Abstract
Background: Retarded gingival healing is the hallmark of bisphosphonate-related osteonecrosis of the jaw (BRONJ) and poses a great challenge to maxillofacial surgeons. Although previous studies have showed that bisphosphonates (BPs) are highly toxic to healthy gingival mesenchymal stem cells (GMSCs) in vitro, there is overall lack of direct evidence demonstrating the regeneration capacity of oral mucosa in BRONJ patients. In present study, we aim to isolate GMSCs from BRONJ patients’ gingiva and assessed their phenotypes and functions in vitro, as well as their therapeutic effects for wound healing in a mice excisional skin model. Methods: BRONJ patients’ gingival samples were used for microarray analysis, histological detection and cell culture. The stem cells isolated from the central gingiva (center-BRONJ GMSCs) and the peripheral lesions (peri-BRONJ GMSCs) were analyzed by Cell Counting Kit-8 (CCK-8), cell adhesion, scratch and flow cytometry. Luciferase/GFP (Green Fluorescent Proteins)-labeled GMSCs combined with Hydrogel were transplanted in a mice excisional skin model, and mice were divided into a hydrogel alone group, a hydrogel/control GMSCs group, a hydrogel/center-BRONJ GMSCs group and a hydrogel/peri-BRONJ GMSCs group. Bioluminescence imaging trace cell survival in vivo. Healing effects were evaluated by wound area measurement, histology, immunohistochemistry (IH) and immunofluorescence (IF). Results: Center-BRONJ GMSCs and peri-BRONJ GMSCs were all fibroblast-like cells, but they became slender and more wrinkled compared control GMSCs. Notably, they exhibited decreased proliferation, adhesion, migration capacities and underwent early apoptosis in vitro. In animal model, BRONJ GMSCs transplantation also displayed lower cell survival rate and poor healing effects than that of control group. Mechanistically, we found that the expression of TGF-β1 signaling pathway was suppressed not only in BRONJ patients’ gingival lesions but also in BRONJ GMSCs transplantation animal model. Conclusions: In BRONJ patients’ microenvironment, the regeneration ability of oral mucosa was dramatically decreased. Our mice skin model demonstrated for the first time that BRONJ GMSCs transplantation displayed poor effects on wound healing mainly via suppressing TGF-β1 signaling pathway. This study provides new insights into the prevention for BRONJ by improving the functions of GMSCs in accelerating gingival wound healing.
Electrostimulation of a 3D in vitro skin model to activate wound healing
