SLIT3/ROBO1 axis contributes to angiogenic-osteogenic coupling in endothelial progenitor cells and peripheral blood mesenchymal stem cells.

BACKGROUND: Early vascularization is one of the limitations of periodontal tissue engineering (PTE) based on mesenchymal stem cells (MSCs). Directed differentiation of endothelial progenitor cells (EPCs) into endothelial cells facilitates the osteogenic effect of MSCs. Therefore, this study constructed EPCs/peripheral blood derived-MSCs (EPCs/PBMSCs) sheets and evaluated their repair value and potential molecular mechanisms in bone regeneration. METHODS: Different ratios of EPCs and PBMSCs were co-cultured to prepare EPCs/PBMSCs sheets and the osteogenic differentiation was assessed. Exploring the bone regeneration properties of EPCs/PBMSC sheets in an animal model of alveolar bone defects. The effect of the SLIT3/ROBO1 axis on angiogenic-osteogenic coupling of EPCs/PBMSCs sheets was explored using exogenous modulation by shRNA lentivirus and neutralizing antibody. RESULTS: EPCs/PBMSCs sheets could form angiogenic-osteogenic coupling, and different ratios of EPCs/PBMSCs sheets had higher angiogenic and osteogenic differentiation properties than EPCs or PBMSCs alone, especially the ratio 4:6. Moreover, EPCs/PBMSCs sheets accelerated bone regeneration in the alveolar bone defect model and the treatment was superior to PBMSCs alone. The expression patterns of SLIT3 and ROBO1 were consistent with the angiogenic-osteogenic coupling of EPCs/PBMSCs sheets. Knockdown of SLIT3 in PBMSCs and/or neutralization of ROBO1 protein in EPCs effectively suppressed calcified nodule formation and markers expression of osteogenic differentiation and angiogenesis (ALP, RUNX2, OCN, Osx, EMCN, and CD31) in EPCs/PBMSCs sheets, and hindered its therapeutic effect in the alveolar bone defect model. CONCLUSION: EPCs/PBMSCs sheets ameliorate the limitations of early vascularization in PTE and the SLIT3/ROBO1 axis mediates the angiogenic-osteogenic coupling of EPCs/PBMSCs sheets, thereby augmenting their osteogenic effects.