Integrin-specific hydrogels for increased vascularization and bone regeneration of critically-sized bone defects

摘要

Introduction: Non-healing bone defects and fractures represent a serious clinical problem with over 600,000 bone replacement procedures performed each year. The lack of functional vasculature following trauma or resection of bone has emerged as a major limitation that prevents full regeneration of the defect. While numerous scaffolds have been engineered to increase regeneration of the vascular network, the majority use growth factors at supraphysiological levels which increase the cost of potential therapies. Engineering scaffolds that inherently promote revascularization based on the material's interaction with surrounding tissue can be an effective strategy for healing the defect. In this study, we investigated how integrin specificity in the biomaterial influences vascularization of defects with the hypothesis that integrin specificity will enhance vascularization. Methods: Hydrogel Synthesis: Integrin-specific hydrogels were engineered by functionalizing 4-arm PEG-maleimide macromers with either the α2β1-specific 'GFOGER' peptide or the αvβ3-targeting 'RGD' peptide followed by functionalization with vascular endothelial growth factor (VEGF) and cross-linked with a protease-degradable peptide. Vasculogenic protein analysis: Human mesenchymal stem cells (hMSCs) were encapsulated within integrin-specific hydrogels and cultured in osteogenic differentiation media for 7 days. Conditioned media was then assayed for VEGF by ELISA. Critically-sized murine radial defect: For in vivo studies, hydrogels were implanted within a 2.5 mm long critically-sized murine radial bone defect. At 8 weeks, animals were perfused with a radiopaque polymer (Microfil) and μCT analysis performed for vasculature. Bone formation was also analyzed by μCT at 4 and 8 weeks. Results and Discussion: hMSCs cultured within GFOGER-functionalized hydrogels in osteogenic differentiation media exhibited increased secretion of VEGF compared to hMSCs within RGD hydrogels (Fig. 1). In vivo, GFOGER-functionalized VEGF-free hydrogels exhibited significantly increased vascular volume (Fig. 2A), density (Fig. 2B) and resulted in a larger number (counts) of thicker blood vessels compared to RGD-functionalized VEGF-free hydrogels (Fig. 2C-D). The levels of vasculogenesis for VEGF-free GFOGER hydrogels were similar to those of RGD hydrogels delivering 50 ng of VEGF. VEGF-free GFOGER hydrogels also exhibited significantly increased bone volume at 4 and 8 weeks compared to VEGF-free RGD hydrogels (0.028 ± 0.008 mm3 vs 0.004 ± 0.001 mm3 and 0.06 ± 0.02 mm3 vs 0.01 ± 0.006 mm3 for 4 and 8 weeks respectively, mean ±SEM, p＜0.05) Conclusion: Whereas growth factors are routinely used to increase vascular regeneration in vivo, their high cost as well as their detrimental effects at supraphysiological levels demands exploration of more viable strategies. Integrin-specificity is a crucial aspect of vascularization and represents a useful tool in engineering constructs for this purpose. Cellular interaction with α2β1-specific hydrogels caused increased secretion of VEGF which con-elated with increased vascularization and bone regeneration of a bone defect in vivo. Future biomaterial should consider integrin specificity to reduce or even avoid the use of growth factors.