Adipose-derived stem cells prevent the onset of bisphosphonate-related osteonecrosis of the jaw through transforming growth factor β-1-mediated gingival wound healing

Abstract

Background: Due to its complex pathogenesis and low clinical cure rate, bisphosphonate-related osteonecrosis of the jaw (BRONJ) poses a substantial challenge for oral and maxillofacial surgeons. Therefore, the treatment of BRONJ should focus on prevention. In clinical studies, primary wound closure can significantly reduce the incidence of BRONJ. Whether local stem cell transplantation can promote primary gingival healing in patients with a medication history and prevent BRONJ has not been reported.

Methods: In this study, animals were divided into a healthy group (non-drug treatment), a BP group, a hydroxyapatite (HA) group, and an adipose-derived stem cell (ADSC) group. All groups except the healthy group were treated with BPs and immunosuppressive drugs once per week for 8 weeks, simulating clinical use for the treatment of cancer patients with bone metastasis, to induce BRONJ-like animals. After the sixth drug treatment, the bilateral premolars were extracted in all groups. In contrast to the healthy and BP groups, the extraction sockets in the HA and ADSC groups were filled with HA or HA + ADSCs simultaneously post extraction to observe the preventive effect of ADSCs on the occurrence of BRONJ. At 2 and 8 weeks post extraction, animals from all groups were sacrificed.

Results: At 8 weeks post transplantation, ADSCs prevented the occurrence of BRONJ, mainly through accelerating healing of the gingival epithelium at 2 weeks post extraction. We also found that ADSCs could upregulate the expression of transforming growth factor β1 (TGF-β1) and fibronectin in tissue from animals with a medication history by accelerating gingival healing of the extraction socket. A rescue assay further demonstrated that TGF-β1 and fibronectin expression decreased in TGF-β1-deficient ADSC-treated animals, which partially abolished the preventive effect of ADSCs on the onset of BRONJ.

Conclusion: ADSCs prevent the onset of BRONJ, mainly by upregulating the expression of TGF-β1 and fibronectin to promote primary gingival healing, ultimately leading to bone regeneration in the tooth extraction socket. Our new findings provide a novel stem cell treatment for the prevention of BRONJ.

Keywords: Adipose-derived mesenchymal stem cells; Bisphosphonate-related osteonecrosis of the jaw; Fibroblast; Fibronectin; Wound healing; Zoledronic acid.

Fig. 1

The treatment schedule used to induce BRONJ-like animals and the ADSC transplantation procedure. a Animals were divided into two groups, the healthy group (non-medication treated) and the BP group. The BP group was treated with zoledronic acid (ZOL) and dexamethasone (DEX). After the sixth dose of ZOL+DEX treatment, the bilateral premolars were extracted in both the healthy and BP groups. At 2 weeks and 8 weeks post extraction, samples were collected. b The procedure for tooth extraction and an extracted tooth (bar = 2 mm). c Animals were divided into two groups: the hydroxyapatite (HA) group and the ADSC group. Both groups were treated with ZOL+DEX once per week and followed the same schedule used to induce BRONJ-like animals. d The two groups were subjected to extraction of the bilateral premolars. Simultaneously, after tooth extraction, the HA group was filled with hydroxyapatite, and the ADSC group was filled with ADSCs that were incubated on HA. Arrows indicate filled HA or ADSC-HA complex

Fig. 2

Induction of BRONJ-like animals by delivery of zoledronic acid and dexamethasone. A The clinical appearance of the healthy group and the BP group. The area in the dotted line shows the extracted tooth area at 2 and 8 weeks post extraction. Arrows indicate exposed jaw bone and unhealed gingiva (bar = 1 mm). B Hematoxylin and eosin (H&E) and trichrome staining (Masson) show bone regeneration and collagen deposition. Regenerated bone (RB). Necrotic bone (NB). The rectangular line indicates the area that was magnified and displayed on the side (a–d, bar = 1 mm; a’–d’, bar = 50 μm). C Quantitative analysis of the proportion of necrotic bone (%), subgingival collagen deposition (%) and unhealed area of the tooth extraction wound (mm²) at both 2 and 8 weeks post extraction and the proportion of new bone formation (%) at 8 weeks post extraction in the healthy and BRONJ groups. *p < 0.05, ^#undetected data. The results are representative of three independent experiments

Fig. 3

Local ADSC therapy promotes primary gingival healing at 2 weeks post extraction and facilitates bone regeneration at 8 weeks post extraction. A Clinical appearance of the healthy, BP, HA, and ADSC groups at 2 and 8 weeks post extraction. Areas in the dotted line show the tooth extraction site. Black arrows indicate jaw bone exposure in the BP group (bar = 1 mm). B H&E staining shows gingival healing in each group at 2 weeks post extraction. Masson staining indicates extraction site subgingival collagen deposition (a–h, bar = 1 mm; a’–d’, bar = 200 μm; e’–h’, bar = 50 μm). The rectangular line shows the magnified area. C Quantification of the unhealed area of the tooth extraction wound (mm²), the proportions of necrotic (%), and collagen deposition (%) in each group at 2 weeks. *p < 0.05. D TRAP-positive cells indicate osteoclasts. The arrows indicate osteoclasts, bar = 50 μm. Quantification of the number of osteoclasts per linear bone perimeter (#/mm), *p < 0.05

Fig. 4

Primary gingival healing by transplantation of ADSCs facilitate socket bone regeneration and upregulate osteogenic genes expression. A Local ADSC therapy facilitates socket bone regeneration. H&E and Masson staining indicate bone regeneration in the ADSC and HA groups (a–d, bar = 1 mm; a’–d’, bar = 50 μm). Quantification of the unhealed area of the tooth extraction wound (mm²), the proportion of necrotic bone (%), new bone formation (%), and extraction socket subgingival collagen deposition (%) in each group at 2 weeks post extraction. *p < 0.05, ^#undetected data. B Computed tomography (CT) examination shows reconstructed three-dimensional (3D) images and quantification of bone volume/total volume (BV/TV) and bone mineral density (BMD) in each group. *p < 0.05. The results are representative of three independent experiments. C Relative expression of osteogenic target genes in each group. *p < 0.05

Fig. 5

Local ADSC transplantation improved suppressed TGF-β1 and fibronectin expression in BRONJ-like rabbits. A Assessment of the target genes in each group. Relative tissue messenger RNA (mRNA) expression levels of target genes in each group at 2 weeks post extraction. *p < 0.05. Immunohistochemical detection of TGF-β1 (B) and fibronectin (FN) (C) abundance and quantification of their expression in the subgingiva of the extraction socket in each group. *p < 0.05; B a–d, bar = 200 μm; a’–h’, bar = 20 μm; C a–d, bar = 200 μm; a’–d’, bar = 20 μm. The rectangular line shows the magnified area. D Immunohistochemistry showing TGF-β1 expression in gingival tissue from a BRONJ patient and quantified expression. An ELISA kit and western blot were used to detect TGF-β1 expression in gingival fibroblasts (HGFs) from three BRONJ patients. *p < 0.05, bar = 20 μm

Fig. 6

TGF-β1 played a pivotal role in ADSC-mediated gingival healing. A Clinical manifestations at the extraction site in the ADSC group and KD group. The area in the dotted line indicates the tooth extraction site. B TGF-β1 and FN relative expression in gingival and bone tissue from the KD group compared to that in the ADSC and healthy groups. *p < 0.05. C H&E and Masson staining show gingival healing. Unhealed area of the tooth extraction wound (mm²) and extraction socket subgingival collagen deposition (%) were quantified. *p < 0.05; a–b, bar = 1 mm; c–f, bar = 200 μm; g–h, bar = 50 μm. Immunohistochemistry to detect TGF-β1 (D) expression in the KD group and ADSC group and quantification of their expression in the subgingiva of the extraction socket. *p < 0.05. D a–b, bar = 1 mm; c–d, bar = 200 μm; c’–d’, bar = 20 μm. The rectangular line shows the magnified area. The results are representative of three independent experiments