired to occur matrix deposition and growth of bone tissue in close contact with the bone . The surface treatments are performed with the aim of increasing the mechanical and chemical bond between the implant and bone.
Dental and orthopedic biomaterials should promote osteoblast adhesion optimizing the process of integration between surgical implants placed and biological tissues.
The results show that the chemical treatment and the roughness of the surface appear to act in shear forces , especially evaluating the implant removal torque . Thus , the authors observed that the changes in the surface of the implants can optimize osseointegration and allow loading and earlier use in areas with lower density or auxiliary application in bone regenerated recentemente.
Surface modification of implants can result in morphological phenomena and physicochemical significant bone response . Another possible explanation for the good results is that changes in the chemical structure of the surface may be more favorable for binding marrow.
Thus, based on histomorphometry and biomechanical data , implants with changes in structure showing an anchorage higher than the unmodified implants . These implants allow greater integration with the bone implants unmodified after a short time of healing.
Key-words: Treatment of surface, Dental implantation.

Hollow activated carbon fiber (ACF) had been fabricated by using kapok through carbonization and activation processes. In the sample preparation, pieces of kapok were washed by HCl to remove surface wax before they were annealed at 600oC in argon for an hour. The samples were further activated for 10 to 35 min at 850oC under constant flow of CO2. During activation, the unstable carbon atoms in the fibers were selectively removed and more pores were created and increasing the surface area. The SEM analysis confirmed that the ACF samples were with morphology similar to that of the original Kapok. The average diameter of the fibers was 15mu;m and the thickness of the wall of the hollow fibers was 100nm. The Raman scattering patterns of the ACF samples showed two peaks at 1339cm-1 and 1596cm-1 corresponding to the disorder and graphitic carbon, respectively. This implied that the samples contained both crystalline and amorphous carbon.
The nitrogen adsorption and desorption isothermals were obtained from the AFC samples which were activated in CO2 for (a) 10, (b) 15, (c) 25, and (d) 35 min. Isotherm (a) showed a sharp increase in the adsorbate upon a slight increase of gas pressure before it achieved a plateau. This indicated that there was a limited pore size range and a monolayer adsorption. Thus, majority of the pores in this ACF sample were micropores. The isotherm (b) for sample after 15 min of CO2 treatment, a gentle shoulder appeared first, indicating that it was a mixed mono- and multi-layer adsorption. In isotherms (c) and (d) of samples after >15 min of CO2 treatment, their shoulders diminished suggesting the presence of larger pores for multi-layer adsorption and a mesoporous structure. All the isotherms exhibited hysteresis which was related to the filling and emptying of mesopores by pore condensation or by capillary, respectively, during the adsorption/desorption processes. Based on these results, we confirmed that these ACF demonstrated a microporous structure after short activation, but developed a mesoporous structure after prolong activation. We also determined that the maximum BET surface area of these ACF samples was 1517m2/g.
The methylene blue (MB) dye adsorption test by using our AFC samples was performed and the concentration of MB in water was monitored at different time. To study the kinetic mechanism of the adsorption, the pseudo first order and second order models were used to interpret our measured results. We found that the behavior of MB adsorption by ACF was consistent with the pseudo-second-order rate model having the correlation coefficients higher than 0.99. The equilibrium absorbance of the dye was as high as 526 mg/g in using the ACF sample treated in CO2 for 25 min. In summary, the kapok fibers are promising precursors for producing ACF. Its superior absorption ability for absorbing organic dye MB had been confirmed in this work.

Although cell adhesion to nanostructured interfaces has been extensively studied, few studies have focused on tuning nanotopographical surfaces to direct cell migration for cell patterning. Using multi-photon ablation lithography, we fabricated arrays of nanoscale craters in quartz substrates with a variety of geometries and spacing (i.e. pitch). Changing the nanocrater diameter (600-1000 nm), depth (110-350 nm), and/or pitch (1-10 um) alters the planar surface area available for cells to establish stable focal adhesions (FAs) and induces migration away from regions of high nanocrater density. This persistent migration can be used to dictate cell patterning (e.g., lines, circles) according to the nanocrater parameters. To further investigate interactions of these surfaces and the cell&’s adhesion mechanisms, we probed the effects of extracellular ligand presentation and intracellular contractile protein (e.g., Talin) activation on patterning. Nanocraters patterned in a gradient array, and presenting a RGD peptide ligand for cell adhesion, promoted cell migration in a ligand density dependent manner that encouraged cell patterning determined by the nanocrater pitch. Additionally, we found that cells that overexpress the N-terminus of Talin-1, which is one of the major proteins responsible for stable focal adhesion formation, lack the necessary balance of integrin activation to quickly spread and migrate from low pitch to high pitch regions. These nanoscale surfaces serve as tools for mechanobiology studies and understanding the attributes of surfaces necessary to physically pattern cells.

Orthopedic implant surfaces are often coated with biologically active agent to improve the surface biocompatibility of materials [1]. Bone morphogenetic protein-2 (BMP-2) has been widely used to accelerate the healing of bone defects and is also used in medical and dental implants. Therefore, many studies have incorporated BMP-2 in implants to stimulate and augment bone formation [2]. However, negative side effects such as heterotopic bone formation, retrograde ejaculation and osteoclast activation are often found when supraphysiologic doses of BMP-2 were delivered over relatively short periods. Various materials have been proposed and investigated for use as BMP carriers [3]. However, in many cases, BMP-2 adsorbed superficially on the metal surfaces is released with an initial burst and diffuses away from the implantation site too quickly. Therefore, a more efficient growth factor delivery strategy is needed to promote better orthopedic implant healing.
It is expected that titanium implants containing and continuously releasing BMP-2 will enhance bone formation at the implant-bone interface and promote faster bone regeneration at the injured site. We proposed this novel drug delivery system in which we utilize a porous metal scaffold as the drug carrier, coat the inner pore walls of scaffold with growth-factors, and compact the scaffold, so that the growth factors get trapped within the interconnected pores of the scaffold. When the scaffold is implanted in the body, the growth factors will be released from the surface initially, and the ones in the inner pores will be released slowly over time.
In this study, we fabricated BMP-2-embedded Ti implants using a novel technique and demonstrated effective embedment and long-term release of growth factors in metal scaffolds. For loading the growth factor, porous Ti specimens were soaked in BMP-2 solution in vacuum, and then air-dried. After drying, the porous Ti discs coated with BMP-2 were pressed uniaxially. We report the results of the first in vitro BMP-2 release studies for a prolonged period as well as those of an accompanying in vivo study. Fabricated titanium implant released growth factors for extended periods of time of up to 5 months. The BMP released from Ti specimens that had been immersed in a PBS solution for several months remained biologically active. In comparison with the conventional growth factor release systems, this approach surmounts the limitations associated with rapid denaturalization and diffusion of growth factors, which potentially reduces the required growth factor dose.
[1] Bae SE et al. The Journal of cell biology 1991;113:681-7.
[2] Jun S-H et al. Journal of Materials Science: Materials in Medicine 2013:1-10.
[3] Kitajima T et al. Journal of Controlled Release 2012;160:676-84.

This talk will give an overview of our research into the development of new materials and materials-based characterisation approaches for regenerative medicine [1-4]. The ability to control topography and chemistry at the nanoscale offers exciting possibilities for stimulating growth of new tissue through the development of scaffolds that mimic the nanostructure of the tissues in the body and advances here will be presented. By applying state of the art materials-based approaches we can better engineer the cell-material interface and can also elucidate disease processes within tissues. Recent examples from our group utilising state of the art analysis to investigate the structure of engineered tissue will be presented.
References
[1] E. T. Pashuck, M. M. Stevens "Designing Regenerative Biomaterial Therapies for the Clinic."
Science Translational Medicine.2013. 4 (160) 160sr4.
[2] S. Bertazzo, E. Gentleman, K. Cloyd, A. Chester, M. Yacoub, M.M.Stevens
“Nano-analytical electron microscopy reveals fundamental insights into human cardiovascular tissue calcification.”
Nature Materials. 2013. 12(6):576-83.
[3] E. Gentleman, R. Swain, N. Evans , S. Boorungsiman , G. Jell , M.Ball , T. Shean , M. Oyen , A. Porter, M. M. Stevens "Comparative materials differences revealed in engineered bone as a function of cell-specific differentiation."
Nature Materials. 2009. 8(9): 763-770.
[4] M. D. Mager, V. LaPointe, M. M. Stevens “Exploring and exploiting chemistry at the cell surface.”
Nature Chemistry. 2011. 3(8): 582-589.

Producing functional biomaterial scaffolds for Tissue engineering applications involves the design and fabrication of three-dimensional (3D) environments that are reminiscent of the native extracellular matrix (ECM): a morphologically, mechanically and chemically rich environment whose biological function is to support and provide a dialogue between the structure and the attendant cells, controlling their function and behaviour (1). Nanofibrous materials yielded by the self-assembly of peptides are rich in potential; particularly for the formation of scaffolds that mimic the landscape of the host environment of the cell. Such scaffolds should be endowed with the capacity to promote endogenous or exogenous cell survival and integration within an injury site for tissue repair. Here, we report on a range of novel approaches for the formation of supramolecular structures presenting desirable amino acid sequences and biological macromolecules via the co-assembly of multicomponent systems.In the first example, we demonstrate an enzyme mediated scaffold formed from short peptides and ECM components (2). In the second approach we present a novel methodology to include bioactive epitopes in rationally designed minimalist peptides that cannot otherwise yield the desired scaffold structures under biologically relevant conditions (3). Through the combination of these approaches, we show that we can rationally design self-assembled structures for the facile fabrication of biochemical and mechanical environments which can directly influence cell behavior.
1)Nisbet, D.R., and Williams, R.J.(2012) Self-assembled peptides: Characterisation and in vivo response, Biointerphases. 7, 1-4.
2) Williams, R.J. et al (2011) The in vivo performance of an enzyme-assisted self-assembled peptide/protein hydrogel, Biomaterials. 32, 22, 5304-5310
3)Rodriguez, A., Parish, C.L, Nisbet, D.R., and Williams, R.J. (2013) Tuning the amino acid sequence of minimalist peptides to present biological signals via charge neutralised self assembly. Soft Matter. 9, 3915-3919

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.

Synthetic hydrogels are promising in situ cell carriers for tissue engineering due to their ease of formation and tunablity. However, nearly all implanted non-biological materials elicit a foreign body reaction (FBR), characterized by an acute inflammatory reaction followed by the formation of a dense, avascular fibrous capsule surrounding the implant. With the promise of synthetic-based hydrogels for in vivo applications, questions arise as to the role the FBR plays in tissue engineering, in particular when cells are present inside a hydrogel. Our group has investigated the FBR to poly(ethylene glycol) (PEG)-based hydrogels and demonstrated that although PEG is considered bioinert and largely non-fouling, a FBR ensues evidenced by the presence of macrophages at the hydrogel-host interface and the formation of a fibrous capsule. We have further shown that creating a softer and more biomimetic PEG hydrogel (e.g., YRGDS incorporation) attenuates the severity of macrophage activation and the FBR, although does not abrogate it. Our recent work has focused on understanding (a) the mechanisms by which macrophages sense and respond to PEG-based hydrogels and (b) how macrophages and the FBR affect and are affected by encapsulated cells. Towards addressing the former, we have investigated the FBR to PEG hydrogels and PEG hydrogels with YRGDS or YRDGS. Our results from in vivo studies indicate that proteins adsorb loosely to all three hydrogels with similar signatures. The fibrous capsule thickness, however, was significantly lower for YRGDS, when compared to YRDGS (p<0.01) and PEG (p=0.057), suggesting that macrophages recognize the underlying peptide and that integrin-mediated events in part modulate the FBR. Towards addressing the latter, we have investigated the FBR to cell-laden hydrogels when the encapsulated cells are dermal fibroblasts, mesenchymal stem cells (MSCs) or osteogenically differentiating MSCs, all derived from C57BL/6 mice, and implanted subcutaneously into C57BL/6 mice. Our findings demonstrate that MSCs are able to attenuate the FBR, while differentiating MSCs have a reduced ability, and differentiated fibroblasts led to a more severe FBR. The latter is attributed to a negative affect of the macrophages on the encapsulated fibroblasts, which in turn contributed to a more severe FBR. Taken together, our findings suggest that the chemical and mechanical nature of the hydrogel dictates the severity of the FBR and when cells are encapsulated in the hydrogels they are affected and can affect the severity of the FBR. Interestingly, MSCs are able to reduce significantly the FBR and our preliminary studies point towards PGE2 as a key immunomodulatory molecule secreted by MSCs. In conclusion, our studies indicate that the FBR will indeed play an important role in tissue engineering.

Tissue engineering is a branch of science that deals with the design and synthesis of biomaterials with properties such that promote tissue regeneration by cell proliferation and differentiation. Biocompatible polymers have been widely investigated for the development of biomaterials due to their low cytotoxicity, together with its ability to form polymeric scaffolds with morphology suitable for mechanical support of the tissue. Also, various techniques have been developed which allow functionalization of polymeric scaffolds in order to improve such mechanical and biological properties, thus yielding, high quality hybrid materials with application in the area of tissue engineering. In this study, graphene oxide sheets (GrO) were functionalized with hydroxyapatite nanoparticles (nHAp) through a simple and effective hydrothermal treatment and a new physicochemical process. Microstructure and crystallinity of the new hybrid nanomaterial GrO/nHap were investigated by Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray diffraction (XRD) and thermo-gravimetric analysis (TGA). Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were performed to characterize the morphology of the functionalized material. To analyze biological properties, the composite material was functionalized using three different GrO:nHap ratios. In order to evaluate the cytotoxicity and cell proliferation the obtained materials were subjected to a MTT assay using the NIH-3T3 cell line. Polymeric scaffolds based in chitosan-polyvinyl alcohol (PVA-Ch) co-polymer were fabricated, integrating the hybrid material GrO/nHap at different concentrations (1-5 wt %), using a physical technique. The morphological characteristics of the resulting biomaterials were observed by SEM. The technique parameters were modified to increase the surface area and pore size of the biopolymer scaffolds. Confocal electron microscopy and SEM were performed to observe cell adhesion on the composite. Cell viability of the polymeric scaffolds reinforced with the hybrid materials was measured by MTT assay, using the same cell line described before. The mechanical and physical properties will be characterized using thermal analysis (TGA and DSC) and mechanical tester. The resulting novel materials combine the biocompatibility of the nHap with the strength and physical properties of the graphene improving the properties of the polymer matrices, thereby obtaining a biomaterial with potential applications in tissue engineering.

Fluorenylmethyloxycarbonyl diphenylalanine (Fmoc-FF) hydrogels have biocompatibility and viscoelasticity comparable to extracellular matrices and commonly used biopolymers. As such, they are proposed as a promising new material for regenerative medicine applications. Fmoc-FF hydrogels self-assemble through molecular stacking of molecules to form three-dimensional networks of ordered fibril structures, ideal for use as tissue engineering scaffolds or biomedical device coatings. Additionally, the self-assembly process is a simple, cost effective method for manufacturing these nanomaterials on a large scale.
The existence of piezoelectric properties could facilitate the further application of Fmoc-FF fibrous networks to applications where electrical or mechanical stimuli can be used to promote tissue regeneration. For example, bone and nerve regeneration have both been identified as being sensitive to piezoelectric properties. The direct piezoelectric effect has been linked with the ability of bone to remodel in response to an applied stress. Piezoelectricity has also been shown to promote in-vitro axonal regeneration following nerve injury.
Here, we report local shear piezoelectricity in self-assembled peptide hydrogels composed of Fmoc-FF nanofibrils, measured by piezoresponse force microscopy (~1-2 pm/V - comparable to collagen ~1-2 pm/V). The nanofibrillar nature of the gel is further confirmed by scanning electron microscopy, transmission electron microscopy, and helium ion microscopy. Also, comparisons of fluorescence emission spectra measured for Fmoc-FF in solvent and in the gel phase suggest that pi-stacking interactions between Fmoc moieties facilitate nanofibril formation. Structural analyses (circular dichroism and attenuated total reflectance-Fourier transform infrared spectroscopy) confirm the Fmoc-FF molecules within the fibrous network are predominantly in a β-sheet conformation, similar to the dominant structure observed in diphenylalanine nanotubes. Therefore, the non-centrosymmetric nature of the β-sheet is likely to be responsible for the observed piezoelectricity in the Fmoc-FF hydrogels as well.

One of the major limitations in tissue engineering is the lack of proper vascularization [1]. Nowadays skin and cartilage grafts are successfully used in-vivo mainly thanks to their low requirement for nutrients and oxygen that can be met by the host vascularization. However this approach fails when applied to complex and massive tissues. The formation of new blood vessels is indeed a slow phenomenon and the deficiency of oxygen and nutrients supply rapidly cause widespread cell death in the graft core. With the aim to overcome this hindrance we developed an innovative technique based on sacrificial elements together with a library of novel synthetic hydrogels [2]. In this approach fluidics channels are deeply embedded within the hydrogel porous scaffold in order to favor biomimetic synthetic vasculature generation.
This approach was demonstrated to be suitable for the creation of densely populated vital tissue constructs with perfusable branching endothelialized channels. In vitro perfusion was efficiently performed using a customized bioreactor, specifically developed for the perfusion of soft hydrogel scaffolds. One key advantage of this technology is that the perfusable scaffold is formed in a one step process simply by filling the empty volume around the template, crosslinking the matrix and then dissolving the template. This rapid method prevents the formation of a necrotic core in thick cellular construct, indicating that this technology could provide a solution to scale current engineered tissue to clinically relevant dimensions.
This technique allows the development of scaffolds with vascular network for treatment of big defects in tissue regeneration based on innovative and complementary technologies which combine material properties, network architecture, rapid prototyping, 3-D stem cells culture and scaffold bio-functionalization.
[1] A. Khademhosseini, J.P. Vacanti, R. Langer, Progress in tissue engineering, Sci Am. 2009, 300(5), 64-71
[2] F. Martello, A. Tocchio, M. Tamplenizza, I. Gerges, V. Pistis, R. Recenti, M. Bortolin, M. Del Fabbro, S. Argentiere, P. Milani, C. Lenardi, Poly(amidoamine)-based Hydrogels with Tailored Mechanical Properties and Degradation Rates for Tissue Engineering, Acta Biomaterialia, in press.

Improved design of efficient drug delivery systems (DDS) capable of responding to biological stimuli within an extended time window are a constant pursue in the field of tissue engineering and regenerative medicine (TERM). Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a natural and biodegradable polymer with piezoelectric properties, i.e. capable of suffering electric polarization due to mechanical stress, and vice-versa. Naturally occurring electric currents are an intrinsic property of human skin tissues, likely to act as an integrator of cells organization, development as well tissue regeneration. Under this context, this work reports an injectable piezoelectric DDS system incorporating hydrophilic and hydrophobic bioactive molecules aiming at modulating defined biological functions and tackle tissue regeneration.
Microparticles of PHBV incorporating a model protein, Bovine Serum Albumin (BSA), and glucocorticoid, Dexamethasone (Dex), were produced by a double emulsification-solvent evaporation method with modifications. Variations of the composition of the organic phase during processing allowed tuning surface topography, size distribution and core porosity of the PHBV microparticles. Likewise, the entrapment efficiency of Dex, but not BSA was modulated by varying the processing method. However, the in vitro release profile studies confirmed an initial burst effect which was followed by a sustained pattern, typical of a first order release kinetics independently of the conditions and the incorporated molecules.
An innovative approach that tackle the reduced residence time of microparticles at the injection site, and takes advantage of its piezoelectric character to release the loaded bioactive molecules was designed. Injectable formulations of Gellan Gum hydrogel, already proposed by us for diverse tissue engineering applications, were considered as carriers. Combined and well integrated systems of PHBV microparticles within GG hydrogel, responsive to electrical stimulation, were successfully achieved. By varying the properties of the hydrogel and the intensity of the provided signal, we were able to design systems with different release profiles, which can then be tuned according to tissue and pathology/injury specific requirements.
In this sense, the development of an injectable piezoelectric drug delivery system, which guarantees a localized delivery and allows the release of biochemical cues with different physicochemical features, as well as its localized deliver, was achieved representing a versatile tool to prepare instructive cell microenvironments towards tissue regeneration in TERM applications.

Cardiovascular disease remains the leading cause of death in the western world. Two major types of cardiovascular disease, myocardial infarction and peripheral artery disease, have few available treatments and therefore numerous patients continue to decline towards heart failure for the former and amputation for the latter. Current clinical trials have focused on cell therapies. It is however largely acknowledged that these cells act via paracrine mechanisms to recruit endogenous cells to help repair and regenerate the tissue. In animal models, it has been established that cellular recruitment to the damaged tissue can also occur via implantation of biomaterial scaffolds. In particular, injectable hydrogels derived from decellularized extracellular matrix (ECM) can provide tissue specific cues to promote endogenous regeneration. Rather than surgical implantation, these materials can be delivered minimally invasively, including via catheter-based injection into the heart. Hydrogels derived from porcine ventricular ECM and skeletal muscle ECM have shown significant promise for treating myocardial infarction and peripheral artery disease, respectively. Recent developments and translational progress with these materials will be discussed.

Fibrin is a provisional matrix during tissue repair and is an attractive biomaterial for cell delivery. Fibrin hydrogel properties can be tuned using a variety of techniques including altering protein concentrations, pH, calcium content, and overall ionic strength. As an alternative to increasing the concentration of clotting proteins, we demonstrated that tailoring the NaCl content in the pre-gel solution alters gel stiffness, pore size, fiber diameter, and permeability. The supplementation of fibrin gels with increasing NaCl concentrations slowed gelation time from 4 minutes to 10 minutes, retaining gelation time within in a clinically acceptable period without risking osmotic shock. We hypothesized that fibrin gels with increased bulk stiffness would provide substrate-mediated cues to entrapped cells, and these cues would instruct cell function. We examined this hypothesis in two distinct applications: neurogenesis and osteogenesis.
Neurite extension from dorsal root ganglia (DRGs) entrapped in fibrin gels correlated with substrate stiffness. We observed significant decreases in neurite length as NaCl content increased from 0.8% (w/v) to 3.5% (w/v). Furthermore, we observed significant reductions in degradation area surrounding the entrapped DRG with increasing salt concentration. We determined that neurite extension within fibrin gels is dependent on fibrinolysis and is mediated by the secretion of serine proteases and MMPs by entrapped DRGs, as confirmed by culturing cells in the presence of inhibitors against these enzymes and real-time-polymerase chain reaction.
We also explored the contribution of fibrin gels with varying NaCl content toward the dual potential of mesenchymal stromal cells (MSC) in bone repair: osteogenic differentiation and trophic factor secretion. Gel volume was better preserved in fibrin hydrogels containing greater NaCl concentrations (which also exhibited higher compressive moduli), while human MSC entrapped in fibrin gels with lower NaCl content rapidly contracted the material. Early MSC proliferation was markedly accelerated (over 200% increase in DNA) in gels containing less NaCl compared to gels with the highest NaCl content. Alkaline phosphatase activity and calcium deposition, early and late indicators of osteogenic differentiation, respectively, also were highest in fibrin gels with more NaCl, yet secretion of vascular endothelial growth factor (VEGF) was lowest. Fibrin gel osteoconductivity was dramatically improved by adding polymeric substrata coated with bone-like mineral, exhibiting near linear increases in both calcium and phosphate entrapment over 21 days.
Collectively, these data demonstrate that the biophysical properties of fibrin gels can be tailored by the simple addition of NaCl. Salt-mediated control of fibrin gel properties is sufficient to significantly direct cell function as it relates to tissue engineering and regenerative medicine.

Human bodies are highly vascularized to transport gaseous molecules, nutrients, and cell metabolites to and from cells residing in tissues and organs. As such, cardiovascular disease resulting from vascular occlusion, leakage, and rupture is the world&’s leading cause of death. Extensive efforts were made to treat these cardiovascular diseases by recreating vascular networks sing regenerative medicine including growth factors and cells. These medicines are often integrated with biomaterial systems that can enhance their therapeutic efficacy. To contribute these efforts, we have been developing various implantable devices that can elevate the quality of vascular regeneration. In this talk, I will introduce a few advanced biomaterial systems including (1) a hydrogel-based “Living” microvascular stamp that can control spatial organization of blood vessels during regeneration and (2) a self-folding hydrogel that can modulate vascular drug release with its shape change. I will also introduce nanomaterials used to engineer stem cell surface for cell delivery ischemic tissue. These material systems will greatly serve to take quality of revascularization therapies to the next level.

Skin is an important organ that forms a protective barrier against the external environment. Once broken, the process of wound healing is immediately set in motion. However, in conditions associated with severe skin loss, normal wound healing cannot reconstitute the barrier, leading to high mortality. To mitigate this, different wound dressings are routinely employed in surgical practice. However (a)more cost-effective dressings that offer shorter recovery times and (b)dressings that better resemble important physiological features of skin with minimal morbidities are needed.
A number of microfluidic approaches have been suggested for drug screening in microscale organ-mimetic platforms, as well as for the continuous formation, assembly and in vivo application of cell populated microfibers. Function of skin cells and wound-healing behavior have been studied using microfluidic platforms, and bioprinting efforts are beginning to make their way towards the repair of burn wounds and cartilages.
Here, we present to our knowledge the first approach for the in-flow formation and in vivo application of cell-populated wound dressings that accurately reproduce key features of human skin using biomaterials. The skin printer consists of a microfabricated cartridge that enables the continuous formation of wound dressings from mosaic hydrogel sheets, with the controlled incorporation of viable human fibroblasts, and the ability to define multilayered and vascularized(i.e.perfusable) hydrogel sheets.
In order to print cell-populated sheets with materials promoting both cell viability and proliferation while easy to handle, two materials are used:(1)providing stiffness to the sheet, (2)for cell printing. Precise control over the combination of these two materials is critical, as the printed patterns size and pitch will directly impact on the sheet stiffness. Different patterns were produced by varying the valve actuation time and inlet gas pressure. Such pattern was repeated on a bilayer system to illustrate the ability to create a structure that would enable the co-localization of keratinocytes and fibroblasts. These patterns can either be hollow to promote vascularization while providing relevant thicknesses, or cell-populated with local control over cell seeding density. As a case study, we printed biopolymer sheets seeded with human fibroblasts and showed their proliferation and attachment in vitro.
By performing excisional skin biopsy on immunodeficient mice, we replaced excised skin with these biopolymer sheets. Preliminary data suggests that our printed biopolymer sheets led to an improved skin regeneration as compared to our control.
We believe the skin printer provides a scalable approach(up to 35mm wide sheets were produced at rates of up to 10mm/s) and uniquely addresses the need to provide skin substitutes at affordable prices that are easy to handle and apply, while potentially reducing wound recovery times along with improving clinical outcomes.

Statement of Purpose: Sintered composite microsphere matrices have shown potential as autograft replacements, but cellular migration is often limited to the periphery in static culture. Fibrous networks of the physical scale of collagen ECM in bone may increase cellular retention and migration leading to improved bone repair. While a fibrous structure alone would have limited clinical utility due to no load-bearing potential, we propose to increase cell migration throughout a mechanically stable microsphere matrix using a secondary, nanofibrous phase within its pore structure. We hypothesize that a nanofiber mesh within the pore structure may increase cell migration, residence, and differentiation throughout the scaffold.
Methods: Sintered, composite microsphere matrices were fabricated according to reported procedure, submerged in three separate concentrations (0.25%, 1%, and 2%) of PLLA in DMF, and cooled to allow thermally induced phase separation to occur. For in vitro studies, bone marrow stromal cells were harvested from bone restricted GFP reporter mice, seeded on three different nanofiber-infused scaffolds, and assayed for DNA content and alkaline phosphatase (ALP) activity at 1, 3, 7, 14, and 21 days. Scaffolds were then implanted for 6 weeks in calvarial defects of transgenic mice carrying the same GFP reporter and the animals received a single injection of alizarin complexone 1 day prior to sacrifice. Cryohistology of the nondecalfied implants were evaluated for TRAP, ALP, GFP, and active mineralizing surfaces.
Results: For in vitro studies, DNA analysis showed lower content on nanofiber-infused scaffolds compared to scaffolds that contain no nanofibers. Raw ALP activity was lower on nanofiber-infused scaffolds compared to scaffolds without nanofibers, but when normalized to DNA content, there was a statistical increase between the 1% scaffold group and control group at day 1. In vivo studies revealed higher levels of bone tissue formation and osteoclast-mediated remodeling in the presence of nanofiber-infused scaffolds compared to control scaffolds.
Conclusions: The lower DNA content of scaffolds that contained nanofibers may be due to hindered cell migration into the scaffolds or that the fibers in the scaffold are supporting earlier differentiation, thereby quieting proliferation of the bone marrow cells. In support the latter idea, the amount of alizarin complexone deposited on active bone forming surfaces as well as bone surface associated AP and GFP reporter activity was significantly greater in the nanofiber-permeated scaffold. The presence of areas of active remodeling by osteoclasts further suggests this deposited tissue supports the idea that this newly formed tissue is active. While promising, the perceived discrepancy between the in vitro and in vivo findings necessitate further study to maximize the regenerative potential of these synthetic hybrid scaffolds.

Towards engineered tissue replacements and tissue models, biological function is closely related to the spatial arrangement of cells and the extracellular matrix (ECM). Various tissues are structured such that the cellular and extracellular material exists in aligned layers, which in turn may be oriented in distinct directions. Cartilage, heart muscle, blood vessels, and the brain are examples of such tissues. Electrospun and aligned nanofibers have been developed to mimic the ECM and influence cellular behaviors through surface topography and controlled adhesion; however, the assembly of these layered scaffolds into higher-ordered structures has been challenging. Specifically, the creation of multilayered substrates where the orientation of aligned fibers can be easily controlled between layers would facilitate regenerative engineering approaches to these tissue types. To address this, we used guest-host interactions between two hyaluronic acid (HA) derivatives, one modified with β-cyclodextrin and methacrylate groups (CD-MeHA) and the other with adamantane (Ad-HA). β-cyclodextrin and adamantane interact with one another to form non-covalent complexes based on hydrophobicity and size. We first electrospun mats of aligned nanofibers of CD-MeHA onto a spinning mandrel and photocrosslinked the fibers with inclusion of an initiator and exposure to ultraviolet light, leading to fibers with an average diameter of ~190 nm. We then held two or more mats in contact with one another in desired orientations as a solution of Ad-HA was applied, which resulted in the physical bonding of the mats through interactions of the β-cyclodextrin on the fibers and the adamantane tethered to HA polymers in solution. The layers of nanofibers remained adhered for longer than seven weeks at 37°C in PBS and were capable of bearing shear stresses on the order of 350 Pa (as determined by tensile adhesion tests of adhered mats), unlike control CD-MeHA mats to which no Ad-HA was applied. Aligned mats were arranged in a variety of orientations and assessed with confocal microscopy and scanning electron microscopy to reveal the hierarchical structure. The stability of bonds between the layered fibers offers the potential for engineering aligned and oriented tissue structures for regenerative medicine.

The challenge in the development of surrogate extracellular matrices (ECMs) is to design and prepare synthetic materials capable of influencing cell differentiation, proliferation, survival, and migration through both biochemical interactions and mechanical cues. Current studies of engineered synthetic ECMs revealed that molecular features such as peptides, proteins and bio-interactive polymers incorporated within insoluble scaffolds play a dual role in cell interaction. The functional moieties act directly on cells while also modifying the hierarchical structural organization and mechanical properties of the resulting material, thus affecting the cellular response indirectly. While the former has been investigated extensively, studies of these structural effects induced by introducing bioactive molecular features are less conclusive.
Here, we present a systematic exploration of the effect of bioactive molecules on the self-assembly of polysaccharide hydrogels and the resulting structures. Bioactive polysaccharide (hyaluronic acid, chitosan and alginate) hydrogels were prepared by covalently binding a peptide containing the cell adhesion ligand RGD to the polymer chain followed by addition of a gelling agent. Their structural and mechanical properties were investigated as a function of polymer concentration, peptide/polymer ratio, and gelling agent concentration. Thorough characterization of the polysaccharides building blocks (both natural and modified) in aqueous solutions using rheology, optical tensiometer, zeta potential, and small angle X-ray scattering (SAXS) revealed that the modifications affect the structural features of the polymer chain, the spatial arrangement of the polymer networks, and the bulk structural properties of the gels. These results suggest that elucidating the key factors involved in the structure-property relationships of these hydrogels will improve our ability to design and prepare tailor-made scaffolds for a variety of applications.

The synthesis of whitlockite (WH: Ca₁₈Mg₂(HPO₄)₂(PO₄)₁₂) has remained a long challenge, even though it is one of the major components of bone tissue in living organism. Until now, it was difficult to synthesize pure WH in aqueous solution without using any additives or buffers and thus understanding of its mechanism of formation or contribution in our body was limited. Therefore, in the present study, we designed and developed a large-scale synthetic technique for pure WH nanoparticles in physiologically relevant condition of ternary Ca(OH)₂-Mg(OH)₂-H₃PO₄ system, based on the systematic approach. From the XRD analysis and FESEM observation, we confirmed that WH was in homogeneous phase with 50 nm of rhombohedral nanoparticles. In addition, FT-IR and TGA analysis demonstrated that our WH nanoparticle was a chemically distinct phase from its synthetic analogue tricalcium phosphate (TCP: Ca₃(PO₄)₂) by clear existence of HPO₄^2- in its structure. In this research, we also investigated a new synthesis mechanism of WH and its stability in various pH conditions by solubility test. Interestingly, the result indicated that WH had higher stability than hydroxyapatite (HAP: Ca₁₀(PO₄)₆(OH)₂), below pH 4.2. Another noteworthy finding was that this synthesized nano-WH showed comparable biocompatibility with HAP from the human osteoblast proliferation test. Furthermore, nano-WH showed higher rate of cell growth than that of bulk TCP that has been widely used as a commercial implant material. In addition, to verify that proliferated bone cells were also active in bone formation process on the surface of WH, quantified gene expressions associated to bone-mineralization process were compared relative to HAP by RT-qPCR analysis. The result showed that WH reinforced osteoblast cell function similar to the bone cells grown on HAP. Based on these positive results, nano WH shows high potential to be applied as a new biocompatible material. We expect that this research will shed light on the understanding of the precipitation mechanism of calcium phosphate compound in physiological systems and suggest a systematic platform for synthesizing important bio-compounds.

Estrogen plays a vital role in the regulation of many physiological conditions, such as cell growth, proliferation and differentiation. It can passively diffuse into the cell nucleus to elicit alternation of gene expression via binding to the nucleus estrogen receptors (hormone genomic effect). However, recently it is also reported that some estrogen receptors widely distributed around cell membranes. By binding those membrane estrogen receptors, estrogen can induce unique biological phenomenon (non-genomic effect). However, it is quite difficult to monitor the estrogen induced non-genomic biological behavior because this effect occurs in extremely transient time scale. As a result, in order to specifically study the estrogen non-genomic effect, one of the strategies is to keep estrogen interacting with mERs but not penetrating into the cell nucleus. The objectives of this study are to develop estrogen-immobilized biosensors for understanding the mechanism of hormone non-genomic effect.
One approach is to immobilize estrogen onto the micropatterned surfaces which were prepared by photolithography. By this top-down method, gold spots around 2 mu;m in diameter were deposited onto a glass substrate and the glass or gold surfaces were modified differently via specific reaction. On the glass surface, a cell adhesive peptide, cyclic RGD was introduced in order to induce the attachment of estrogen receptor over expressed human breast cancer cells (MCF-7 cells) to the surfaces. On the gold surface, estrogens were also successfully immobilized which SPR study was performed to monitor the whole reaction process in situ. In the cell ELISA study, we found that the ERK phosphorylation level which is an important non-genomic marker was significantly higher in the estrogen presence group compared with the control group. Another approach is that estrogen was covalently conjugated onto the biocompatible polymer chitosan films by bottom-up method. The chitosan film was prepared by simply drop casting chitosan aqueous solution onto a glass substrate. After drying, estrogen was conjugated onto these chitosan films. Similarly, we found that estrogen conjugated chitosan films significantly enhanced the ERK phosphorylation level in the MCF-7 cell line as well. In the DAF imaging based nitric oxide release study, we found that the estrogen immobilized chitosan film significantly stimulated NO production in the estrogen receptor positive endothelial cell line (EA.hy926) and this stimulation could also be blocked by estrogen receptor inhibitor ICI 182,780. The results above demonstrated that the surface immobilized estrogen could provide protective effects for the cardiovascular disease. In sum, the present study gave strong evidence that the surface immobilized estrogen by different strategies can specially induce estrogen non-genomic effect and hence be useful for biomedical applications.

The research and development of β-titanium alloys as bone fixation and bone replacement materials is of great interest. By alloying titanium with 40 weight percent niobium (Ti-40Nb) a low young&’s modulus is achieved which should prevent the well-known stress shielding effect. To improve the biocompatibility of titanium alloys a rough surface with a thick oxide layer is essential. Therefore usually chemical etching (e.g. H₂O₂ etching) and anodic oxidation with acid based electrolytes are used. Low temperature plasma oxidation is a beneficial alternative because residues from liquid electrolytes are mostly harmful and need to be removed before implantation. In this work we use a radio frequency oxygen plasma with additional bias voltage to clean, oxidize and sterilize metal implant surfaces in a single device. By varying the process parameters we induce stable or unstable oxide growth as shown by [Vennekamp2005].
Disks of Ti-40Nb were oxidized in a self-constructed plasma setup. The sample temperature, oxygen gas pressure and additional bias voltage of the process were varied. The plasma treated samples were characterized by confocal laser microscopy, SEM, TEM, EBSD, XPS and ToF-SIMS. Surface energy was determined by contact angle measurements using the OWRK method. After culturing of human mesenchymal stromal cells (hMSC), metabolic activity was determined after 24 hours using a MTS assay with cp-Ti as control.
Oxide layers consisting of a mixed titanium-niobium oxide with thicknesses between 50 and 150 nm were grown. Surface roughness values and microstructure indicate that the growth mode of the oxide can be well controlled by the sample temperature and oxygen gas pressure. Rough and defective surfaces with high surface energies were obtained by applying bias potentials higher than 50 V. Metabolic activity of samples with stable and unstable oxide growth is as high as for cp-Ti. With our method we were able to control the growth mode and surface energy of the growing metal oxide layer while maintaining the metabolic activity of hMSCs compared to cp-Ti.
[Vennekamp2005] Vennekamp, M. and Janek, J. (2005). Control of the surface morphology of solid electrolyte films during field-driven growth in a reactive plasma. Phys. Chem. Chem. Phys. 7, 666-677

Collagen is widely used as a scaffolding material for both soft and hard tissue engineering applications due to its good biological properties, in addition to its low cytotoxicity and immunogenicity. Currently, the main sources of collagen for clinical applications are from cows, pigs and sheep. However, the outbreak of diseases such as bovine spongiform encephalopathy, foot-and-mouth disease and transmissible spongiform encephalopathy has limited the clinical applications of these materials. [1,2] Therefore, it is necessary to discover an alternative safer source of collagen.
In this study, collagen was successfully isolated from bullfrog skin, a food processing waste product. For the first time, we report the extraction of bullfrog skin-derived acid-soluble collagen (ASC) and the subsequent fabrication and characterization of ASC-based three-dimensional scaffolds. Results showed that bullfrog skin-derived ASC was type I collagen, which consisted of α1(I)-, α2(I)-, β- and γ-chains that is similar to the type of collagen found in the skin of other species. The extracted ASC was subsequently fabricated into 3D scaffolds and further crosslinked with 1,4-butanediol diglycidyl ether (BDE) to evaluate the effect of crosslinking on the scaffold properties. BDE-crosslinked ASC scaffolds were found to have different morphology and material properties compared to untreated ASC scaffolds. Untreated ASC scaffolds had a greater pore size range, porosity and swelling ratio than BDE-crosslinked ASC scaffolds. As BDE concentration increases, the compressive moduli and denaturation temperatures of the scaffolds also increased. Cell culture studies showed that the untreated ASC scaffold was more effective in supporting the cell proliferation of MSCs. However, an in vitro degradation study showed that BDE-crosslinked ASC scaffolds had greater stability than untreated ASC scaffolds, and the stability increased with increasing BDE crosslinking concentrations. In addition, the various types of ASC scaffolds exhibited good biocompatibility in a two-week in vivo implantation study.
Overall, this study demonstrated the possibility of using bullfrog skin as an alternative source of collagen for the fabrication of tunable ASC scaffolds suitable for tissue engineering purposes.
[1] Li B, Liu L, Gao H, Chen H.. Studies on bullfrog skin collagen. Food Chem. 2004;84:65-69.
[2] Wang L, An X, Yang F, Xin Z, Zhao L, Hu Q. Isolation and characterisation of collagens from the skin, scale and bone of deep-sea redfish (Sebastes mentella). Food Chemistry 2008;108(2):616-23.

Regenerative medicine aims to replace or repair tissues within the body to improve and replicate biological function. This can be achieved by seeding and differentiating stem cells within a biocompatible scaffold and implanting the resulting tissue type into a defect site within the body. However, human tissue is a complex and hierarchical composite material, made of different cell types of varying gradients and patterns. Building these complex 3D tissue structures while ensuring adequate vascularisation is a huge challenge in tissue engineering. This work details two novel methodologies for engineering complex, composite tissues: responsive scaffold construction and 3D droplet printing.
Currently, most commercial scaffolds play a purely structural role in growing new tissue in vitro. However, our results show that commercial cell scaffolds can be covalently cross-linked with proteins to produce a biomaterial that performs both structural and functional roles. The synthesis is facile and versatile, with extensive coverage of the scaffold fibres, as demonstrated by confocal microscopy. We have also shown that cell adhesion can be significantly increased using cationic proteins. We are now applying these techniques to generate oxygen reservoirs that increase both oxygen delivery and adhesion, and to biochemical growth factors to promote angiogenesis and differentiation.
In addition, we have developed a 3D droplet printing methodology for scaffold-free tissue engineering. Here, we show layer-by-layer printing of individual, viable mesenchymal stem cells contained in picolitre cell media droplets with precise spatial arrangements, for the design of complex hierarchical tissue structures that include patterning, gradients and vascularisation.

Introduction: Hydrogel-based scaffolds are widely used for culturing cells in 3D due to their tissue-like water content and tunable biochemical and physical properties. Most conventional hydrogels lack the macroporosity desirable for efficient cell proliferation and migration and have limited flexibility when subject to mechanical load. We have recently reported development of gelatin-based, microribbon-like elastomers that can photocrosslink into macroporous and highly flexible scaffolds. The goal of this study is to examine the potential of microribbon-based scaffolds for supporting osteogenic differentiation of human adipose-derived stromal cells (hADSCs) in 3D.
Materials and Methods: The gelatin based microribbons were synthesized as we previously reported using wet-spinning. We then coated the microribbons with methacrylate anhydride to enable photocrosslinking among microribbons, pre-fixed the microribbons by a trace of glutaraldehyde, and finally neutralize the aldehyde residues by lysine. The wet-spinning rate, selection of drying agent (acetone vs. methanol), drying temperature and the level of glutaraldehyde crosslinking were adjusted to control the mechanical properties of individual microribbons, which present mechanical cues to cells. Upon photocrosslinking, the microribbon formed a highly macroporous scaffolds with porosity tunable by microribbon density. To study the osteogenic differentiation of hADSCs, cells were mixed at 10 Million cells per ml among the microribbons, and the mixtures were photocrosslinked for direct cell encapsulation. hADSCs were also encapsulated in conventional gelatin-based hydrogels(HG). Cell-free scaffolds alone were also included as controls. All samples were cultured in osteogenic medium for 21 days. Outcomes were analyzed using mechanical testing, gene expression, and cell proliferation. Extracellular matrix production was measured using biochemical assays and histology.
Results and Discussion: Live dead assay confirmed that ADSCs retained high viability after being encapsulated within microribbon-based scaffolds. DNA content showed ADSCs proliferated 100% more within microribbon-based scaffolds than HG control over 3 weeks of culture. Biochemical assays and mechanical testing demonstrated that the microribbon scaffold supported the deposition of extracellular matrix (ECM) components, which resulted in a 200% increase in compressive moduli in cell-containing constructs compared to acellular groups after 21 days. Histology demonstrated that macroporosity of the microribbon-based scaffold supported homogeneous cell proliferation and ECM deposition throughput the construct. In conclusion, our study has demonstrated the potential of microribbon-based scaffolds for supporting stem cell osteogenesis. Coupled with the advantages of enhanced nutrient diffusion and mechanical property, such microribbon-based scaffolds may provide a promising platform for regeneration of large bony defects.

As the world&’s population ages, regenerative medicine continues to rise in importance. An important aspect of this is developing the ability to reconstitute load bearing tissue such as articulating cartilage, which often cannot replenish themselves or gradually fail to do so in old age. To rectify this, much research has focused on developing hydrogels either as replacement for lost tissue or to serve as a cellular matrix encouraging the production of native tissue. However, hydrogels are naturally a weak and brittle material that cannot withstand the load demands of the human body. Tough hydrogels have been investigated in recent years to compensate for the material&’s mechanical weakness. These hydrogels have the ability to dissipate deformation energy by one of several mechanisms, thus resisting loading and fracture at values similar to those of native tissue. However, no tough hydrogel has been developed that is capable of both being 3D printed and encapsulating cell culture.
Here, we report a novel tough hydrogel that is biocompatible for cell encapsulation as well as a unique method of rapid prototyping allowing for complete shape control of the hydrogel. Our hydrogel is comprised of two interconnected polymer networks. The “backbone” network is a poly(ethylene glycol) diacrylate (PEGDA) matrix that forms crosslinks when exposed to ultraviolet (254nm) light. This network works to maintain the shape of the hydrogel during deformation and gives it strength. A secondary network of alginate chains weaves throughout the PEGDA. These chains are ionically bonded via Ca2+ ions; these bonds easily break and reform, allowing them dissipate energy and give the hydrogel its toughness. It should be noted that this hydrogel is completely biocompatible and we demonstrate its ability to encapsulate healthy eukaryotic cells. The other novel contribution we make is in the shaping of the hydrogel. We apply rapid prototyping technology to form this tough hydrogel into three-dimensional shapes. To do this we mix the PEGDA-alginate gel (containing eukaryotic cells) together with Ca2+ ions and UV crosslinking agent in a syringe that is extruded by a desktop 3D printer capable of reading standard computer-aided design (CAD) files. Due to the short curing time, the cells are not harmed and give our method great promise for the production of tough hydrogels capable of regenerating load-bearing tissues.

Catechol is the organic molecule that can be easily found in nature. Catechol and its derivatives have drawn much attention due to their unique physical and chemical characteristics. When catechol molecule is oxidized, it becomes highly reactive quinone and the formation of quinone allows a variety of reactivity toward amines, hydroxyls, and thiols in chemical reaction.
Inspired by mussel adhesive proteins that contain multiple catechol molecules, dopamine, one of catechol derivatives, has been utilized as a universal catechol-amine surface modifier to generate self-polymerized thin films. Single-molecule study using AFM (Atomic Force Microscope) has confirmed that catechol can generate a covalent bonding with primary amines and thiols. This reactivity of catechol toward amines and thiols has been utilized to crosslink hydrophilic polymers to generate hydrogels for tissue engineering. Due to specific covalent bond formation during the reaction of catechol and primary amine, it has drawn our attention to investigate catechol as the pH sensitive and site specific chemical linker for the PEG (polyethyleneglycol) and polysaccharide modification of peptides and proteins.
PEGylation - covalent attachment of PEG to proteins - is a widely used method to increase the therapeutic effect of pharmaceutical proteins. Benefits of PEGylation include physical protection of the proteins against enzymatic proteolysis, improved solubility of the PEGylated drugs, and significant increase in blood half-life in vivo. Thus, PEGylated proteins have increasingly been in the spotlight as the pharmaceutical protein drug market matures. Studies of PEGylation have modified a variety of biological macromolecules: proteins, oligonucleotides, and antibody fragments. However, PEGylation often results in decrease in intrinsic biological activities, which is due to lack of selectivity of functional groups in the existing PEGylated chemistries. Thus, to maximally retain original activity of PEGylated molecules, it is critical to develop a method that can modify a specific residue of proteins, in other words, site-specific PEGylation.
Herein, we describe a novel N-terminal selective PEGylation and chemical reaction inspired by the chemistry of catechol. The reaction occurs in a mild neutral condition, which is suitable for maintaining activities of PEGylated proteins. This PEGylation strategy is generally applicable to a wide variety of proteins such as G-CSF (granulocyte-colony stimulating factor), bFGF (basic fibroblast growth factor), EPO (erythropoietin), and lysozyme as well as a peptide named hinge-3. The PEGylated protein exhibited increases stability with the original activity of proteins. Not only PEGylation, but also catechol modified hyaluronic acid had a great ability of N-terminal specific peptide modification. Therefore, we believe that the catechol-involved site-specific protein modification method can potentially contributes to develop new pharmaceutical proteins.

Hydroxyapatite (HA) is a crystalline calcium phosphate that is the primary mineral component of teeth and bone, and the osteoconductive properties of synthetic HA has led to its widespread use as a coating or additive in bone grafts, scaffolds, and orthopedic implants. Electrical polarization of HA was achieved in previous studies by ion migration at elevated temperature (>300°C) driven by a strong applied electric field (>1 kV/cm), resulting in permanent stored charge near room temperature. It has been demonstrated that electrical polarization of HA has the abilities to enhance bioactivity and osteointegration. However, traditional polarization method is low efficient because of high ion migration resistance in solid HA crystals. We demonstrate a method to retain strong electrical polarization in HA coatings on titanium from aqueous solution. The stored charge is larger by more than an order of magnitude than any previously reported electret or ferroelectret material. The polarized HA coatings on titanium display improved bioactivity, indicating promising potential in orthopedic implants. The very high stored charge may enable new applications of the HA coatings in electret generators, filters or energy storage.

The success of the bone regeneration approach not only requires the appropriate cells, but also an appropriate osteoactive microenvironment. A novel approach to enhance bone regeneration provided by transplantation of bone marrow derived cells involves rapid concentration and selection of the osteoblastic progenitor population in the graft using selective attachment to the matrix surface. Microelectromechanical systems technology and related microfabrication techniques can be used to create precisely defined surface microscale topographies that can selectively stimulate cells on the surface of scaffolds to enhance osteoprogenitor cell growth and guide subsequent bone formation.
The purpose of this study is to investigate the influence of precise defined surface topographies on osteogenesis in vitro and in vivo by examining the proliferation and differentiation characteristics of a class of adult stem cells and their progeny, collectively known as human bone marrow derived stromal cells (BMSCs).
The polydimethysiloxane (PDMS) substrates comprising surface post microtextures (10 micrometer in height, 10 micrometer in diameter, and 10 micrometer separation between posts) were fabricated using soft lithography-based techniques, and cultured with BMSCs in vitro. Subsequently, the performance of PDMS constructs with surface microtopographies was investigated in mice models to test the osteogenic capacity of the subcutaneous biomaterial implants for bone tissue formation.
The biological performance of BMSCs with respect to proliferation and osteoblastic differentiation is significantly modified by interaction with post microtextures when compared with smooth surfaces, as assessed by cell morphology, cell retention, matrix deposition, and osteoblastic differentiation in vitro and in vivo. Particularly, osteoblastic gene expressions, such as Collagen I, Collagen X, Bone sialoprotein and osteocalcin, were up-regulated (over 280%) cells on post microtextures in vivo compared to those of on smooth surface and in vitro (100%). Human BMSCs on precise defined surface topographies provide osteoconductive stimuli for bone tissue regeneration and enhanced fracture repair characteristics.

The skin is the body&’s largest organ, and every year millions of people suffer from the effects of skin disease and degeneration. Solar ultraviolet (UV) radiation is one of the most ubiquitous conditions the body encounters and, although necessary for vitamin D production, is responsible for a host of negative skin responses, including inflammation and infection due to compromised barrier function. The outermost layer of skin, the stratum corneum (SC), protects the body from harmful environmental conditions such as UV exposure by serving as a selective barrier. We have previously shown that solar UV radiation poses a double threat to the SC by increasing the driving force for cracking while simultaneously decreasing the SC&’s resistance to cracking by significantly reducing cellular cohesion, largely dominated by the intercellular lipids and corneodesmosomes, thereby impairing the critical barrier function of the skin. To protect against the harmful effects of solar UV exposure and induce regenerative repair after injury, inorganic UV-blocking micron- and nano-sized zinc oxide (ZnO) and titanium dioxide (TiO2) particles are commonly applied to the SC. These diffuse and partition into the SC intercellular boundaries, and the resulting construct is highly effective in preventing erythema from solar UV radiation. However, it remains unclear if these treatments can prevent degeneration in the biomechanical barrier of the SC.
We explored the interaction of zinc oxide and titanium dioxide particles with SC in the presence of UV radiation. We used substrate curvature techniques to characterize the drying stresses, and hence the driving force for damage, that occur with UV exposure and the ability of inorganic particles to mitigate this damage. We also explored the ability of the particles to protect the innate SC resistance to corneocyte separation, which has been shown to decrease under UV exposure. We found that the inorganic UV inhibitors protected the SC&’s mechanical properties under relatively large doses of UV radiation. We also compared efficacy of the inorganic molecules to chemical (UV absorbing) sunscreens. Clinical implications of this work include prevention and treatment of sunburn and long term skin damage such as photo-aging.

Local and abrupt changes in mechanical forces play a fundamental role in control of tissue and organ development. While some investigators have varied material properties of tissue engineering scaffolds to influence cell behavior, no biomaterials have been developed that harness mechanical actuation mechanisms to induce new tissue formation. Here, we describe the development of mechanically-actuatable polymers that induce tissue differentiation by harnessing the physical induction mechanism that drives tooth organ formation in the embryo. The formation of many epithelial organs is triggered when sparsely distributed mesenchymal cells abruptly pack closely together and undergo a “mesenchymal condensation“ response. For example, in tooth development, the associated physical compression and rounding of dental mesenchymal cells is sufficient to induce whole organ formation in vitro and in vivo*. Inspired by this developmental induction mechanism, we fabricated an artificial, shrink-wrap like polymer scaffold that can stimulate tooth tissue differentiation by abruptly inducing physical compaction of cells cultured within it when warmed to body temperature. A porous, GRGDS-modified, hydrogel scaffold was fabricated from poly(N-isopropylacrylamide) (PNIPAAm), which remains in an expanded form in the cold, and rapidly contracts volumetrically when placed at body temperature. When undifferentiated embryonic dental mesenchymal cells were seeded within this hydrogel sponge and polymer shrinkage was thermally induced by warming, the cells became physically compressed and exhibited a more compact, rounded morphology, as they do when they undergo mesenchymal condensation during tooth organ development in the embryo. This physical change in cell shape stimulated tooth differentiation, as measured by the induction of key odontogenic transcription factors in vitro and stimulation of mineralization in vivo. This polymer-based mechanical actuation mechanism represents a new bioinspired approach to induce organ-specific tissue differentiation that could be useful for stem cell biology, tissue engineering and regenerative medicine.
*T. Mammoto, A. Mammoto, Y. S. Torisawa, T. Tat, A. Gibbs, R. Derda, R. Mannix, M. de Bruijn, C. W. Yung, D.Huh, D. E. Ingber, Dev. Cell 2011, 21, 758-69.

Recent developments in nanotechnology brought us innovations in bio-engineering materials and bio-device fabrications. There are a lot of challenges for chemists to play an important role in this area since the synthesis of novel bio-materials by following nanotechnologic approaches is also a part of the chemical domain. Hydrogels have been widely investigated and used for bio-applications. Functional hydrogels have been synthesized by a microfluidic technique by designing novel microfluidic reactor and mixer. Microfluidic synthesis has taken an intensive attraction in many areas, including clinical applications since microfluidic reactors allow us to produce specific advantages that conventional synthesis can&’t achive. We introduced a novel microfluidic reactor, which synthesized functional hydrogels. We also present here a microfabrication of hydrogels to develop bio-devices.

Polyurethane has become one of the most widely used plastics for various applications such as insulation, automotive parts, seating materials, and artificial leather because of its good flexibility, elasticity, and damping ability. However, the starting materials for preparation of polyurethane are strongly dependent on petroleum as a feedstock, and this fact became problematic because of dramatic depletion of fossil oils, continuous fluctuations in the oil price, and environmental concerns. Therefore bio-renewable materials for polyurethane became highly desirable in the industrial field.
In this research, castor oil and polycaprolactone (PCL) were used as the bio-renewable feedstocks for polyurethane. The castor oil is an excellent polyol candidate because it is nonedible, chemically stable, and biodegradable. The PCL is also biodegradable materials and has linear structure, leading to flexibility enhancement of polyurethane when it is incorporated. The castor oil/PCL based polyurethane was prepared by the one-step polymerization process with different castor oil contents. The castor oil/PCL based polyol reacted with 4,4'-diphenylmethane diisocyanate (MDI). The PCL and MDI acted as soft and hard segments, respectively, and castor oil acted as dendritic point in the castor oil/PCL based polyurethane structure. The castor oil/PCL based polyurethane was synthesized successfully and the obtained polymers were soluble in common organic polar solvents. The chemical structure, crystallinity and molecular weight of synthesized castor oil/PCL based polyurethane were investigated. The molecular weight and crystalline peak intensity of castor oil/PCL based polyurethane were decreased with increasing castor oil contents because PCL contents with higher molecular weight were decreased relatively with increasing castor oil. The thermal and mechanical properties of castor oil/PCL based polyurethane were also investigated. This research was supported by National Research Foundation of Korea. (Project No. 2013-055716)

We investigated the influence of a specific microstructure and organic matrix distribution pattern on the material properties of biological carbonates with high-resolution EBSD, modulus mapping, selective etching of the mineral and dynamic nanoindentation of dry and wet skeletal elements. We examined four distinct microstructures of two carbonate phases, implemented in the shells of two major marine organisms: the gastropod Haliotis glabra (Haliotis) and the bivalve Mytilus galloprovincialis (Mytilus). Mytilus consists of fibrous calcite and nacreous aragonite, while Haliotis is formed of prismatic and nacreous aragonite. A transition zone separates the microstructures: it is pronounced in Mytilus and is located between the two carbonate polymorphs, while in Haliotis it is less obvious and is between the prismatic and nacreous aragonitic shell portions. This study comprises three major aims: (1) the investigation of the entire skeleton, not only its nacreous portion, (2) performance of nanoindenation testing in dry and more natural, wet, conditions, (3) the comparison of structure-property relationships between gastropod and bivalve hard tissues.
Selective etching reveals two biopolymers: (1) membranes present as organic sheaths around fibers and tablets and (2) an occluded network of fibrils within the fibers and the tablets. EBSD shows the exquisite co-orientation of Mytilus&’ calcite in the fibers, that is reduced in Haliotis&’ prismatic aragonite. Stacks of highly co-oriented tablets were identified and highlight a super-structure. This new hierarchical level indicates a strong correlation between mechanical properties and the microstructure as it was observed by both modulus mapping and EBSD, respectively. Nanoindentation performed under dry conditions revealed an increased hardness and modulus (H asymp; 3.5 GPa, E asymp; 95 GPa) of the nacreous aragonite structure and matched well for both species. In contrast, prismatic aragonite and fibrous calcite of Haliotis and Mytilus differed significantly. The former showed an increased hardness and modulus, while for the latter mechanical properties were slightly reduced.
It is shown for the first time, for Haliotis and Mytilus, that the shell&’s different microstructures give rise to distinct mechanical performances. Haliotis forms hard and stiff outer aragonite prisms but tougher aragonitic nacre. In contrast, Mytilus&’ tabular arrangement is slightly harder and stiffer than its fibrous shell portion, which is unexpectedly tougher than inner nacre. Predation avoidance on the substrate requires a hard outer shield that protects softer shell portions and the soft tissue of the animal. However, adaptation to specific habitats results in the hard outer rim of Haliotis, while for Mytilus a soft and pliant outer shell serves its requirements. Mytilus&’ fibrous microstructure exhibiting similar toughness as the nacreous arrangement may facilitate new artificial ultratough materials with alternative architectures.

Nowadays a great interest of application of biopolymers such as cellulose or lignin derivatives in the development of electrochemical devices can be observed; they are rather environmentally friendly, renewable and easy accessible materials. Their huge occurrence in nature and low cost improves also their attractiveness from the economical point of view.
Taking into account electrochemical demands, particularly in the field of energy storage, sensor applications and electrocatalytic properties, lignin derivatives seems to be especially profitable. Lignosulfonates being a water soluble by-products of pulp and paper industry, recently were used as a electroactive materials and showed strong electrocatalytic behavior toward biomolecule. This activity is attributed by existence of many phenolic and methoxyphenolic functional groups that can be easily converted into reversible quionone/hydrquinone redox moieties. Moreover, due to possession of negatively charged sulfonic groups may be treated as a polyanions and used as a dopants for conducting polymers. Thus, they open up new possibilities for the production of cost efficient, environmentally friendly, up - scalable and lightweight energy storage systems as well as enhanced electrochemical sensors.
The aim of present work is a synthesis of conducting composites based on lignosulfonates for electrocatalysis, potentiometric sensors and energy storage applications. For this purpose, relatively simple functionalization of conducting polymers such as: polyaniline, poly - 3, 4 ethylenedioxythiophene (PEDOT), polypyrrole has been applied, where during galvanostatic polymerisation, lignosulfonate acts as a doping agent. In all cases, significant improvement of surface confined redox signals has been observed, because of development of reversible couple assignable to quinone/hydroquinone system. Such designed materials can be used as electrocatalytic mediators for dihydronicotinamide adenine dinucleotide (NADH) and ascorbic acid. Moreover, the possibilities of using this composites as potentiometric sensors are widely discuss. The abilities to store an electric energy in redox active lignin derivatives are presented.

The ability to easily remove any organic and inorganic contaminants on surfaces is of fundamental importance to a broad range of energy, marine, and biomedical applications related to anti-fouling and drag-reduction. This led to enormous interest to the development of surface coatings that possess self-cleaning ability. To this end, nature has provided inspirations on the design of self-cleaning surfaces, with examples such as lotus leaves, Tokay gecko, and shark skin [1]. Here, we introduce a new class of bioinspired self-cleaning surfaces that are inspired by the Nepenthes pitcher plants. These novel self-cleaning surfaces, termed as Slippery Liquid-Infused Porous Surfaces (SLIPS) [2, 3], are capable of removing both inorganic and organic contaminants through a unique self-cleaning mechanism that is distinguished from other well-known mechanisms. Specifically, we have studied the influence of size, geometries, and surface chemistry of the surface contaminants to the effectiveness of the cleaning process. Owing to the omniphobic nature of SLIPS, one can utilize a broad range of cleaning fluids to remove surface contaminants, which is in contrast to other bioinspired self-cleaning surfaces. Fundamental study of the self-cleaning mechanisms of SLIPS and other bioinspired surfaces will be discussed in the meeting.
References
[1] Kesong Liu and Lei Jiang, “Bioinspired Self-Cleaning Surfaces”, Annual Review of Materials Research, vol. 42, pp. 231 - 263 (2012).
[2] Tak-Sing Wong, Sung Hoon Kang, Sindy K. Y. Tang, Elizabeth J. Smythe, Benjamin D. Hatton, Alison Grinthal & Joanna Aizenberg, Bioinspired self-repairing slippery surfaces with pressure-stable omniphobicity. Nature, vol. 477, pp. 443 - 447 (2011).
[3] Tak-Sing Wong, Taolei Sun, Lin Feng, and Joanna Aizenberg, "Interfacial materials with special wettability', MRS Bulletin, vol. 38, pp.366 - 371 (2013).

The behavior of a Ti6Al7Nb biomaterial with a nanostructured HA-type coating was studied in vitro and in vivo conditions. The metallic material used like substrate alloy for layer deposition was a Ti6Al7Nb alloy obtained by double electron beam melting furnace. In order to obtain a nano-crystalline HA-coating first sodium titanate layer was obtained on the surface and then the implant was immersed in Ringer solution with additional PAW1 biovitroceramic (particles under 20 mu;m). Three different pH Ringer solutions were used (2.5, 7.0 and 9.0) and different deposition times (5, 10 and 19 days) were employed. Microscopy analysis and corrosion tests of the implants relives that the nanostructured HA layer after 19 days of immersion shows promising results as regarding the implant employ in preclinical experiments. After a complex design based on bone radiography there has been manufactured two different types of devices for the metallic implants: a metallic plate and a pin. He implants were in contact with the bone for 6 months. Analysis results seem to show that HA deposited layers illustrate a quite regular aspect of HA growth with some small pores. The HA deposited layer after 19 days shows promising results as regarding implant using in preclinical experiments. The obtained results are important for the safe long-term in vivo application of the Ti-6Al-7Nb alloy since niobium has the characteristics of an immunologically inert metal.

Self assembly of multidomain peptides (MDP) into nanofibers which mimic extracellular matrix allows for unique control of in vitro and in vivo responses. These functional properties are dependent on amino acid sequence, supramolecular peptide folding and structure, incorporation of biomimetic moieties and sustained drug release. Control of biodegradation, cell adhesion and angiogenic sequences were afforded by tailoring peptide sequence. Bio-interactivity was promoted by incorporation of an MMP-2 cleavage sequence, integrin cell adhesion moiety and an angiogenic moiety. Folding of peptides into stable anti-parallel beta sheets stabilized by reversible hydrogen bonds and ionic interactions afforded shear thinning and mechanical recovery of hydrogels. Drug release of MCP-1 (a potent stimulator of monocyte/macrophage recruitment) and IL-4 (a stimulator of Th2 immune response) over short (24-48 hr) and long term (12-16 day), respectively, was demonstrated. Human monocytic leukemia cell line (THP-1) differentiation was dependent on cytokine loading and release from hydrogels. In vivo rat subcutaneous injections showed that hydrogels with cell adhesion moieties show no measurable fibrous capsule formation. MCP-1 loaded scaffolds show significantly greater CD68+ macrophage recruitment, compared to IL-4 scaffolds which had greater vWF+ endothelial cells - commonly associated with a pro-healing M2 macrophage response. Pro-angiogenic peptides showed greatest blood vessel formation with stable vessels lined with endothelial cells and smooth muscle cells. Overall, this design strategy has allowed for the development of a novel class of polymers that uniquely or combinatorially allow for modulation of inflammation, tissue healing and angiogenesis.

Shear-thinning hydrogels afford direct injection or catheter delivery to tissues without the use of triggers such as chemical initiators or heat which may cause potential premature gel formation and delivery failure. Hydrogels based on physical crosslinking mechanisms (e.g., ionic and hydrophobic interactions, chain entanglement) have been widely explored, and we have recently reported on the development of shear-thinning hydrogels based on the guest-host interactions of adamantane modified hyaluronic acid (guest macromer, Ad-HA) and β-cyclodextrin modified hyaluronic acid (host macromer, CD-HA). Mixing of the two macromer components resulted in rapid formation of a hydrogel composed of non-covalent bonds. To illustrate ease of delivery, materials were injected into a porcine left ventricular explant via syringe (27G 1/2 in needle) or catheter (4F 60cm with fixed 21G ½ in needle). Observation by MRI (9.4T) demonstrated that materials were retained at the injection site and exhibit minimal spreading into the surrounding tissue. Hydrogel morphology is maintained at 1 week post-injection with minimal swelling (24.4±2.1%) as determined from 3D MRI reconstructions. These results demonstrate the utility of guest-host hydrogels developed as injectable materials for therapeutic interventions.
In addition to the ease of initial delivery mechanism, the properties of the hydrogel following injection are of great interest as they may be highly application dependent. Shear-thinning hydrogels may be limited in this respect, as the physical crosslinking mechanisms typically result in hydrogels having low mechanical strength. Thus, we have engineered the shear-thinning HA hydrogels with the addition of secondary covalent crosslinking via a Michael-addition reaction. To afford covalent crosslinking, HA was modified by addition of thiol groups or various Michael-acceptors: methacrylates (Me), acrylates (A), vinyl sulfones (VS), or maleimides (Ma). To identify macromers and conditions with gelation on timescales necessary for ease of delivery, crosslinking kinetics were monitored by real-time rheological time-sweeps (1Hz, 0.5%strain) following mixing of the two macromers. Gel times were observed to decrease with reactivity of the Michael-acceptor (Ma

Heart disease is a major clinical problem in the United States and post myocardial infarction (MI), left ventricular (LV) remodeling ensues and leads to geometric changes that result in dilation and thinning of the myocardial wall. This increases stress in the infarct and healthy tissue and can ultimately result in heart failure. Injectable biomaterials are being investigated to address this clinical problem, including to alter stresses in the infarct region when injected as an array and to deliver biologics, such as stem cells and biomolecules. We are interested in a class of hydrogels based on the molecule hyaluronic acid (HA). HA is found during cardiac development and is involved in numerous biological functions, such as morphogenesis and wound healing, and importantly, can be modified with reactive groups (e.g., methacrylates) to form hydrogels. We have synthesized variations of HA macromers that form hydrogels with a range of mechanical properties and degradation (from a few weeks to stable over many months) and that form hydrogels through either photoinitiated or red-dox initiated radical polymerizations. This tunability in properties allows us to investigate how material properties (e.g., mechanics and degradation) influence the ability of injectable HA hydrogels to alter stress profiles and LV remodeling and to deliver therapeutic molecules (e.g., stromal-derived factor 1-alpha and TIMP-3, to alter progenitor cell homing to and matrix remodeling within infarcts, respectively). Furthermore, we are now designing self-assembling and shear-thinning hydrogels for percutaneous delivery to the heart. Our ability to design materials with controlled properties and degradation is allowing us to investigate how engineered hydrogels can be used to alter cardiac outcomes by adjusting endogenous signals. Overall, these hydrogels provide us the opportunity to investigate diverse and controlled material properties for a range of biomedical applications.

Electrospun nanofibers have shown promise as tissue engineering scaffolds to replicate the size, mechanical structure, and anisotropy found in native extracellular matrix (ECM); however, there has been little work towards engineering nanofibrous scaffolds that degrade through proteases involved in the cell-mediated degradation of native ECM such as matrix metalloproteinases (MMPs). In particular, recent studies with MMP-sensitive hydrogels have demonstrated the importance of MMP susceptibility in temporally modulating cell-material interactions. Thus, incorporating MMP sensitivity into a synthetic macromer capable of nanofiber formation would allow for tissue engineering scaffolds that mimic ECM structural features and biologically relevant degradation mechanisms in response to MMPs.
Towards this goal, we introduce a modified hyaluronic acid (HA, a linear polysaccharide found throughout the ECM that is known to influence cell behavior) macromer, Methacrylated Peptide HA (MePHA), that is both photopolymerizable and MMP sensitive to create nanofibrous scaffolds. Synthetic peptides with MMP sensitivity were functionalized with a photopolymerizable methacrylate group and conjugated to a maleimide functionalized HA backbone to yield the final macromer, MePHA. In bulk hydrogel studies, rheology data indicated that gelation occurs within 5 minutes when 2-3 wt% MePHA solutions were exposed to UV light (365 nm) in the presence of a photoinitiator (I2959) with maximum storage moduli ranging from 1-6kPa, depending on the degree of HA functionalization, intensity of UV light, and wt% of MePHA. For fiber formation, a 1 wt% solution of MePHA with 4.5% polyethylene oxide as a carrier polymer was electrospun into a dry mat and crosslinked with exposure to UV light in the presence of I2959. To visualize hydrated fibers, fluorescent peptides were synthesized and also conjugated to the macromer prior to electrospinning. Dry fibers were 0.32 ± 0.1 mu;m in diameter, regular in shape, and swelled to 2.0 ± 0.5 mu;m upon placement in TTC buffer (TritonX-100: Tris-HCl: CaCl2). Electrospun scaffolds (1mm thick) were swollen and equilibrated in TTC buffer and then placed into either 500 U/ml Type II Collagenase or TTC buffer. Scaffolds degraded in response to Collagenase within 14 days while scaffolds retained over 80% of their mass within TTC Buffer alone. Furthermore, scaffolds were modified with RGD peptides and supported the adhesion of endothelial cells with greater than 84% viability after one week of culture.
Here, we developed an MMP sensitive, photopolymerizable scaffold that can be further tuned to alter mechanics (degree of crosslinking), cellular adhesion sites (RGD conjugation), and fiber alignment (rate of collection on spinning mandrel). These tunable properties are important in applications exploring cellular morphogenesis as a function of scaffold biochemical and biophysical properties.

Introduction. Smooth muscle cells (SMCs) play important roles in maintaining structures and functions of various tissues including urethra and blood vessels. Cell-based therapy provide a promising strategy for repairing lost smooth muscle tissues, however, injecting cells alone often suffer from low viability and engraftment efficiency. Hydrogels could provide an injectable cell delivery matrices to enhance cell viability, and provide physical and chemical cues to promote desirable cellular fates after delivery. The goal of this study is to develop an injectable and in situ crosslinkable hydrogel platform with muscle tissue-mimicking niche cues for delivering cells to repair smooth muscle tissues.
Materials and Methods. To allow in situ crosslinking of hydrogels, we chose thio-ene michael addition which supports hydrogel formation under physiological conditions without any catalyst. To control the mechanical cues of hydrogel matrix, multi-arm PEG with acrylate or thiol end groups was used. To support cell adhesion, gelatin, a digested form of collagen, was modified with acrylate groups to allow chemical crosslinking with PEG network. Smooth muscle cells (human aortic, Invitrogen) were encapsulated in hydrogels at the concentration of 2.5 million cells/ml, and cultured in growth medium for 3 weeks before analyses. Outcomes were analyzed using mechanical testing, live-dead assay, cell proliferation, bioluminescence imaging and gene expression.
Result and Discussion. By changing the molecular weight between crosslink points, the mechanical stiffness of the hydrogel could be changed from 15 kPa to 25 kPa, which mimics muscle tissues. Live/dead staining showed high cell viability after gelation. Hydrogels supported SMC cells to retain their phenotype and maturation in 3D culture, as shown by increased smooth muscle cell gene expression and retained MMP activity. Hydrogels also supported SMC cell proliferation, and DNA content increased 30% over 21 days of culture. Furthermore, hydrogel encapsulation did not interfere with GFP/luciferase expression by the cells, which support tracking cell in vivo post-injection. In sum, here we report the development of an injectable and in situ crosslinkable hydrogel platform with muscle tissue-mimicking niche cues, and can be used for delivering cells to aid regeneration of lost smooth muscle tissues.

In the search of synthetic materials to mimic the extracellular matrix hydrogel-based materials have widely been investigated due to their excellent biocompatibility in terms of their physical and chemical properties. Elastin gives connective tissue its elastic properties. The elastin structure is composed of elastic protein fibers, which are rich in hydrophobic amino acids and are cross-linked by the reaction of lysine side chains, which form the desmosine core. In this work we designed a short, rigid cross-linker to form elastic hydrogels inspired by elastin and its natural cross-linker desmosine. We chose hyaluronan as our hydrogel backbone, which unlike elastin, is a readily available and easily modifiable biopolymer found in all connective tissues. The developed new class of cross-linkers mimics key features of desmosine such as the pyridinium core structure with the positive charge, and the connectivity between the backbone in elastin. The obtained semi-synthetic hydrogels are form-stable, can withstand repeated stress, and have a large linear-elastic range characteristic for rubbery materials and elastin networks. Further these gels show strain stiffening at higher stress, a characteristic of many other biopolymers such as actin, collagen and fibrin. The stiffness, swelling ratio and bioactivity of these hydrogels can be tuned by changing the cross-linking density and the charge on the cross-linker over a range, which is interesting for soft tissue engineering. Finally these hydrogels are also stable for a long time under cell culture conditions and have good biocompatibility.

Advances in the fields of micro- and nanoscale technologies have attracted significant attention for a wide range of applications from biomedicine to tissue engineering and stem cell bioengineering. Moreover, using these technologies, it has been possible to develop microengineered three dimensional (3D) in vitro tissue constructs for drug and cytotoxicity testing, disease modeling and high throughput cell based screening. To date, a wide variety of hydrogel-based biomaterials mimicking the biological cues of natural matrices, have been developed for cell and tissue based bioengineering applications. Engineering the physical and biochemical properties of these biomaterials have been paramount interest to fabricate biocompatible scaffolds for specific tissue engineering applications. In the past few years, our lab has been actively involved in a wide spectrum of research areas from biomaterials design to microfabrication and tissue engineering. We have developed highly interdisciplinary strategies to integrate biomimetic features within hydrogel-based biomaterials using micro- and nanoscale technologies to address the major challenges in tissue engineering. In this talk, I will outline our work in modulating cell-microenvironment interactions within microfabricated hydrogel constructs for numerous tissue engineering applications to mimic the physiological, structural, physical, and topographical features of the native tissues. Our approach is mainly based on “bottom-up” and “top-down” microfabrication processes, to tailor the properties of hydrogel constructs. Bottom-up strategies rely on small, cell-laden microgel modules that are assembled into larger constructs, whereas top-down strategies are typically centered on cell seeded and encapsulated porous hydrogel scaffold to form an extracellular matrix like microenvironment and create biomimetic vascularized structures. We have also developed nano- and biotechnological approaches to engineer tissue constructs and bioactuators with improved structural, electrical, and mechanical integrity using nanomaterials and bioinspired elastomeric hydrogels. In this talk, I will outline the recent findings in our lab covering both biomaterials design and tissue engineering aspects. I will also discuss our major biological findings related to tissue engineering and stem cell differentiation using miniaturized systems.

Tissue engineering may be a powerful approach to repair osteochondral defects - damage extending from cartilage to subchondral bone. Hydrogels have been widely used a 3-D scaffolds to create an environment in which living cells can attach, proliferate, differentiate and produce new extracellular matrix. In a “materials-guided” tissue engineering approach, the scaffold&’s chemical and physical properties alone guide cell behavior. In this work, we prepared a hydrogel scaffold system by combining an inorganic, hydrophobic macromer [methacrylated star polydimethylsiloxane; PDMSstar-MA] and an organic macromer [poly(ethylene glycol) diacrylate; PEG-DA]. We observed that PDMSstar-PEG hydrogels were bioactive and osteoinductive. In addition, several parameters were tuned to tailor chemical and physical properties. First, the total concentration and wt% ratio of the two macromers were varied. Second, the use of solvent induced phase separation (SIPS) to alter PDMS distribution and other scaffold properties was explored. Poration of SIPS hydrogels was also studied. Finally, to recapitulate the interfacial nature of osteochondral tissue, the hydrogels were prepared as continuous gradients. The impact of composition and fabrication variables on morphology, PMDS distribution, hydration, modulus and bioactivity were evaluated. Based on their tunable properties, these PDMSstar-PEG scaffolds are predicted to be useful for osteochondral regeneration.

One of the key barriers in engineering stem cell based tissue in vitro is to mimic and understand the cell environment in vivo taking into account the dynamic interplay between cells and the extracellular matrix (ECM). Laminin-111 is among the first ECM proteins expressed during early embryogenesis, and together with collagen-IV, nidogen, perlecan and other proteins it is assembled into the basement membrane, where it provides not only physical support, but also the capacity to modulate ECM function when modified by other proteins. The interactions between matrix metalloproteinases (MMPs) and ECM proteins have been extensively studied in the past few decades. The cellular expression of MMPs is precisely regulated such that ECM molecules are processed at different cell stages. Recent insights into the existence of “cryptic” ECM interaction sites, sequences usually hidden within the tertiary structure of the protein, have enabled a greater understanding of how cells interact with the ECM. The ECM can no longer be thought of as just a passive scaffold and physical support for cells in vivo or as a more or less adequate cell culture substrate in vitro, the ECM and its modification is now known to act as one of the key constituents in cell regulation. Therefore, to successfully differentiate cells in vitro, it is necessary to consider cell-ECM interactions. In this work, we address the question of whether MMP2 modifies laminin-111 and whether such modifications act to regulate cell behaviour like the epithelial-to-mesenchymal transition (EMT). The EMT is an essential process in a variety of different cellular changes such as phenotype and migratory capacity, and most importantly is responsible for the formation of fibrotic tissue. Here we report a biologically active Laminin-111 fragment generated by MMP2 processing, which is not active in pluripotent cells, but highly up-regulated during differentiation. We show that the fragment binds cells via α3β1-integrins, thereby triggering the down-regulation of MMP2 in mouse and human embryonic stem cells (ESCs). Additionally, the expression of MMP9 and E-cadherin is changed in mouse ESCs; key players in the EMT. This study reveals a novel role of Laminin-111 in early stem cell differentiation that goes far beyond basement membrane assembly and a new mechanism by which an MMP2-cleaved laminin fragment regulates the expression of E-cadherin, MMP2 and MMP9. This fragment can be incorporated into materials thereby modulating the EMT during cell differentiation.

Engineered tissue with a dense extracellular matrix, such as bone or cartilage, can exhibit cell necrosis at the centre of the tissue mass. This is attributed to restricted oxygen diffusion, and as a result, the problem is exacerbated when engineering large, clinically relevant tissue constructs. We have addressed this issue through the development of a novel cell-functionalisation technology based on the construction of hybrid protein-surfactant nanoconstructs. Specifically, we have primed adult human mesenchymal stem cells (hMSCs) with myoglobin to act as an oxygen reservoir capable of alleviating hypoxic stress during the engineering of large cartilage constructs.
The nanoconstructs exhibited near-native protein structure and function, while the amphiphilic surfactant corona facilitated the association of the bioconjugate with the cytoplasmic membrane of the hMSCs. The process is non-cytotoxic, the cells remain labelled for up to a week in culture, and retain their ability to proliferate and differentiate. We primed a population of hMSCs with myoglobin-surfactant nanoconstructs and used the functionalised cells to engineer large constructs (5 mm x 6 mm) of hyaline cartilage. When compared to control samples grown from untreated stem cells, the myoglobin-primed cells produced tissue constructs with a superior biochemical composition, and crucially, a significant reduction in cell necrosis and tissue degradation at the centre of the engineered cartilage.

Control over the presence of biologics (e.g. stem cells, growth factors) is a common theme in natural tissue formation, and also an emerging theme in functional tissue engineering strategies. However, a persistent challenge in tissue engineering approaches has been to effectively incorporate biologics into tissue engineering devices while maintaining optimal physical and chemical properties of the device. In particular, there is often a design trade-off between effective biologic delivery and optimal scaffold physicochemical properties. This talk will present a series of coating strategies we have used to deliver genes, growth factors, and stem cells from tissue engineering devices. Fundamental mechanisms of affinity binding are used to stabilize incorporated biologics and enable uniquely high stability and biological activity. Controllable nucleation and growth of coatings also allow for spatial and temporal control over biologic delivery. In addition, coatings can be formed on a variety of tissue engineering devices, ranging from custom-designed macroporous scaffolds to injectable microparticles. Importantly, these coatings can be independently optimized for intended biologic delivery without influencing bulk properties of the underlying device. This “modular” approach results in devices that have optimized physical and biochemical properties from the macroscopic scale to the molecular scale. Our recent studies also demonstrate that enhanced throughput, array-based strategies can “select” coating chemistries for specific biological or biomedical goals. Examples include coatings that optimize long-term protein stabilization, autologous biologic capture, stem cell differentiation, and non-viral transfection.

3D cell culture strategies have been developed to bridge the gap between 2D cell culture and animal studies in the past two decades since 3D culture approaches enable abundant cell-cell and cell-ECM interactions that closely mimic the in vivo cell milieu. Importantly, aggregate culture of stem cells shows promise for application in tissue engineering, where the aggregates can be used as building blocks for tissue regeneration. However, control over stem cell differentiation within aggregates still remains challenging due to the inherent diffusion barrier for biological mediators (e.g. growth factors) throughout the 3D multicellular aggregates. Non-viral transfection approaches are an attractive approach to control stem cell fate, as it is possible to deliver genes encoding key biological mediators. Although initial successes of cell transfection have been achieved on 2D culture systems using nanoparticulate vectors (e.g. liposomes, polyplexes), it is difficult to translate these delivery systems from 2D to 3D stem cell culture systems. The goal of this work was to develop a microparticle based gene delivery approach to transfect stem cells within multicellular aggregates with high efficiency and uniformity throughout the aggregates. We hypothesize that incorporation of plasmid DNA (pDNA) laden mineral coated microparticles (MCMs) into hMSC aggregates would enable efficient pDNA delivery to human mesenchymal stem cells (hMSC), resulting in efficient cell transfection within the 3D aggregates. We show that the transfection efficiency increased 4-fold in the hMSC aggregates incorporated with pDNA bound MCMs when compared to standard protocols for non-viral transfection. The distribution of transfected cells was found to be homogenous throughout the stem cell aggregates. Finally, we used the microparticle approach to deliver a gene encoding the functional growth factor bone morphogenetic protein-2 (BMP-2) to induce osteogenic differentiation within the aggregates. The technology described herein may have utility as a tool to achieve scaffold-free 3D cell transfection with high efficiency and uniformity.

Translating information from two-dimensional (2D) in vitro experiments into three-dimensional (3D) systems has been a major hurdle in the use of biopolymers for tissue repair applications. In order to design improved culture environments and responsive architectures for neuronal repair, our goal is to advance the understanding of how cells of the nervous system respond to 3D environments. We have demonstrated that a 3D culture platform can invoke characteristic transformations of neuronal and glial morphology, overcoming the loss of these essential cellular milestones that occurs on 2D substrates. We demonstrate that growth factor and integrin signaling as well as extracellular matrix synthesis are drastically altered in 3D vs 2D cultures with 3D more closely mimicking the in vivo scenario. These findings have been integrated into the design of synthetic hydrogel systems with defined cell-scaffold, solute-scaffold and degradation characteristics. We further demonstrate that the synthetic hydrogels can be used to direct neural progenitor cell differentiation in materials designed for cell transplantation into sites of neuronal injury or degeneration.

Interactions between mesenchymal stem cells and extracellular matrix have a regulatory function on differentiation of multipotent stem cells. Cues presented by native matrix can be exploited to design engineered scaffolds to induce in vitro differentiation. Hyaluronic acid is a native component of cartilage and mesenchymal stem cells interact via several cell surface receptors with hyaluronic acid. These interactions play role in cellular signaling and subsequent cartilage tissue development. Here, first we design multivalent glyconanostructures using high-aspect-ratio self-assembled peptide nanofibers emulating hyaluronic acid function. Then, in vitro chondrogenic differentiation was analyzed on hyaluronic acid mimetic peptide nanofibers. Our results showed that peptide nanofibers induced early chondrogenic differentiation with respect to control groups. This early chondrogenesis was achieved through CD44, a primary receptor for hyaluronic acid, revealed by antibody inhibition assay. These results show that hyaluronic acid mimetic peptide nanofiber system provides a promising platform for stem-cell based cartilage regeneration.

Scaffolding for engineered biological tissues is formed by polymeric fibers arranged in multilayers. Understanding microstructure-property relationships of such multilayers is critical to developing reliable biological tissues for many applications, including heart valves repair. In this presentation, we address two issues. First, we develop a simple equation relating the fiber density and orientation density function to the linear and areal node densities. These relationships are then used to develop a random walk algorithm that allows one to construct geometric models for fibrous microstructures with desired geometric properties. Second, we examine microstructural evolution under various prescribed deformation states and identify key microstructural parameters governing the mechanical response.

The extracellular matrix (ECM) is a nanofibrillar network of proteins and other molecules that mechanically integrates cells into tissues and acts as an insoluble signaling network. Recent work has demonstrated that decellularized ECM from different organs can serve as a scaffold to regrow tissues from dissociated cells by providing instructive cues. This impressive result has spurred substantial research in this area. However, this is a top-down approach, requiring an existing organ to be decellularized first. We asked, why not build the ECM from the bottom-up just like cells do during embryogenesis or wound healing?
To do this, we have developed a range of fabrication strategies to engineer ECM protein-based materials from the nanometer to centimeter length-scales. At the nanometer scale we have developed a biomimetic process that recapitulates aspects of receptor-mediated ECM assembly in cells. Termed surface-initiated assembly, we can be build nanofibers and 2-dimensional (2D) basement membrane-like sheets of ECM composed of fibronectin, laminin, collagen type I and/or collagen type IV. Using soft lithography techniques, we have precise control over fiber dimensions and composition. At the micrometer to millimeter scale we have developed a micromolding technique to create fibrillar scaffolds from fibrin, collagen, alginate and hyaluronic acid, which can be integrated with the ECM protein nanofibers to create hierarchically structured scaffolds. At the millimeter to centimeter scale we have devised a new 3D bioprinting approach that enables soft hydrogels based on ECM proteins and polysaccharides to be formed into complex 3D structures such as blood vessels. In combination, these approaches enable the multiscale engineering of ECM scaffolds, which we are currently developing to (i) study basic biomechanics and mechanobiology of the ECM, (ii) fabricate 2D and 3D protein scaffolds and (iii) applying these scaffolds in cardiac and ophthalmic tissue engineering applications. To rapidly evolve our designs, we are using confocal and multiphoton imaging to create 3D computational models of the ECM structure and composition in tissues that we hope to recreate and using these to directly fabricate biomimetic scaffolds. For example, we are using the ECM in the embryonic heart as a template for developmentally-staged fibronectin scaffolds for cardiac tissue engineering. Similarly, we are using the ECM in Descemet&’s Membrane in the cornea as a template for bioengineering a corneal endothelium suitable for keratoplasty. In summary, we have implemented a range of technologies that enable us to start to put the ECM back together from the bottom-up and build ever more complex ECM structures from simple 1D nanofibers, to 2D basement membranes to 3D matrices. This compliments the top-down approach used for ECM scaffolds from decellularized organs by providing a reductionist system where complexity can be engineered back into the system.

Cell-surface interactions are influenced by various factors and two of those are surface toughness and surface nano-/micro-topography. Concerning surface topography, materials are often used which are considered very rigid as far as cells are concerned. It is known that cell-surface interactions and cellular behavior like proliferation and alignment greatly depends on the stiffness of the surface and hence nanostructures cannot just be used in combination with “hard” surfaces. Translation of nanostructures from “hard” materials to “soft” materials is therefore an important approach to further investigate “fine-tuning” and controlling of cell-surface interactions and cellular behavior. Here we use a lithography-free fabrication of nanostructured multi-responsive hydrogel surfaces. We use Poly(dimethylsiloxane) (PDMS) wrinkled surfaces as a template to polymerize thermo- and pH-responsive hydrogels which are tethered covalently to the surface. N-Isopropylacrylamide (NIPAam) is used to induce the thermo responsiveness and Vinylimidazole (VIM) for pH-responsiveness. and the surface dimensions as well as the responsiveness are characterized via atomic force microscopy (AFM) in a controlled aqueous environment. These nanostructures, the hard PDMS wrinkles and the soft hydrogel-winkles are used as a template for directed cell growth. Through the different mechanical properties of the substrates, we investigate the cell-growth and alignment behavior of hard bone cells (U2OS) and softer skin cells (HskF) and correlate cell toughness to thoughness of nanostructured surfaces.

Grafting such as autograft or xenograft transplantation used in tissue engineering is often related to the limited supply and risk of disease transmittance or immune respond. Therefore, synthetic materials such as polymers, metals and etc. have been widely used as a substitution of implantation scaffolds due to excellent material properties (Wei and Ma 2004; Lee and Shin 2007). On the other hand, the hydrophobicity of certain materials limit the cell attachment and cell proliferation onto the scaffolds (Choong, Yuan et al. 2012). Therefore, immobilization of hydrophilic compound is widely performed onto the surface to improve on the functionality of the scaffold. Surface modification is done by atom-transfer-radical-polymerization (ATRP) process. In this work, novel collagen extracted from natural waste materials (fish scales and bullfrog skin) is conjugated onto polyvinylidene fluoride (PVDF) films to improve the cell-material interaction of hydrophobic PVDF and thus its biocompatibility.
Acid solubilization method was used for collagen extraction in this study. PVDF was chosen as substrate due to its excellent inert stability and the fluorine end group which could induce direct surface-initiated ATRP. The ATRP process was done by grafting of 2-hydroxyethyl methacrylate (HEMA) which is hydrophilic in nature, followed by 1,1-carbonyldiimidazole (CDI)-mediated acylation to ease the immobilization of the collagen. The protein/collagen extracted was identified by SDS-PAGE and western blot shown that collagen Type I is successfully extracted from both fish scales and bullfrog skin. For the ATRP process, attenuated total reflection infrared spectroscopy (ATR-FTIR) was done to identify the successful grafting of the pHEMA and collagen through different chemical functional group present of each complete process, followed by water contact angle (WCA) measurements to compare the change in surface hydrophilicity of PVDF samples. Subsequently, the biocompatibility of collagen-conjugated PVDF surfaces was investigated by analyzing cell attachment and cell proliferation using human umbilical vein endothelial cells (HUVECs). The improved hydrophilicity of the surface and the antifouling properties resulted in enhanced HUVEC cell attachment and cell proliferation indicating improved cell-material interactions and biocompatibility.
References:
Choong, C., S. Yuan, et al. (2012). "Optimization of poly (ε-caprolactone) surface properties for apatite formation and improved osteogenic stimulation." Journal of Biomedical Materials Research Part A 100(2): 353-361.

Lee, S.-H. and H. Shin (2007). "Matrices and scaffolds for delivery of bioactive molecules in bone and cartilage tissue engineering." Advanced Drug Delivery Reviews 59(4-5): 339-359.

Wei, G. and P. X. Ma (2004). "Structure and properties of nano-hydroxyapatite/polymer composite scaffolds for bone tissue engineering." Biomaterials 25(19): 4749-4757.

Gellan Gum (GG) hydrogels are attractive biomaterials for regenerative medicine and tissue engineering applications since they can be easily prepared by temperature decrease below critical solution temperature, and by the addition of ions. This has been advantageous for the encapsulation of viable cells within a structure that gellifies at room temperature ^[1]. However, GG hydrogels do not present cell adhesive properties ^[1] and are slowly degraded by hydrolysis. Considering these constrains, GG polymer was chemically functionalized with divinyl sulfone (DVS) groups by a one-step reaction, followed by dialysis and diethyl ether precipitation, obtaining degrees of substitution up to 95%. Divinyl sulfone functionalized gellan gum (GG-DVS) was reacted with different amounts of a thiol crosslinker sensitive to human metalloproteinase 1 (MMP-1), under physiological conditions, obtaining stable GG-DVS hydrogels with tuned mechanical properties and MMP-1 degradation kinetics. Polymer modifications were analyzed by NMR, rheology, sol fraction, swelling and MMP-1-driven degradation. In order to improve endothelial cell adhesion, thiol terminated peptides (e.g., C16 or T1) were reacted with GG-DVS. The T1 peptide exists in the third domain of the pro-angiogenic CCN1 protein and is known to mediate vascular cell adhesion, proliferation and neovascularization ^[2], while C16 is a peptide sequence which exists in laminin, which is known to promote the differentiation of cells into capillary-like structures ^[3]. In vitro studies with GG-VS hydrogels containing different crosslinker concentrations, and with either T1 or C16 at different concentrations, promoted Human Umbilical Vein Endothelial cells (HUVECs) spreading, proliferation and differentiation. Functionalized GG-based hydrogels showed tunable physical properties and degradability, as well as cell-binding properties that improved cell endothelial performance. There are promising novel materials for tissue regeneration purposes, specifically for improved angiogenesis via the direct interaction with endothelial progenitor cells either seeded or derived from the host after implantation.
[1] J. T. Oliveira, L. Martins, R. Picciochi, P. B. Malafaya, R. A. Sousa, N. M. Neves, J. F. Mano, R. L. Reis, J Biomed Mater Res A 2010, 93, 852.
[2] K. Grote, G. Salguero, M. Ballmaier, M. Dangers, H. Drexler, B. Schieffer, Blood 2007, 110, 877.
[3] M. Ponce, M. Nomizu, H. Kleinman, FASEB J. 2001, 1389.

Polydimethylsiloxane (PDMS) is a widely used biomaterial mainly due to its biocompatibility, non-toxicity and chemical resistance. It is easy to fabricate and can assume various bulk properties. Another characteristic of PDMS is its hydrophobic nature. For the purpose of using PDMS as a substrate for cellular studies for microfluidics and lab on chip devices, it is important to improve the surface hydrophilicity. In the current study, PDMS films were surface modified using various surface modification methods, resulting in three different groups (i) Plasma treatment + Polyvinyl(PVA) dip coating, (ii) Plasma treatment+ Potassium hydroxide(KOH) dip coating and (iii) Surface graft polymerization using 2-Methacryloyloxyethyl phosphorylcholine. Characterization of the modified surfaces was done by Fourier Transform Infrared Spectroscopy (FTIR), Contact angle measurement and Scanning Electron Microscopy (SEM). A protein (BSA) adsorption profile was carried out to check for protein adsorption capability of the surfaces. Subsequently, different types of cells including 293FT fibroblasts and Human umbilical vein endothelial cells (Huvecs) were cultured on these modified PDMS and studied for cell attachment and cell proliferation. Unmodified PDMS was used as a control. The Plasma treated PDMS with PVA coating showed lowest contact angle measurements. Plasma treatment is an effective yet short-lived method for surface modification. PVA coating stabilizes the hydrophilicity of the surface created by the plasma treatment. Cell culture studies showed higher cell attachment and proliferation on modified surfaces as compared to the unmodified PDMS. However, the cell proliferation profiles for both the different types of cells were distinctively different. This suggests that different types of cells behave differently even when subjected to the same environment. Overall, the surface modification of PDMS allows for the optimization of cell-material interactions.

Nanoparticulate composites of hydroxyapatite (HAp) and chitosan were synthesized by ultrasound-assisted sequential precipitation and characterized for their microstructure at the atomic scale, surface charge, drug release properties and combined antibacterial and osteogenic response. Crystallinity of HAp nanoparticles was reduced due to the interference of the surface layers of chitosan with the dissolution/reprecipitation-mediated recrystallization mechanism that conditions the transition from the as-precipitated amorphous calcium phosphate phase to the most thermodynamically stable one - HAp. Embedment of 5 - 10 nm sized, narrowly dispersed HAp nanoparticles within the polymeric matrix mitigated the burst release of the small molecule model drug, fluorescein, bound to HAp by physisorption, and promoted sustained release kinetics throughout the three weeks of release time. The addition of chitosan to the particulate drug carrier formulation, however, reduced the antibacterial efficacy against S aureus. Excellent cell spreading and proliferation of osteoblastic MC3T3-E1 cells evidenced on microscopic conglomerates of HAp nanoparticles in vitro also markedly diminished on HAp/chitosan composites. Mitochondrial dehydrogenase activity exhibited normal values only for HAp/chitosan particle concentrations of up to 2 mg/cm2 and significantly dropped, by about 50 %, at higher particle concentrations (4 and 8 mg/cm2). The gene expression of osteocalcin, a mineralization inductor, and the transcription factor Runx2 was downregulated in cells incubated in the presence of 3 mg/cm2 HAp/chitosan composite particles, while the expression of osteopontin, a potent mineralization inhibitor, was upregulated, further demonstrating the partially unfavorable osteoblastic cell response to the given particles. The peak in the expression of osteogenic markers paralleling the osteoblastic differentiation was also delayed most for the cell population incubated with HAp/chitosan particles. Overall, the positive effect of chitosan coating on the drug elution profile of HAp nanoparticles as carriers for the controlled delivery of antibiotics in the treatment of osteomyelitis was compensated for by the lower bacteriostatic efficiency and the comparatively unviable cell response to the composite material, especially at higher dosages.

Introduction
Gene therapy provides a powerful tool for enhancing tissue regeneration by upregulating desirable cellular processes. Minicircle (MC) DNA are supercoiled DNA molecules free of bacterial plasmid backbone elements, and have been reported to enhance prolonged gene expression compared to conventional plasmids. Despite the great promise of MC DNA for gene therapy, there are no methods available for scaffold-mediated delivery of MC DNA in situ. Here we report the development of a scaffold-mediated, BMP-2 MC DNA delivery platform for enhancing bone regeneration. To avoid the use of organic solvents during scaffold fabrication, we have chosen supercritical CO2 to transform poly(DL-lactide-co-glycolide) (PdLGA) into a 3D macroporous scaffold, while simultaneously encapsulating MC DNA during the fabrication process. The efficacy of scaffold-mediated delivery of BMP-2 MC DNA for enhancing in vivo bone regeneration was then examined using a mouse critical size calvarial defect model.
Materials and Methods
To encapsulate MC DNA into a scaffold, PdLGA (85:15, 70 mg) was combined with MC (160 mu;g) in a teflon mold, lyophilized, then placed under scCO2 (12.4 MPs, 35 °C) for 1 hour. In vitro release was determined by incubating MC DNA-containing scaffolds in PBS at 37 °C, and supernatant was collected regularly over 55 days. DNA release was quantified using a PicoGreen (Invitrogen) assay. For in vivo bone regeneration, scaffolds containing MC DNA encoding luciferase, GFP or BMP2 (4.5 mm diameter x 0.5 mm thick) were placed into a critical sized (4.5 mm) calvarial defect in mice. In vivo luciferase production was monitored using bioluminescence imaging up to 60 days. Bone formation was monitored using micro-CT up to 91 days. At day 91, animals were sacrificed and implants were harvested for histological analyses to evaluate bone formation.
Results and Discussion
Porous PdLGA scaffolds containing MC DNA were fabricated in a one-step process in the presence of scCO2. Our release data demonstrated sustained delivery of MC DNA in vitro for at least 55 days, and detectable luciferase protein expression in vivo for at least 60 days. When implanted in a critical size mouse calvarial defect model, PdLGA scaffolds containing BMP2 MC DNA resulted in robust bone regeneration by 12 weeks, whereas no bone formation was observed using PdLGA containing GFP MC (control). A similar trend was observed in histological analyses, with extensive mature bone formation observed throughout the scaffolds releasing BMP-2 MC, whereas only a thin fibrous capsule observed in the GFP MC group. Together, our results highlight the promise of scaffold-mediated MC delivery in situ using scCO2 for scaffold fabrication, which may provide a valuable tool for broad applications of MC DNA-based gene therapy to enhance regeneration of a broad range of tissue types.

Tissue loss or organ failure is one of the most devastating and costly problem in health care. Current treatment options for patients include organ/tissue transplantation of autografts/allografts, delivery of bioactive agents, and utilization of synthetic replacements composed of metals, polymers, and ceramics. However, each strategy suffers from a number of limitations. The early attempts to overcome these drawbacks led to the emergence of tissue engineering that provided viable tissue substitutes using a combination of biomaterials, cells, and factors. This approach was ideally suited to repair damaged tissues; however the substitution and regeneration of large tissue volumes and multi-level tissues such as complex organ systems integrated into a single phase required more than optimal combinations of biomaterials and biologics.
The future is ‘Regenerative Engineering&’ aimed at creating large and complex tissue systems incorporating the advances in material science, stem cell technology and developmental biology. We believe that recent breakthrough technologies in advanced materials science and nanotechnology have allowed us to recapitulate native tissues. The novel designer polymers provide bioactivity and physical features specific to a tissue regeneration application. Structural cues especially over the nanoscale have enabled better control over cellular behaviour. Overall, they afford selective control of cell sensitivity and temporal and spatial control of stimulatory cues. Advanced materials science is emerging as a driver of stem cells toward multiple lineages allowing distinct tissue types to regenerate next to one another. Spatiotemporal control enabled by advanced biomaterials guide tissue development through the release of varying morphogens. We aim to build multi-level systems such as organs through location-specific topographies and physico-chemical cues incorporated into a continuous phase using a combination of classical top-down tissue engineering approach with bottom-up strategies used in regenerative biology.
Musculoskeletal tissues are critical to the normal functioning of an individual and following damage or degeneration they show extremely limited endogenous regenerative capacity. This talk will cover the development of material and structural platforms to modulate stem cell behaviour to enhance tissue regeneration including hard and soft musculoskeletal tissues.

Fabricating porous structure is one of the challenges in designing materials. Porous scaffolds are attracting increasing interest in bone tissue engineering, because they can provide a favorable environment for bone ingrowth while providing a stable long-term fixation [1]. Magnesium (Mg) has been recognized as a very promising biomaterial for bone implants because of its excellent mechanical properties and its favorable characteristics of being biodegradable and bioresorbable [2]. Porous magnesium structure has the potential to serve as a degradable scaffold in bone substitute applications. However, only a few papers reported the synthesis and characterization of porous Mg and previously reported studies showed poor mechanical and corrosion properties because of low strength of magnesium and high surface area of porous structure. In our study we propose a feasible approach in which we can obtain porous Mg scaffolds with high strength and corrosion resistance by adding a reinforcing phase to form Mg composites and to coat the porous Mg scaffold with a stable materials
Mg /Alumina composites was mixed uniformly with sodium chloride powder (60vol.%) and sintered using spark plasma sintering (SPS) in low vacuum state [3]. The temperature was increased to 585°C in 5 minutes and maintained for 2 hours. After sintering, NaCl was dissolved in 1M sodium hydroxide (NaOH) solution. For the MgF2 coating process, the fabricated porous were immersed in hydrofluoric acid at room temperature for 24 h. For this purpose, biological and mechanical properties experiments are carried out by using cell tests and compression test. Significant increases in strength characteristics were observed for porous Mg by adding alumina powder. Also corrosion rate and cellular response of porous Mg was well controlled with MgF2coating by solution treatment. Our results show that Porous Mg is a potential alternative to existing load-bearing implant materials.
[1] Hollister SJ. Porous scaffold design for tissue engineering. Nature Materials 2005;4:518-524.
[2] Staiger MP, Pietak AM, Huadmai J, Dias G. Magnesium and its alloys as orthopedic biomaterials: a review. Biomaterials 2006;27:1728-1734.
[3] Kang M-H, Jung H-D, et al. Production and Bio-corrosion Resistance of Porous Magnesium with Hydroxyapatite Coating for Biomedical Applications. Materials Letters 2013;108:122-124

Micrometer-sized hydrogel particles (microgels) represent an important class of biomaterials for applications in regenerative medicine[1] and controlled therapeutic biomolecule delivery, because they have similar mechanical properties as many objects in living systems. This makes them ideal scaffolds to encapsulate, stabilize, culture, and deliver living cells, proteins, and oligonucleotides. Recently, our group reported the preparation of microgel particles that contain living cells with exquisite control through the use of droplet-based microfluidics and bio-inert polymers such as polyethyleneglycol (PEG) and hyperbranched polyglycerol (hPG). The microgel gelation is achieved via bio-orthogonal thiol-ene click reaction of dithiolated PEG macrocrosslinkers and acrylated hPG building blocks and does not require any initiator.[2] Microgel properties can be optimized to obtaining mammalian cell viabilities of up to 90%. These microgels are stable at acidic pH and show only slow degradation of several weeks at pH 7.4. However, for several applications in regenerative therapy and for efficient cell analysis stimuli-responsive microgel degradation with precise control over the release rates must be achievable to liberate encapsulated cells on demand. For this purpose, reversibly crosslinked supramolecular polymer gels are an extraordinarily interesting class of materials. We follow this approach and prepare supramolecular crosslinked cell-laden microgels that consist of PEG-bipyridine crosslinked by complexation of iron(II)-sulfate.[3] These microgels can be degraded by dilution on a timescale of hours or by addition of decomplexing agents on a timescale of minutes. Another approach to achieve microgel degradation is the controlled cleavage of covalent bonds located in the constituent polymer network. Among various hydrogel degradation mechanisms, hydrolysis at acidic pH is very promising for controlled release applications in inflamed and tumor tissue (pH <7.0). We have developed a microgel construction kit for bioorthogonal cell encapsulation by strain promoted azide-alkyne cycloaddition (SPAAC) and programmed cell release triggered by benzacetal hydrolysis.[4] The use of different substituted benzacetals as pH-cleavable linkers allow us to control the release kinetics in the interesting pH range between 6.0 and 7.4 with no effect on the excellent cell viabilities (~95%) and the cell spreading. [1] B. V. Slaughter, S. S. Khurshid, O. Z. Fisher, A. Khademhosseini, N. A. Peppas, Adv. Mater. 2009, 21, 3307-3329. [2] T. Rossow, J. A. Heyman, A. J. Ehrlicher, A. Langhoff, D. A. Weitz, R. Haag, S. Seiffert, J. Am. Chem. Soc. 2012, 134, 4983minus;4989. [3] T. Rossow, S. Bayer, R. Albrecht, C. C. Tzschucke, S. Seiffert, Macromol. Rapid. Comm. 2013, 34, 1401-1407. [4] D. Steinhilber, T. Rossow, S. Wedepohl, F. Paulus, S. Seiffert, R. Haag, Angew. Chem. Int. Ed. 2013, DOI: 10.1002/anie.201308005; Angew. Chem. 2013, DOI: 10.1002/ange.201308005.

While bioelectric currents have been demonstrated to have roles in directing cell migration¹ and mesenchymal stem cell differentiation², their potential use in tissue engineering applications remain relatively untapped. We have fabricated electroactive polycaprolactone (PCL)-based substrates by coating heparin-doped polypyrrole (PPy) onto electrospun PCL nanofibrous meshes via in situ template polymerization. This thin coating of PPy-heparin was verified by EDX analysis, morphological examination under the SEM and the toluidine blue assay. Electrical conductivity and mechanical properties conferred by heparin-doped PPy onto the PCL nanofibers were significantly higher than undoped, camphorsulfonic acid (CSA)-doped or chloride-doped PPy. The use of heparin dopant in PPy not only enhanced the electroactivity of PPy, but also allowed utilization of its bioactive properties in vascular tissue engineering. On PCL substrates conjugated with heparin, endothelial cell proliferation was enhanced and plasma recalcification times demonstrated anti-coagulative properties. As heparin is a binding partner of vascular endothelial growth factor (VEGF)³, our hybrid electroactive scaffold can also be developed into an electronically-controlled smart scaffold for VEGF release.
1. Zhao M. et al; Nature. 442(7101), 457, 2006
2. Creecy CM. et al; Tissue Engineering Part A, 2012
3. Martino MM. et al; PNAS. 110(12), 4563, 2013

Osteoarthritis (OA) is a debilitating disease that affects the articular cartilage (AC) and the underlying bone in joints. It is the leading cause of both disability and chronic pain in elderly people, with approximately 35 to 40 million Europeans currently affected with OA. Trauma and abnormal joint loading also contribute to AC defects. Due to the limited self-repair ability of adult AC, defects that are not treated properly can develop into secondary (late-stage) OA. Numerous surgical interventions and biomaterials have been introduced over the last decades but none has been successful in restoring the structure, composition and biomechanical function of AC to its native state. Our research aims to provide a long-term solution for the treatment of AC defects by investigating a temporary structural support that is able to sustain in vivo joint loading forces, and hence, assist the body to regenerate functional competent AC. Three dimensional scaffolds were produced by electrospinning biocompatible polyhydroxyalkanoates blends, i.e. poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) [P(3HB)/P(3HO)] into nanometre-sized fibrils that exhibit a similar structure compared to the collagen fibril meshwork in native cartilage. Structure of the electrospun P(3HB)/P(3HO) fibrils was stable in liquid condition due to “pseudo crosslinking” represented by the hydrogen bonding formed between the included water molecules and carbonyl groups of the polyhydroxyalkanoates backbone. The stiffness of P(3HB)/P(3HO) nanofibrous scaffolds can be adjusted by controlling the composition of P(3HB) and P(3HO) in the polymer blends. Indentation-type atomic force microscopy (IT-AFM) employing hard borosilicate glass spherical probes revealed a stiffness of the P(3HB)/P(3HO) nanofibrous scaffold of 0.87 ± 0.54 MPa for a ratio of P(3HB)/P(3HO) = 1:0.25, which is a factor of two lower compared to the stiffness of 1.86 ± 0.50 MPa of native AC and needs to be further optimised. After culturing the scaffold with human articular chondrocytes for 3 weeks, the stiffness of the construct increased to 1.06 ± 0.60 MPa. Matrix formation within the construct was confirmed by Alcian blue and Sirius red staining of the proteoglycan and total collagen content, respectively. Higher total collagen and less proteoglycan contents were produced on the stiffer P(3HB)/P(3HO) scaffolds compared to those on the softer scaffolds. The nanofibrous structure provides a large surface area that promotes good chondrocytes attachment and the cells were observed to successfully penetrate into the voids between fibrils. A major advantage of employing polyhydroxyalkanoates blends compared to similar approaches in cartilage repair is that the polymer scaffolds are biodegradable, which allows ingrowth and formation of new cartilage into the defect site. Our future work is to investigate the in vivo performance of these constructs by implanting them into the joints of immunosuppressed mice.

The concept of bone replacement has been dominant in the development of metallic implants and the biodegradable metals with the potential to stimulate bone formation have recently captured the attention of scientists. Among these metals, magnesium alloy is an attractive biodegradable material with unique set of properties. The corrosion of magnesium is accompanied by hydrogen evolution and a local increase in pH, which impose constraints on many potential biomedical applications. We have overcome these limitations and created a road map to the next generation of metallic biodegradable implant materials with the addition of completely biocompatible elements. Along with the addition of Ca, which is a biocompatible element that plays major in bone formation and remodeling, excellent material properties were achieved through the in-house built special mechanical extrusion machine. The state of the art method to synchronize the corrosion potentials of two constituent phases (Mg + Mg2Ca) with the selective doping of Zn into Mg2Ca was developed to control the corrosion rate. Furthermore, mechanical extrusion broke the connectivity of the Mg2Ca phases, which prevented continuous corrosion and the formation of a galvanic circuit that caused severe corrosion of the Mg-Ca alloy. Animal studies confirmed the large reduction in hydrogen evolution and revealed good tissue compatibility with increased bone deposition around the newly developed Mg alloy implants. Newly developed set of KIST magnesium alloys have the mechanical strength, ability to stimulate bone growth and controlled slow degradation rate to be considered as an ideal candidate for biodegradable implant applications.

With all the extensive in vivo experience using developed magnesium alloy material over the past 3 years, we were able to receive an approval from Korea Food and Drug Administration (KFDA) for the first human clinical trial of orthopedic biodegradable metallic implant devices. Working closely with major hospitals in Korea, we have performed several cases of small bone fixation screws so far and the results seem promising.

A major challenge in tissue regeneration is to direct the assembly of cells and their extracellular matrix (ECM) into arrangements that possess the unique physical and mechanical properties of the native tissue. We have developed a unique template that directs alignment of confluent cells and ECM across an entire two-dimensional surface. The template is a reactive, two-component interface that is synthesized in three steps in nanometer thick, micron-scaled patterns on silicon and on several biomaterial polymers. In this method, a volatile zirconium alkoxide complex is first deposited at reduced pressure onto a surface pattern that is prepared by photolithography; the substrate is then heated to thermolyze the organic ligands to form surface-bound zirconium oxide patterns. The thickness of this oxide layer ranges from 10 to 70 nanometers, which is controlled by alkoxide complex deposition time. The oxide layer is treated with 1,4-butanediphosphonic acid to give a patterned monolayer whose composition and spatial conformity to the photolithographic mask are determined spectroscopically. NIH 3T3 fibroblasts and human bone marrow-derived mesenchymal stem cells attach and spread in alignment with the pattern and maintain alignment as they grow to form a confluent monolayer across the entire substrate surface. Confluent cells assemble an ECM, in which the fibronectin fibrils are highly aligned. Decellularization yields a composite material with spatially aligned matrix attached to a synthetic polymer surface. We illustrate biologic function by oriented neurite outgrowth along the aligned matrix fibrils. Our results support the goal of developing aligned matrices to serve as platforms for integrating directed cell behavior with synthetic devices. This combination of simple chemistry with autologous stem cells could be a powerful tool for generating a naturally assembled ECM for spatially predetermined tissue repair.

A single pulse of 1.5 kJ/0.7g of atomized spherical Ti powders from 300 mF capacitor was applied to produce a microporous-surfaced Ti implant compact by electro-discharge-sintering (EDS). A solid core surrounded by microporous layer was self-consolidated by a discharge in the middle of the compact. Coatings with hydroxyapatite (HA) on the microporous Ti compact were first conducted by electrostatic-spray-deposition (ESD). The precursor solution for the HA coating was prepared by mixing nano-scaled HA powders with ethyl alcohol. As-deposited HA coating was in the form of porous structure and consisted of HA particles which were uniformly distributed on the Ti compact. By heat-treatment at 700 °C, HA particles were agglomerated each other and melted to form a highly smooth and homogeneous HA thin film consisted of equiaxed sub-micro grains. Microporous-surfaced Ti implant compacts coated with highly crystalline apatite phase were successfully obtained by using the EDS and ESD.

Thermoresponsive shape memory polymers (SMPs) are stimuli-sensitive materials that return to their permanent shape from a temporary shape in response to heat. Porous SMP foams exhibit distinct properties that make them well suited for certain applications in the biomedical field. Yet, current SMP foams are restricted to a limited group of organic polymer systems. In this study, porous biodegradable inorganic-organic scaffolds are prepared via rapid photochemical cure of a macromer comprised of poly(dimethyl siloxane) (PDMS) and organic poly(ε-caprolactone) (PCL) segments, diacrylated PCLn-block-PDMSm-block-PCLn. High pore interconnectivity is achieved via a revised solvent-casting/particulate-leaching (SCPL) method in which the precursor solution is crosslinked over a fused NaCl template. By varying macromer concentration, salt size, and percent salt fusion, the impact on pore size, porosity, shape memory ability, compressive modulus and degradation will be systematically studied. We anticipate that these SMP foams could be used as self-fitting bone defect scaffolds, as they could be triggered to expand and conformably fit into bone defects with the application of warm saline before cooling and hardening at body temperature.

Recent decades have seen a huge turn in implantology and biomaterial development towards regenerative medicine. The approach in orthopedic surgery is no longer to just replace damaged tissue by a passive implant that evokes the least possible interference with biological tissue, but rather to provide active stimulation and actuation. FePd ferromagnetic shape memory alloys are promising smart materials with a unique set of properties. They are superelastic and respond to external magnetic fields with deformations and strains up to 5%. Additionally, they are very ductile and, as we showed earlier, biocompatible [1].
To determine, how cellular behavior can be mediated by common protein coatings, such as fibronectin, laminin and poly-L-lysine, we conducted in vitro experiments with NIH 3T3 fibroblasts, MCF 10A epithelial cells and primary HOB osteoblasts on functionalized vapor-deposited single crystalline Fe70Pd30 thin films. Depending on the cell type, improvement in cellular adhesion and morphological changes could be achieved.
A more detailed investigation was carried out on the amino acid sequence RGD, which is a component of fibronectin and the selective binding motif to integrin receptors on the cell surface mediating cellular adhesion. In addition to cell tests, self-designed delamination tests, in which the interaction force of RGD ligands with FePd films was probed, showed a strong link between substrate and coating. Theoretical ab initio considerations via density functional theory explain these results in a fundamental physical way: The adhesion is mainly mediated by coordinate bonds between iron atoms of the films and nitrogen/oxygen atoms of the RGD [2]. The strength of this connection exceeds the interaction forces of RGD with integrin receptors, meaning that cells are unable to peel of the coating during adhesion - an important prerequisite for sustainable surface functionalization.
[1] U. Allenstein, Y. Ma, A. Arabi-Hashemi, M. Zink, S.G. Mayr. Fe-Pd based ferromagnetic shape memory actuators for medical applications: Biocompatibility, effect of surface roughness and protein coatings. Acta Biomater. 2013.
[2] M. Zink, F. Szillat, U. Allenstein, S.G. Mayr. Interaction of Ferromagnetic Shape Memory Alloys and RGD Peptides for Mechanical Coupling to Cells: from Ab Initio Calculations to Cell Studies. Adv Funct Mater. 2012.

Patient-specific induced pluripotent stem (iPS) cell-derived cardiomyocytes (iPS-CMs) have been shown to provide valuable models of congenital cardiomyopathies with genetic mutations. Long QT syndrome type 3 (LQT3) results in delayed repolarization and increased risk of fatal arrhythmia. Developing a “disease-specific” three-dimensional (3D) cardiac model with highly controllable cellular microenvironments will offer a significant advancement for understanding disease mechanisms in vitro. Using two-photon initiated polymerization (TPIP), we created a biomimetic cardiac tissue model with a cohort of 3D filamentous matrices that precisely regulated the structural alignment of CMs and adjusted the cellular mechanical environment. The filamentous matrices were fabricated via the TPIP system based on a femtosecond laser beam irradiated vertically to the photoresist, a UV-curable organic-inorganic hybrid polymer (ORMOCER®, Micro resist technology). Fibers with diameters of 5 µm and 10 µm were created with powers of 2.6 mW for 0.7 s exposure and 6.4 mW for 1 s exposure respectively. Different fiber spacing within the matrices (25 µm, 50 µm and 75 µm) were controlled by the X-Y-Z stage with high positioning precision operated by a PC. By quantifying the cell alignment and tissue formation of iPS-CMs growing on the filamentous matrices, we found that CMs growing on 50 µm spacing matrices formed condensed and aligned 3D cardiac tissue better than the matrices with 75 µm and 25 µm spacing. Since the stiffness of 10 µm fibers (598 ± 86 MPa) was 4 times stiffer than 5 µm fiber (158 ± 9 MPa), iPS-CMs can only bend the fiber with lower stiffness. By measuring the displacement of 5 µm fibers due to the iPS-CMs contraction, we were able to calculate the contraction force of iPS-CMs (~ 35 nN) according to classical beam theory. Using such thin fibers, we were able to study the iPS-CM maturation by monitoring the increase of force generation. Using multielectrode array analysis, LQT3 iPS-CMs exhibited prolongation of field potential duration (~ 400 ms) compared to WT iPS-CMs (~ 250 ms), which verify that LQT3 iPS-CMs can faithfully recapitulate the LQT3 electrophysiological abnormality in vitro. In drug testing experiments, our findings indicated that LQT3 iPS-CMs became more susceptible to drug-induced cardiac toxicity (40% higher risk of uncoordinated beating) growing on the matrices with low stiffness compared to the ones with high stiffness and the pharmaceutical industry standard 2D surface, suggesting that there might be false-negative readouts during toxicity testing if only traditional 2D cell culture systems are used for drug screening.

Peptide amphiphiles (PA), which belong to an emerging class of molecules that have been shown to self-assemble into novel nanostructures such as nanofibers in hydrogel, are of significant interest due to their potential applications in regenerative engineering. A representative PA molecule is comprised of a hydrophobic alkyl tail, a short peptide sequence for intermolecular hydrogen bonding, a group of charged amino acids for enhanced solubility, and a region for bioactive signals to be transduced via cells or proteins. Smart biomaterials that are self-assembled from such PA molecules are known to undergo morphological transitions in response to specific physiological stimuli. Using a novel coarse-grained peptide/polymer model, which has been validated by comparison of equilibrium conformations from atomistic simulations, we have performed large-scale molecular dynamics simulations to examine the spontaneous self-assembly process. Starting from random configurations, these simulations result in the formation of nanostructures of various sizes and shapes as a function of electrostatics and temperature. At optimal conditions, the self-assembly mechanism for the formation of cylindrical nanofibers is deciphered involving a series of steps: (1) PA molecules quickly undergo micellization whose driving force is the hydrophobic interactions between alkyl tails; (2) neighboring peptide residues within a micelle engage in a slow ordering process that leads to the formation of β-sheets exposing the hydrophobic core; (3) spherical micelles merge together through an end-to-end mechanism to form cylindrical nanofibers that exhibit high structural fidelity to the proposed structure based on experimental data. As the temperature and electrostatics vary, PA molecules undergo alternative kinetic mechanisms, resulting in the formation of a wide spectrum of nanostructures. A phase diagram in the electrostatics-temperature plane is constructed delineating regions of morphological transitions in response to external stimuli. doi: 10.1002/adhm.201370049