Bisphosphonates are frequently used to treat osteoporosis, bone metastasis of various cancers, and other diseases. However, bisphosphonate related-osteonecrosis of the jaw (BRONJ) is an intractable disease because it often recurs after surgery or is exacerbated following conservative treatment. Therefore, an alternative approach for treating BRONJ is needed. In this study, we developed a bone marrow-derived multipotent mesenchymal stromal cell (MSC) sheet to treat BRONJ and investigated the effect of MSC sheet transplantation in a rat model of BRONJ-like disease. The MSC sheet transplantation group showed wound healing in most cases, while only minimal healing was observed in the control group and MSC intravenous injection group. Our results suggest that the MSC sheet is a promising alternative approach for the treatment of BRONJ.

IntroductionLarge animal experiments are important for translational research in regenerative medicine. Recently, mini pigs have been used in large animal studies and surgical training. The use of multipotent mesenchymal stromal cell (MSC) sheets for the treatment of many diseases is increasing. The purpose of the present study was to establish optimal methods for generating mini pig MSC sheets from various tissues and to compare the properties of MSCs in these sheets.MethodsMSCs were isolated from the bone marrow, adipose, periodontal ligament, gingiva, or periosteum of mini pigs. The proliferation, markers, and mRNA expression of these MSCs were examined. Colony-forming and differentiation assays were performed. MSCs were seeded onto temperature-responsive culture dishes to develop MSC sheets.ResultsMSCs derived from bone marrow (BMSCs), adipose (ASCs), periodontal ligament (PDLCs), gingiva (GMSCs), and periosteum (PSCs) were positive for MSC-related markers. BMSCs and PSCs showed increased proliferation compared with other MSCs. The osteogenic potential of PDLCs and the adipogenic potential of PSCs were the highest among these MSCs. The expression levels of COL1A1 and COL3A1 in BMSCs and PSCs were significantly higher than those in other MSCs. The expression levels of FGF2, VEGFA, ICAM-1, and TIE-1 in GMSCs were significantly higher than those in other MSCs. PSCs showed the highest levels of TGF-β1 and ANG-1 expression among all MSC types. We succeeded in developing MSC sheets from BMSCs, ASCs, and PSCs.ConclusionsWe developed methods to generate MSC sheets from various tissues of mini pigs, and these methods are useful to pursue regenerative translational research using mini pigs.

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

IntroductionMany cases of bisphosphonate-related osteonecrosis of the jaw (BRONJ), which is an intractable disease, have been reported. Although a general intravenous injection of multipotent mesenchymal stromal cells (MSCs) may be effective for treating BRONJ, it has some severe problems. Therefore, our aim was to develop a treatment of locally administered MSCs. In this study, we investigated the effect of MSC sheet transplantation in the mandibular bone healing in beagle dogs, which were administered zoledronate and dexamethasone.MethodsMSCs isolated from subcutaneous fat were seeded onto temperature-responsive culture dishes to produce MSC sheets. Zoledronate and dexamethasone were administered to beagle dogs. Then, the parts of mandibular cortical bones were removed, and MSC sheets were transplanted to cover those bone defects (MSC sheet transplant side) or not (Control side). The specimens were evaluated in micro CT, histology, and immunohistochemistry.ResultsFour weeks after surgery, redness and swellings were observed in the mucosal wounds of the control sides of 2 of 3 dogs. In contrast, the mucosal wounds of the MSC sheet transplant sides of all dogs completely healed. Histological images showed some free sequestrums and many bacterial colonies, and Immunohistological analysis showed some cathepsin K-positive multinuclear cells detached from jaw bone surfaces in the control sides.ConclusionsMSC sheet transplantation promotes healthy healing of wounds caused by zoledronate and dexamethasone in canine mandibular bones. And the injured canine mandibular bones administered zoledronate and dexamethasone showed BRONJ-like findings.

No abstract