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<p>Poor tendon-bone healing is a major challenge after anterior cruciate ligament reconstruction (ACLR). TFRD, the total flavonoids of Rhizoma Drynariae, has pharmacological effects in promoting bone formation, but it remains less understood whether it accelerates tendon-bone healing by regulating the osteogenic differentiation of tendon-derived stem cells (TDSCs). TDSCs were isolated from the Achilles tendons of rats and characterized for their stem cell properties through flow cytometry and multilineage differentiation assays. The effects of TFRD on TDSC proliferation, migration, and osteogenic differentiation were evaluated using the cell counting kit-8 assay, scratch wound healing assays, Alizarin Red S staining, and Western blot analysis. Gene and protein expression levels associated with the transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) signaling pathway were assessed by real-time quantitative polymerase chain reaction and Western blot. A rat ACLR model was created and divided into four experimental groups: Sham, ACLR, TFRD, and TFRD + Noggin. Tendon-bone healing was analyzed using hematoxylin and eosin staining, Masson’s trichrome staining, bone mineral density measurements, and immunohistochemistry. Noggin was used to inhibit BMP signaling for pathway validation. TFRD (50–100&#xa0;μg/mL) significantly enhanced the proliferation, migration, and osteogenic differentiation of TDSCs, and increased the expression of key osteogenic markers, including runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP). Mechanistically, TFRD activated the TGF-β/BMP signaling pathway, leading to elevated levels of TGF-β1, BMP-2, and phosphorylated Smad1/5/8. Moreover, TGF-β1 extended the duration of BMP-2 signaling and suppressed Noggin expression. The osteogenic effects of TFRD were diminished when Noggin was applied, indicating its role in modulating the BMP pathway. In vivo, TFRD significantly improved collagen fiber alignment, calcification, and bone mineral density at the tendon-bone junction, while also upregulating RUNX2 and ALP expression. TFRD promotes osteogenic differentiation of TDSCs by activating the TGF-β/BMP signaling pathway, improves tendon-bone healing after ACLR, and provides experimental support for its clinical application.</p>

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Total flavonoids of Rhizoma Drynariae promote osteogenic differentiation of tendon-derived stem cells through the TGF-β/BMP signaling pathway, thereby improving bone regeneration after anterior cruciate ligament reconstruction in rats

  • 期刊论文

  • 发表日期：

    2026年7月22日

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  • ShangYi Dai,
  • LiXin Cao,
  • XueFeng Wang

摘要

Poor tendon-bone healing is a major challenge after anterior cruciate ligament reconstruction (ACLR). TFRD, the total flavonoids of Rhizoma Drynariae, has pharmacological effects in promoting bone formation, but it remains less understood whether it accelerates tendon-bone healing by regulating the osteogenic differentiation of tendon-derived stem cells (TDSCs). TDSCs were isolated from the Achilles tendons of rats and characterized for their stem cell properties through flow cytometry and multilineage differentiation assays. The effects of TFRD on TDSC proliferation, migration, and osteogenic differentiation were evaluated using the cell counting kit-8 assay, scratch wound healing assays, Alizarin Red S staining, and Western blot analysis. Gene and protein expression levels associated with the transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) signaling pathway were assessed by real-time quantitative polymerase chain reaction and Western blot. A rat ACLR model was created and divided into four experimental groups: Sham, ACLR, TFRD, and TFRD + Noggin. Tendon-bone healing was analyzed using hematoxylin and eosin staining, Masson’s trichrome staining, bone mineral density measurements, and immunohistochemistry. Noggin was used to inhibit BMP signaling for pathway validation. TFRD (50–100 μg/mL) significantly enhanced the proliferation, migration, and osteogenic differentiation of TDSCs, and increased the expression of key osteogenic markers, including runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP). Mechanistically, TFRD activated the TGF-β/BMP signaling pathway, leading to elevated levels of TGF-β1, BMP-2, and phosphorylated Smad1/5/8. Moreover, TGF-β1 extended the duration of BMP-2 signaling and suppressed Noggin expression. The osteogenic effects of TFRD were diminished when Noggin was applied, indicating its role in modulating the BMP pathway. In vivo, TFRD significantly improved collagen fiber alignment, calcification, and bone mineral density at the tendon-bone junction, while also upregulating RUNX2 and ALP expression. TFRD promotes osteogenic differentiation of TDSCs by activating the TGF-β/BMP signaling pathway, improves tendon-bone healing after ACLR, and provides experimental support for its clinical application.