Sonic hedgehog (Shh) has the dual function of promoting angiogenesis and osteogenesis, However, whether Shh treatment can accelerate alveolar bone regeneration by promoting the coupling of type H vessel formation and osteogenesis within periodontitis has not been verified. A series of angiogenesis-related assays (including cell counting kit-8, transwell migration, scratch wound healing, and tube formation) were established to explore the proliferation, migration, and tube formation abilities of endothelial cells (ECs) in response to Shh. Alizarin Red S staining and alkaline phosphatase activity assay were subsequently conducted to determine the effect of Shh on the osteogenic differentiation of periodontal ligament stem cells (PDLSCs). Additionally, the expression levels of key factors of Slit3/Robo4/HIF-1α pathway, and angiogenesis- and osteogenesis-related genes/proteins were assessed. In addition, a mouse periodontitis model was employed, along with micro-CT and immunofluorescence staining analyses, to explore the effects of Shh administration on type H vessel formation and alveolar bone regeneration. Shh treatment increased the proliferation, migration, tube formation and CD31&EMCN intensity of ECs, and enhanced the osteogenic differentiation potential of PDLSCs in vitro. Mechanism studies found Shh treatment up-regulated the expression levels of Slit3, Robo4 and HIF-1α in ECs. Besides, silencing of Slit3 reserved the promoted angiogenic activity of ECs in the presence of Shh, and significantly down-regulated the expression levels of Robo4 and HIF-1α. In vivo studies verified that Shh administration markedly promoted type H vessel formation, increased the proportion of HIF-1α staining cells, and eventually attenuated bone loss within periodontitis model. Shh administration accelerates type H vessel formation by the Slit3/Robo4/HIF-1α signaling, to further alleviate alveolar bone resorption within periodontitis.
Zhou et al. (Tue,) studied this question.