Hyunjoon Kong

Cell-encapsulated hydrogels with complex three-dimensional (3D) structures were fabricated from photopolymerizable poly(ethylene glycol) diacrylate (PEGDA) using modified 'top-down' and 'bottoms-up' versions of a commercially available stereolithography apparatus (SLA). Swelling and mechanical properties were measured for PEGDA hydrogels with molecular weights (M(w)) ranging from 700 to 10 000 Daltons (Da). Long-term viability of encapsulated NIH/3T3 cells was quantitatively evaluated using an MTS assay and shown to improve over 14 days by increasing the M(w) of the hydrogels. Addition of adhesive RGDS peptide sequences resulted in increased cell viability, proliferation, and spreading compared to pristine PEG hydrogels of the same M(w). Spatial 3D layer-by-layer cell patterning was successfully demonstrated, and the feasibility of depositing multiple cell types and material compositions into distinct layers was established.

It is widely assumed that coupling the degradation rate of polymers used as cell transplantation carriers to the growth rate of the developing tissue will improve its quantity or quality. To test this hypothesis, we developed alginate hydrogels with a range of degradation rates by gamma-irradiating high-molecular-weight alginate to yield polymers of various molecular weights and structures. Decreasing the size of the polymer chains increased the degradation rate in vivo, as measured by implant retrieval rates, masses, and elastic moduli. Rapidly and slowly degrading alginates, covalently modified with RGD-containing peptides to control cell behavior, were then used to investigate the effect of biodegradation rate on bone tissue development in vivo. The more rapidly degrading gels led to dramatic increases in the extent and quality of bone formation. These results indicate that biomaterial degradability is a critical design criterion for achieving optimal tissue regeneration with cell transplantation.

An 8-mm rat segmental defect model was used to evaluate quantitatively the ability of longitudinally oriented poly(L-lactide-co-D,L-lactide) scaffolds with or without growth factors to promote bone healing. BMP-2 and TGF-beta3, combined with RGD-alginate hydrogel, were co-delivered to femoral defects within the polymer scaffolds at a dose previously shown to synergistically induce ectopic mineralization. A novel modular composite implant design was used to achieve reproducible stable fixation, provide a window for longitudinal in vivo micro-CT monitoring of 3D bone ingrowth, and allow torsional biomechanical testing of functional integration. Sequential micro-CT analysis showed that bone ingrowth increased significantly between 4 and 16 weeks for the scaffold-treated defects with or without growth factors, but no increase with time was observed in empty defect controls. Treatment with scaffold alone improved defect stability at 16 weeks compared to nontreatment, but did not achieve bone union or restoration of mechanical function. Augmentation of scaffolds with BMP-2 and TGF-beta3 significantly increased bone formation at both 4 and 16 weeks compared to nontreatment, but only produced bone bridging of the defect region in two of six cases. Histological evaluation indicated that bone formed first at the periphery of the scaffolds, followed by more limited mineral deposition within the scaffold interior, suggesting that the cells participating in the initial healing response were primarily derived from periosteum. This study introduces a challenging segmental defect model that facilitates quantitative evaluation of strategies to repair critically sized bone defects. Healing of the defect region was improved by implanting structural polymeric scaffolds infused with growth factors incorporated within RGD-alginate. However, functional integration of the constructs appeared limited by continued presence of slow-degrading scaffolds and suboptimal dose or delivery of osteoinductive signals.

Mechanical stiffness and degradability are important material parameters in tissue engineering. The aim of this study was to address the hypothesis that these variables regulate the function of myoblasts cultured in 2-D and 3-D microenvironments. Development of cell-interactive alginate gels with tunable degradation rates and mechanical stiffness was established by a combination of partial oxidation and bimodal molecular weight distribution. Higher gel mechanical properties (13 to 45 kPa) increased myoblast adhesion, proliferation, and differentiation in a 2-D cell culture model. Primary mouse myoblasts were more highly responsive to this cue than the C2C12 myoblast cell line. Myoblasts were then encapsulated in gels varying in degradation rate to simultaneously investigate the effect of degradation and subsequent reduction of mechanical properties on cells in a 3-D environment. C2C12 cells in more rapidly degrading gels exhibited lower proliferation, as they exited the cell cycle to differentiate, compared to those in nondegradable gels. In contrast, mouse primary myoblasts illustrated significantly higher proliferation in degradable gels than in nondegradable gels, and exhibited minimal differentiation in either type of gel. Altogether, these studies suggest that a critical balance between material degradation rate and mechanical properties may be required to regulate formation of engineered skeletal muscle tissue, and that results obtained with the C2C12 cell line may not be predictive of the response of primary myoblasts to environmental cues. The principles delineated in these studies may be useful to tailor smart biomaterials that can be applied to many other polymeric systems and tissue types.