Total flavonoids of Rhizoma Drynariae enhances CD31hiEmcnhi vessel formation and subsequent bone regeneration in rat models of distraction osteogenesis by activating PDGF‑BB/VEGF/RUNX2/OSX signaling axis

Int J Mol Med. 2022 Sep;50(3):112. doi: 10.3892/ijmm.2022.5167. Epub 2022 Jul 7.

Abstract

Total flavonoids of Rhizoma Drynariae (TFRD), extracted from the kidney‑tonifying Traditional Chinese medicine Rhizoma Drynariae, can be effective in treating osteoporosis, bone fractures and defects. However, the pharmacological effects of TFRD on the specific vessel subtype CD31hiEmcnhi during distraction osteogenesis (DO) remains unclear. The present study aimed to investigate the effects of TFRD on CD31hiEmcnhi vessels in a rat model of DO. In the present study, tibial DO models were established using 60 rats with a distraction rate of 0.2 mm per day for 20 days. Co‑immunofluorescence staining of CD31 and endomucin (Emcn) was conducted to determine CD31hiEmcnhi vessels. Radiographic, angiographic and histological analyses were performed to assess bone and vessel formation. Tube formation, alkaline phosphatase (ALP) and Von Kossa staining assays were performed to test angiogenesis of endothelial precursor cells (EPCs) and osteogenesis of bone marrow‑derived mesenchymal stem cells (BMSCs). Additionally, expression levels of platelet‑derived growth factor (PDGF)‑BB, VEGF, runt‑related transcription factor 2 (RUNX2) and Osterix (OSX) were determined by western blotting and reverse transcription‑quantitative PCR. The in vivo assays demonstrated that TFRD markedly promoted CD31hiEmcnhi vessel formation during DO, whereas PDGF‑BB neutralizing antibody suppressed vessel formation. Furthermore, the ALP, Von Kossa staining and tube formation assays indicated that TFRD notably elevated the angiogenic capacity of EPCs and osteogenic capacity of BMSCs under stress conditions, which was significantly suppressed by blocking PDGF‑BB. The protein and mRNA levels of PDGF‑BB, VEGF, RUNX2 and OSX were upregulated by TFRD, but downregulated by blocking PDGF‑BB. Thus, TFRD could facilitate CD31hiEmcnhi vessel formation and subsequently enhance angiogenic‑osteogenic coupling to regenerate bone defects during DO via the PDGF‑BB/VEGF/RUNX2/OSX signaling axis, which indicated that CD31hiEmcnhi vessels could be a potential novel therapeutic target for DO, and TFRD may represent a promising drug for promoting bone regeneration in DO by increasing CD31hiEmcnhi vessels.

Keywords: CD31hiEmcnhi vessels; bone regeneration; distraction osteogenesis; platelet‑derived growth factor‑BB; total flavonoids of Rhizoma Drynariae.

MeSH terms

  • Animals
  • Becaplermin / metabolism
  • Becaplermin / pharmacology
  • Bone Regeneration
  • Core Binding Factor Alpha 1 Subunit / genetics
  • Flavonoids / pharmacology
  • Neovascularization, Physiologic
  • Osteogenesis, Distraction*
  • Polypodiaceae* / metabolism
  • Rats
  • Sialomucins
  • Vascular Endothelial Growth Factor A / metabolism

Substances

  • Core Binding Factor Alpha 1 Subunit
  • Flavonoids
  • Runx2 protein, rat
  • Sialomucins
  • Vascular Endothelial Growth Factor A
  • Becaplermin

Grants and funding

The present study was supported by the National Natural Science Foundations of China (grant no. 81774337), the Basic Research Project of Science and Technology Department of Yunnan Province (grant nos. 202101AZ070001-123, 202201AU070120), the Doctoral Fund Project of Kunming Municipal Hospital of Traditional Chinese Medicine, and Kunming Health Science and Technology Talent Cultivation Project and ‘Ten Hundred Thousand’ talent project [grant no. 2020-SW (Reserve Personnel)-52].