Induced membrane-mediated DBM in combination with PRP: an autologous bone graft substitute therapy with remarkable...

While the induced membrane technique effectively addresses bone defect reconstruction, its reliance on autografts remains a significant limitation. Building upon the well-established osteogenic properties of platelet-rich plasma (PRP) and demineralized bone matrix (DBM), this study systematically investigated their combined potential as a functional alternative to autografts within the induced membrane microenvironment.

PRP was extracted and DBM was prepared from New Zealand White rabbits. PRP gel and a composite gel incorporating DBM into PRP were subsequently formulated. The extract solutions derived from PRP, DBM, and PRP/DBM composites were, respectively, collected to induce osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Alkaline phosphatase (ALP) staining was performed on day 5, while mineralization was assessed via Alizarin red staining and osteogenic gene (OCN, OPN, Runx-2, and Col-1) mRNA expression was quantified by quantitative real-time polymerase chain reaction on Days 14 and 21. The rabbits (n = 48) were randomly assigned into four experimental groups (n = 12 per group): empty defect control (no graft material), PRP gel alone, DBM alone, and PRP/DBM composite gel graft. A 20-mm large segmental bone defect was surgically created in the bilateral radius of each animal, followed by initially filling with polymethyl methacrylate spacers to maintain the defect space. After an 8-week period, the spacers were removed, and the resulting membranous cavities were implanted with the respective graft materials according to group assignment. Bone regeneration was evaluated at 6 and 12 weeks post-grafting through radiography, micro-CT imaging, fluorescence-labeled dynamic histomorphometry, histochemical staining, and immunofluorescence analysis.

BMSCs treated with PRP/DBM composites demonstrated the most pronounced ALP activity and mineralization capacity, alongside the strongest upregulation of osteogenic genes. Minimal new bone formation, restricted primarily to the defect peripheries, was observed in both control and PRP groups at 6- and 12-week timepoints. In contrast, the DBM and PRP/DBM groups exhibited progressive, time-dependent osteogenic activity. Notably, the PRP/DBM group demonstrated superior bone regeneration, with compact trabeculae and well-organized collagen fully bridging the defect area, accompanied by robust vascularization. In comparison, the DBM group showed less mature bone formation, characterized by loose trabecular structures, immature collagen deposition, and sparse blood vessels. Furthermore, the PRP/DBM group displayed the highest mineral apposition rates and the most pronounced expression of the angiogenic marker CD31. Additionally, the PRP/DBM composite, mediated by the induced membrane, elicited stronger immunomodulatory effects within the local microenvironment and a more robust activation of the hypoxia-inducible factor/vascular endothelial growth factor pathway during bone repair compared to the other groups.

Within the induced membrane microenvironment, PRP synergistically enhances the osteogenic capacity of DBM. The resulting PRP/DBM composite represents a highly promising autologous candidate for bone graft substitution.