"Introduction. Coupling of angiogenesis and osteogenesis is crucial to generate vascularized bone grafts. Semaphorin 3a (Sema3a) regulates osteoblasts and osteoclasts to promote bone synthesis through Neuropilin-1 receptor (NP1). We previously found that: 1) short-term delivery of Vascular Endothelial Growth Factor (VEGF) in osteogenic grafts dose-dependently decreases bone formation by increasing resorption and impairing progenitor differentiation; 2) in skeletal muscle VEGF dose-dependently inhibits endothelial Sema3a expression, impairing recruitment of Neuropilin-1-expressing monocytes (NEM), TGF-b1 production and SMAD2/3 activation. Here we investigated whether: a) VEGF impairs bone formation by inhibiting endogenous Sema3a expression; b) Sema3a treatment could improve both bone formation and vascularization in engineered bone grafts.
Methods. Fibrin matrices were decorated with recombinant VEGF or Sema3a proteins that were engineered with a transglutaminase substrate sequence (TG-VEGF and TG-Sema3a) to allow cross-linking into fibrin hydrogels. Osteogenic grafts were prepared with human bone marrow mesenchymal cells (BMSC) and hydroxyapatite granules in a fibrin hydrogel containing TG-VEGF, TG-Sema3a or both at ratio of 1:1 and implanted ectopically in nude mice. Sema3a blockade was achieved with a specific antibody (anti-NP1A) that prevents Nrp1 binding to Sema3a, but not to VEGF.
Results. 100 mg/ml of TG-VEGF (high dose) caused severe bone loss and significant downregulation of endogenous Sema3a expression. 0.1 µg/ml of TG-VEGF (low dose), instead, preserved both bone formation and Sema3A expression. Loss-of-function experiments showed that blocking Sema3a/NP-1 signaling significantly impaired bone tissue development, increased osteoclasts recruitment and, interestingly, also decreased vascular invasion both in the absence and presence of low TG-VEGF. Further, Sema3a/NP-1 blockade significantly reduced both human progenitor survival and endogenous Sema3a expression, as well as phospho-SMAD 2/3 activation in both human progenitors and host endothelial cells. These data are consistent with a positive feed-back loop between Sema3a and TGF-b1 signaling, as we previously described in skeletal muscle. Conversely, in gain-of-function experiments, TG-Sema3a co-delivery was able to prevent bone loss induced by high TG-VEGF, while preserving efficient vascular growth. Notably, TG-Sema3a alone could increase both the amount of mineralized matrix and vascular invasion in the absence of any TG-VEGF.
Conclusion. These data suggest that Sema3a: 1) is required for intramembranous ossification in osteogenic grafts; and 2) provides a key molecular link coupling angiogenesis and bone formation. These data identify Sema3a as a promising target to generate vascularized bone grafts in a clinical setting."