Cytotherapeutic Use of Allogeneic Mesenchymal Stem Cell Sheets for Bisphosphonate-related Osteonecrosis of the Jaw in a Rat Model

Nobuyuki Kaibuchi DDS, Department of Oral and Maxillofacial Surgery, Tokyo Women’s Medical University, School of Medicine, Tokyo, Japan
Takanori Iwata DDS, PhD, Department of Oral and Maxillofacial Surgery, Tokyo Women’s Medical University, School of Medicine, Tokyo, Japan
Kaoru Washio D.D.S., Ph.D., Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan
Yuka Kato MD, Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan
Toshihiro Okamoto DDS, PhD, Department of Oral and Maxillofacial Surgery, Tokyo Women’s Medical University, School of Medicine, Tokyo, Japan
Masayuki Yamato Ph.D., Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan
Teruo Okano PhD, Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan
Tomohiro Ando DDS, PhD, Department of Oral & Maxillofacial Surgery, Tokyo Women's Medical University, Tokyo, Japan
Statement of the problem

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a severe complication in patients who receive bisphosphonates. Although the number of patients is increasing day by day, there is no definitive treatment or prevention for BRONJ. Recently studies suggested that intravenous infusion of mesenchymal stem cells (MSCs) was effective for bone exposure in BRONJ-like disease models1, 2). However, it is suggested that the stability of injected MSCs at the diseased area is relatively low, resulting in pulmonary embolism or even death in some cases. To overcome the problem, our laboratory introduced “Cell Sheet Engineering” using temperature-responsive culture dishes in which intact cells and extracellular proteins can be harvested as a sheet by simple temperature reduction. In this study, we investigated the effect of bone marrow derived MSC (BMMSC) sheet transplantation on a BRONJ-like disease model in rats.

Materials and methods

MSCs were isolated from bone marrow of enhanced green fluorescent protein (EGFP) SD rats. BMMSCs at passage 3 were seeded on temperature-responsive culture dishes (UpCell®, Cell Seed, Tokyo, Japan) with 50μg/ml ascorbic acid and cultured for 7 days to produce cell sheets. Zoledronate and dexamethasone were administered to SD rats for 4 weeks, then, maxillary first molars were extracted to induce BRONJ-like disease. After surgical debridement, BMMSC sheets were transplanted to the lesion of bone exposure (transplant group) or sutured only (control group).

Methods of data analysis

Evaluations consisted of micro CT (R_mCT2, Rigaku, Tokyo, Japan), histology, and immunohistochemistry at 2 weeks after the transplantation. The transplanted alveolar bone areas were examined by hematoxylin and eosin (H&E) staining or tartrate-resistant acid phosphatase (TRAP) staining. Anti-RECA1 antibody was used immunohistochemically to assess the existence of endothelial cells. Statistical analyses were performed using Student’s t test. P values less than 0.05 were considered significant.

Results

Two weeks after the transplantation, the transplant group showed wound healing in all cases (100%; 8/8). In contrast, the control group showed the presence of bone exposures without soft tissue (71.4%; 5/7). Micro CT analysis showed bone regeneration in the extraction socket of the transplant group and delayed bone healing in the control group. TRAP staining showed that the average number of osteoclasts per mm2 was significantly increased in the transplant group compared with the control group (p < 0.05). In immunohistochemical analysis showed RECA1-positive signals were observed at the GFP-positive cells in the transplant area.

Conclusions

Our results indicate that allogeneic BMMSCs sheet transplantation promoted wound healing, bone regeneration, and angiogenesis in BRONJ-like model of rats. It is suggested that BMMSCs sheet transplantation can be an alternative approach to treat BRONJ.

References

1) Kikuiri T, Kim I, Yamaza T, et al: Cell-based immunotherapy with mesenchymal stem cells cures bisphosphonate-related osteonecrosis of the jaw-like disease in mice. J Bone Miner Res 25: 1668, 2010

2) Y Li, J Xu, L Mao, et al: Allogenic mesenchymal stem cell therapy for bisphosphonate-related jaw osteonecrosis in swine. Stem cells dev 22: 2047, 2013

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