Poly(lactic-co-glycolide) polymer constructs cross-linked with human BMP-6 and VEGF protein significantly enhance rat mandible defect repair

We have previously shown that the combined delivery of mesenchymal stem cells (MSCs), vascular endothelial growth factor (VEGF) and bone morphogenetic protein 6 (BMP-6) induces significantly more bone formation than that induced by the delivery of any single factor or a combination of any two factors. We now determine whether the exogenous addition of VEGF and BMP-6 is sufficient for bone healing when MSCs are not provided. Poly(lactic-co-glycolic acid) (PLAGA) microsphere-based three-dimensional scaffolds (P) were fabricated by thermal sintering of PLAGA microspheres. The scaffolds were chemically cross-linked with 200 ng recombinant human VEGF (P-VEGF) or BMP-6 (PBMP-6) or both (PVEGF+BMP-6) by the EDC-NHS-MES method. Release of the proteins from the scaffolds was detected for 21 days in vitro which confirmed their comparable potential to supply the proteins in vivo. The scaffolds were delivered to a critical-sized mandibular defect created in 32 Sprague Dawley rats. Significant bone regeneration was observed only in rats with PVEGF+BMP-6 scaffolds at weeks 2, 8 and 12 as revealed by micro-computer tomography. Vascular ingrowth was higher in the PVEGF+BMP-6 group as seen by microfil imaging than in other groups. Trichrome staining revealed that a soft callus formed in P-VEGF, PBMP-6 and PVEGF+BMP-6 but not in P. MSCs isolated from rat femurs displayed expression of the bone-specific marker osteocalcin when cultured with P-VEGF, PBMP-6, or PVEGF+BMP-6 but not with P. Robust mineralization and increased alkaline phosphatase gene expression were seen in rat MSCs when cultured on PVEGF+BMP-6 but not on P, P-VEGF, or PBMP-6. Thus, unlike the delivery of VEGF or BMP-6 alone, the combined delivery of VEGF and BMP-6 to the bone defect significantly enhanced bone repair through the enhancement of angiogenesis and the differentiation of endogenously recruited MSCs into the bone repair site.