Efficacy of total flavonoids of Rhizoma drynariae on the blood vessels and the bone graft in the induced membrane

Total flavonoids of Rhizoma drynariae (TFRD), a Chinese medicine, is widely used in the treatment of orthopedic diseases. However, there are few basic and clinical studies on the effect of TFRD on induced membrane technique (Masquelet technique).This trial is to explore effects of TFRD on vascularization of the induced membrane, and mineralization of the bone graft in rats with femoral bone defects.Forty-eight Sprague-Dawley rats were randomly divided into high dose group (H-TFRD), medium dose group (M-TFRD), low dose group (L-TFRD) and control group (control). The segmental bone defects were established with 12 rats in per group. The polymethyl methacrylate (PMMA) spacer was implanted into the femoral bone defect of rats in the first-stage surgery. About 4 weeks after first-stage surgery, induced membranes of 6 rats in each group were selected. The blood vessels and angiogenesis-related factors in the induced membrane were analyzed by hematoxylin-eosin (HE) and masson staining, western blot, qPCR and immunohistostaining. The remaining rats in per group underwent second-stage surgery (bone grafting). Twelve weeks after the bone grafting, the bone tissues was examined by X-ray, micro-computed tomography (Micro-CT), HE staining and enzyme-linked immunosorbent assay (ELISA) to evaluate the growth of the bone graft. Meanwhile, the TFRD-containing serum was collected from rats to culture osteoblasts in vitro. Cell Counting Kit-8 (CCK-8) method, Alizarin Red S (ARS) staining, western blot and immunofluorescence were used to detect effects of TFRD on the osteoblasts' proliferation and BMP-SMAD signaling pathway.Compared with the L-TFRD and control groups, the number of blood vessels and the expression of angiogenesis-related factors (VEGF, TGF-β1, BMP-2, PDGF-BB and CD31) were higher in the H-TFRD and M-TFRD groups. The Lane-Sandhu X-ray score, bone mass and growth rate of the bone graft in the H-TFRD and M-TFRD groups were significantly better than those in the L-TFRD and control groups. In addition, medium and high doses of TFRD significantly increased the expression of BMP-SMAD pathway proteins (BMP-2, SMAD1, SMAD4, SMAD5 and RUNX2) in rat serum and bone graft. In vitro, after osteoblasts were intervened with TFRD-containing serum from the H-TFRD and M-TFRD groups, the cell viability, the number of mineralized nodules and the phosphorylation of BMP-SMAD pathway proteins were markedly increased.TFRD could promote the formation of blood vessels and the expression of angiogenesis-related factors during the formation of the induced membrane. During the growing period of bone graft, it could facilitate the growth and mineralization of bone graft in a dose-dependent manner, which is partly related to the activation and phosphorylation of BMP-SMAD signaling pathway.