Original Full Length ArticleEvaluation of the therapeutic effects of conditioned media from mesenchymal stem cells in a rat bisphosphonate-related osteonecrosis of the jaw-like model
Introduction
Bisphosphonates (BPs) were originally developed as a drug for the treatment of bone-resorbing diseases, such as multiple myeloma and bone metastasis, whose typical origins are breast cancer and prostate cancer, as well as tumor-related hypercalcemia [1], [2], [3], [4], and then to prevent pathological fracture in patients with osteoporosis. Lately, reports in the literature have suggested that a rare but potentially severe side-effect is BP-related osteonecrosis of the jaw (BRONJ) [5], defined as an exposed bone in the maxillofacial region that persists for more than 8 weeks in patients with present or previous BP treatment without a history of radiation therapy to the jaw [6]. Specific target cells of BPs are osteoclasts and mesenchymal stem cells (MSCs) (or osteoblasts), which play a central role in physiological and pathological bone formation and bone resorption [7], [8], [9], [10].
Risk factors for BRONJ include invasive dental procedures, infections, mechanical trauma to the jawbone, and length of exposure to BPs, as well as the concomitant use of immunosuppressive and chemotherapeutic drugs [6], [11]. Because most patients with cancer were receiving multiple immunosuppressant drugs, including dexamethasone (Dex) and chemotherapeutic agents, and thus experiencing some degree of impaired immunity, it is likely that immunosuppression contributes to an increased susceptibility to BRONJ.
Patients with BRONJ present various jaw symptoms, including pain, swelling, infection and, in some severe cases, pathologic fracture [5], [6], [11]. Histologically, several tissue alterations are observed in BRONJ, including necrotic bone honeycombed with residual vital bone, inflammatory cellular elements, and hypernucleated osteoclasts and fibrous tissues [10], [11], [12]. Many attempts to control this disorder have been unsuccessful, and standard osseous sequestrectomy usually results in further enlargement of the bone defects [13], [14]. Therefore, conservative non-surgical approaches to the management of BRONJ have been recommended that slow its deterioration but do not cure the disease [15], [16], [17]. The development of an effective approach to the prevention and treatment of BRONJ is an urgent issue for patients using BPs.
Some studies have reported that an intravenous injection of MSCs improved BRONJ [18], [19], but tumorigenesis of the cells is also possible [20], [21]. Moreover, recent studies of MSC transplantation demonstrated that the implanted MSCs did not survive for a long time [22]. Furthermore, it has been established that MSCs secrete a variety of growth factors and cytokines [23], [24], [25], [26], [27], [28], and the paracrine effects of growth factors and cytokines secreted from the implanted MSCs may promote tissue repair or have antiapoptotic effects [23], [24], [25], [26], [27], [28] and prevent BRONJ [18], [19]. The paracrine factors secreted by MSCs can accumulate in conditioned media during cell culture [23], [24], [25], [26], [27], [28]. The serum-free conditioned media from human MSCs (MSC-CM) have been reported to serve multiple positive functions [29], [30]. In particular, study results to date have supported the theory that MSC-CM is included in the paracrine factors important for the turnover of local bone status [23], [24], [25], [26], [27], [28].
In late years various rat BRONJ-like models are reported [31], [32], [33], [34]. Here we made a rat BRONJ-like model, using zoledronate (Zol) and Dex which are used clinically widely, that recapitulates major clinical and radiographic manifestations of the human disease, including its characteristic features of delayed healing displayed orally as an open alveolar socket without mucosal coverage, exposed necrotic bone or sequestra, increased inflammatory infiltrates, osseous sclerosis, and radiopaque alveolar bone in the jaw.
In this in vitro study, to understand how MSC-CM alters the turnover of local bone status and the inflammatory response of alveolar socket healing, we investigated the effects on MSCs and osteoclasts of MSC-CM. In an in vivo study, we investigated whether MSC-CM injection can have therapeutic effects on BRONJ by using a rat BRONJ-like model.
Section snippets
Cell preparation
All animal experiments undertaken in this study were performed in strict accordance with the protocols approved by the Guidelines for Animal Experimentation of the Nagoya University School of Medicine (approval nos. 25374 and 26063). Human MSCs (hMSCs) were purchased from Lonza, Inc. (Walkersville, MD, USA) and cultured in MSC basal medium (Lonza, Inc.) containing MSCGM SingleQuots (Lonza, Inc.) at 37 °C in 5% CO2/95% air. After primary culture, the cells were subcultured at a density of
Growth factors present in MSC-CM
After performing the cytokine array (RayBio® Human Cytokine Antibody Array G-Series 2000) which compared MSC-CM with DMEM(−), six representative growth factors included more than five times were picked up (Table 2). The concentrations of the growth factors IGF-1, MCP-1, VEGF-A, Ang, IL-6, and M-CSF, released by hMSCs into MSC-CM, were evaluated by ELISA analysis. These growth factors were not detected in DMEM(−). However, MSC-CM contained IGF-1, MCP-1, VEGF-A, Ang, IL-6, and M-CSF at
Discussion
In this study, we showed that BRONJ-like lesions exhibit characteristics similar to those of human diseases involving mucosal ulceration or open sockets, exposed necrotic bone, and radiopaque alveolar bone in the jaw, as demonstrated by micro-CT and histological studies. The clinically obvious BRONJ, specifically open sockets with exposed necrotic bone and no mucosal lining, persisted beyond 2–3 weeks, and the normal course of healing was observed in non-treated control rats. In our rat BRONJ
Acknowledgments
We thank the members of the Department of Oral and Maxillofacial Surgery, Nagoya University, for their encouragement to complete this study as well as Prof. Tatsushi Kawai of the Graduate School of Dentistry, Aichi Gakuin University. We also thank the Division of Experimental Animals and Medical Research Engineering, Nagoya University Graduate School of Medicine, for housing the rats. This work was supported in part by Grants-in-Aid for Scientific Research (Nos. 21791985 and 23592883) from the
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2021, BoneCitation Excerpt :The curative treatments for MRONJ (n = 13) included several similar approaches to those for preventive treatments (Supplemental Table 1). These included PTH [109,141–143], injection of platelet-rich plasma into the extraction socket [144,145], and local transplantation of MSCs, endothelial progenitor cells, or molecular products of MSCs [146–149]. In addition, 3% of the studies (n = 5) focused on investigating mechanisms that could be involved in the pathophysiology of MRONJ.
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