[Effect of demineralized bone matrix modified by laminin α4 chain functional peptide on H-type angiogenesis and osteogenesis to promote bone defect repair].

OBJECTIVE:Based on the cell-extracellular matrix adhesion theory in selective cell retention (SCR) technology, demineralized bone matrix (DBM) modified by simplified polypeptide surface was designed to promote both bone regeneration and angiogenesis. METHODS:Functional peptide of α4 chains of laminin protein (LNα4), cyclic RGDfK (cRGD), and collagen-binding domain (CBD) peptides were selected. CBD-LNα4-cRGD peptide was synthesized in solid phase and modified on DBM to construct DBM/CBD-LNα4-cRGD scaffold (DBM/LN). Firstly, scanning electron microscope and laser scanning confocal microscope were used to examine the characteristics and stability of the modified scaffold. Then, the adhesion, proliferation, and tube formation properties of CBD-LNα4-cRGD peptide on endothelial progenitor cells (EPCs) were detected, respectively. Western blot method was used to verify the molecular mechanism affecting EPCs. Finally, 24 10-week-old male C57 mice were used to establish a 2-mm-length defect of femoral bone model. DBM/LN and DBM scaffolds after SCR treatment were used to repair bone defects in DBM/LN group ( n=12) and DBM group ( n=12), respectively. At 8 weeks after operation, the angiogenesis and bone regeneration ability of DBM/LN scaffolds were evaluated by X-ray film, Micro-CT, angiography, histology, and immunofluorescence staining [CD31, endomucin (Emcn), Ki67]. RESULTS:Material related tests showed that the surface of DBM/LN scaffold was rougher than DBM scaffold, but the pore diameter did not change significantly ( t=0.218, P=0.835). After SCR treatment, DBM/LN scaffold was still stable and effective. Compared with DBM scaffold, DBM/LN scaffold could adhere to more EPCs after the surface modification of CBD-LNα4-cRGD ( P<0.05), and the proliferation rate and tube formation ability increased. Western blot analysis showed that the relative expressions of VEGF, phosphorylated FAK (p-FAK), and phosphorylated ERK1/2 (p-ERK1/2) proteins were higher in DBM/LN than in DBM ( P<0.05). In the femoral bone defect model of mice, it was found that mice implanted with DBM/LN scaffold had stronger angiogenesis and bone regeneration capacity ( P<0.05), and the number of CD31 hiEmcn hi cells increased significantly ( P<0.05). CONCLUSION:DBM/LN scaffold can promote the adhesion of EPCs. Importantly, it can significantly promote the generation of H-type vessels and realize the effective coupling between angiogenesis and bone regeneration in bone defect repair.