Geometric Mismatch Promotes Anatomic Repair in Periorbital Bony Defects in Skeletally Mature Yucatan Minipigs.

Porous tissue-engineered 3D-printed scaffolds are a compelling alternative to autografts for the treatment of large periorbital bone defects. Matching the defect-specific geometry has long been considered an optimal strategy to restore pre-injury anatomy. However, studies in large animal models have revealed that biomaterial-induced bone formation largely occurs around the scaffold periphery. Such ectopic bone formation in the periorbital region can affect vision and cause disfigurement. To enhance anatomic reconstruction, geometric mismatches are introduced in the scaffolds used to treat full thickness zygomatic defects created bilaterally in adult Yucatan minipigs. 3D-printed, anatomically-mirrored scaffolds are used in combination with autologous stromal vascular fraction of cells (SVF) for treatment. An advanced image-registration workflow is developed to quantify the post-surgical geometric mismatch and correlate it with the spatial pattern of the regenerating bone. Osteoconductive bone growth on the dorsal and ventral aspect of the defect enhances scaffold integration with the native bone while medio-lateral bone growth leads to failure of the scaffolds to integrate. A strong positive correlation is found between geometric mismatch and orthotopic bone deposition at the defect site. The data suggest that strategic mismatch >20% could improve bone scaffold design to promote enhanced regeneration, osseointegration, and long-term scaffold survivability.

3D-printed oxygen-releasing scaffolds improve bone regeneration in mice.

Low oxygen (O2) diffusion into large tissue engineered scaffolds hinders the therapeutic efficacy of transplanted cells. To overcome this, we previously studied hollow, hyperbarically-loaded microtanks (µtanks) to serve as O2 reservoirs. To adapt these for bone regeneration, we fabricated biodegradable µtanks from polyvinyl alcohol and poly (lactic-co-glycolic acid) and embedded them to form 3D-printed, porous poly-ε-caprolactone (PCL)-µtank scaffolds. PCL-µtank scaffolds were loaded with pure O2 at 300-500 psi. When placed at atmospheric pressures, the scaffolds released O2 over a period of up to 8 h. We confirmed the inhibitory effects of hypoxia on the osteogenic differentiation of human adipose-derived stem cells (hASCs and we validated that µtank-mediated transient hyperoxia had no toxic impacts on hASCs, possibly due to upregulation of endogenous antioxidant regulator genes. We assessed bone regeneration in vivo by implanting O2-loaded, hASC-seeded, PCL-µtank scaffolds into murine calvarial defects (4 mm diameters × 0.6 mm height) and subcutaneously (4 mm diameter × 8 mm height). In both cases we observed increased deposition of extracellular matrix in the O2 delivery group along with greater osteopontin coverages and higher mineral deposition. This study provides evidence that even short-term O2 delivery from PCL-µtank scaffolds may enhance hASC-mediated bone tissue regeneration.

Myoblast maturity on aligned microfiber bundles at the onset of strain application impacts myogenic outcomes.

Engineered skeletal muscle grafts may be employed in various applications including the treatment of volumetric muscle loss (VML) and pharmacological drug screening. To recapitulate the well-defined structure of native muscle, tensile strains have been applied to the grafts. In this study, we cultured C2C12 murine myoblasts on electrospun fibrin microfiber bundles for 7 days in custom-built bioreactor units and investigated the impact of strain regimen and delayed onset of tensile straining on myogenic outcomes. The substrate topography induced uniaxial alignment of cells in all (strained and unstrained) groups. The engineered grafts in strained groups were subjected to 10% strain amplitude for 6 h per day. We found that both static and cyclic uniaxial strains resulted in similar morphological and gene expression outcomes. However, relative to 0% strain groups, there were stark increases in myotube diameter, myosin heavy chain (MHC) coverage, and expression of key myogenic genes (Pax 7, Troponin, MHC I, MHC IIb, MHC IIx) only if strain was applied at Days 5-7 rather than Days 3-7. This finding suggests that a critical indicator of myogenic improvement under strain in our system is the phenotype of the cells at the onset of strain and suggests that this is a key parameter that should be considered in studies where myoblasts are subjected to biophysical stimulation to promote tissue formation. STATEMENT OF SIGNIFICANCE: This is the first report on the impact of the timing of the initial application of mechanical strain for improving the myogenic outcomes of 3D engineered skeletal muscle grafts. In this work, immature skeletal myoblasts were grown on topographically aligned, electrospun fibrin microfiber bundles and we applied 10% uniaxial static or cyclic strain. We concluded that the maturity of myoblasts prior to strain application, rather than strain waveform, was the primary predictor of improved myogenic outcomes, including myogenic gene expression and myotube morphology. Elucidating the optimal conditions for strain application is a vital step in recapitulating physiological myogenic properties in tissue engineered skeletal muscle constructs, with applications for treating volumetric muscle loss, disease modeling, and drug testing.