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

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

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

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

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

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

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