SEMAPHORIN3A COUPLES OSTEOGENESIS AND ANGIOGENESIS IN TISSUE-ENGINEERED OSTEOGENIC GRAFTS

Jun 30, 2022, 2:00 PM
10m
Room: S1

Room: S1

Speaker

Grosso, Andrea (Department of Biomedicine, University of Basel)

Description

"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."
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