Human amniotic mesenchymal stem cell-derived small extracellular vesicles repair of jaw osteoradionecrosis

Abstract

BACKGROUND

Mesenchymal stem cells (MSCs) and their secretome offer a promising approach for treating osteoradionecrosis of the jaw (ORNJ); however, their efficacy remains controversial and the underlying molecular mechanisms require elucidation.

AIM

To investigate the role of small extracellular vesicles derived from human amniotic MSCs (hAMSCs-sEVs) in ORNJ.

METHODS

A Sprague-Dawley rat model of ORNJ received local hAMSCs-sEVs after mandibular molar extraction and the socket healing was assessed via micro computed tomography, hematoxylin and eosin, Masson trichrome, and immunohistochemical staining. In vitro, bone marrow MSCs (BMSCs) and human umbilical vein endothelial cells (HUVECs) were irradiated (12 Gy). The effects of hAMSCs-sEVs on osteogenic differentiation and angiogenesis were evaluated using alkaline phosphatase (ALP), Alizarin Red S, tube formation, and migration assays. Gene and protein expression were analysed by quantitative real time polymerase chain reaction and western blot. RNA sequencing identified the involved signaling pathways, which were validated with specific inhibitors.

RESULTS

In vivo, hAMSCs-sEVs enhanced socket healing in ORNJ rats, with micro computed tomography showing increased bone volume (bone volume/total volume). Histology revealed mature bone formation and higher expression of collagen-1, ALP, CD31, and vascular endothelial growth factor in the hAMSCs-sEVs group vs controls. In vitro, 12 Gy irradiation significantly inhibited osteogenic differentiation in BMSCs and angiogenesis in HUVECs. hAMSCs-sEVs were internalized by irradiated cells and reversed these inhibitions in a concentration-dependent manner. A concentration of 10¹⁰ particles/mL significantly upregulated osteogenic markers (collagen-1, ALP, runt-related transcription factor 2, osteopontin) in BMSCs and angiogenic markers (vascular endothelial growth factor, CD31) in HUVECs. RNA sequencing revealed that hAMSCs-sEVs reactivated the phosphatidylinositol 3-kinase/protein kinase B pathway in BMSCs and the Wnt/β-catenin pathway in HUVECs, which were inhibited by radiation. These effects were blocked by pathway inhibitors Ly294002 and MSAB, respectively.

CONCLUSION

hAMSCs-sEVs promote the repair of ORNJ by partially reversing radiation-induced inhibition of osteogenesis and angiogenesis, potentially through reactivation of the phosphatidylinositol 3-kinase/protein kinase B and Wnt/β-catenin signaling pathways.

Keywords: Human amniotic mesenchymal stem cells; Small extracellular vesicles; Osteoradionecrosis of the jaw; Osteogenesis; Angiogenesis; Phosphatidylinositol 3-kinase/protein kinase B signaling pathway; Wnt/β-catenin signaling pathway

Core Tip: The small extracellular vesicles from human amniotic mesenchymal stem cells (hAMSCs-sEVs) enhanced tooth socket healing in rats with osteoradionecrosis of the jaw, increasing bone formation and expression of osteogenic (collagen-1, alkaline phosphatase) as well as angiogenic (CD31, vascular endothelial growth factor) markers. In vitro, hAMSCs-sEVs reversed the inhibition of osteogenesis and angiogenesis by reactivating the phosphatidylinositol 3-kinase/protein kinase B and Wnt/β-catenin signaling pathways, respectively, as confirmed by pathway inhibitors. These findings indicate that hAMSCs-sEVs facilitate osteoradionecrosis of the jaw bone healing by reversing radiation-impaired osteogenesis and angiogenesis via key signaling pathways.