Elsevier

Bone

Volume 74, May 2015, Pages 95-105
Bone

Original Full Length Article
Evaluation of the therapeutic effects of conditioned media from mesenchymal stem cells in a rat bisphosphonate-related osteonecrosis of the jaw-like model

https://doi.org/10.1016/j.bone.2015.01.011Get rights and content

Highlights

  • MSC-CM contained various growth factors, e.g., IGF-1, VEGF, angiogenin, IL-6, and M-CSF.

  • MSC-CM protected rMSCs and rOSCs and inhibited osteoclast apoptosis and maintained resorptive activity of osteoclasts against zoledronate.

  • MSC-CM enhanced osteogenic and angiogenesis marker gene expression.

  • MSC-CM intravenous injection improved rats' BRONJ-like lesions.

Abstract

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is defined as an exposed necrotic bone in the oral cavity that does not heal after appropriate intervention for > 8 weeks with present or previous bisphosphonate treatment in the absence of radiotherapy. Until now, although several risk factors, including invasive dental procedures, infection, mechanical trauma to the jawbone, and concomitant use of immunosuppressive and chemotherapy drugs have been implicated in the etiology of BRONJ, its underlying mechanisms and treatments remain largely unknown. A study recently showed that intravenous administration of mesenchymal stem cells (MSCs) improved BRONJ, and it was hypothesized that paracrine effects by secretomes from MSCs are the main constituent. Here we used rat BRONJ models to examine the therapeutic effects with serum-free conditioned media from human MSCs (MSC-CM), including various secretomes. We showed that MSC-CM has protected rat MSCs and rat osteoclasts. MSC-CM enhanced the expression of osteogenic-related genes and neovascularization-related genes by real-time reverse-transcriptase polymerase chain reaction analysis in in vitro study. In in vivo study, 5-week-old Wistar/ST male rats received zoledronate (35 μg/kg/week) and dexamethasone (1 mg/kg/day) subcutaneously for 2 weeks. Unilateral maxillary molars were then extracted. Two weeks later, rats were divided into non-treatment, serum-free Dulbecco's modified Eagle's medium, and MSC-CM groups. In the MSC-CM group, the open alveolar sockets in 63% of the rats with BRONJ healed with complete soft tissue coverage and socket bones, whereas the exposed necrotic bone with inflamed soft tissue remained in the other groups. Histological analysis showed new bone formation and the appearance of osteoclasts in the MSC-CM group. Osteoclasts were significantly reduced in the non-treatment group. Thus, we concluded that the antiapoptotic and antiinflammatory effects of MSC-CM dramatically regulated the turnover of local bone and indicated therapeutic effects on BRONJ.

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

References (54)

  • V. Viereck et al.

    Bisphosphonates pamidronate and zoledronic acid stimulate osteoprotegerin production by primary human osteoblasts

    Biochem Biophys Res Commun

    (2002)
  • M.S. Kim et al.

    MCP-1 is induced by receptor activator of nuclear factor-kappa B ligand, promotes human osteoclast fusion, and rescues granulocyte macrophage colony-stimulating factor suppression of osteoclast formation

    J Biol Chem

    (2005)
  • M. Tsubaki et al.

    Bisphosphonate- and statin-induced enhancement of OPG expression and inhibition of CD9, M-CSF, and RANKL expressions via inhibition of the Ras/MEK/ERK pathway and activation of p38MAPK in mouse bone marrow stromal cell line ST2

    Mol Cell Endocrinol

    (2012)
  • Y. Li et al.

    Insulin-like growth factor 1 enhances the migratory capacity of mesenchymal stem cells

    Biochem Biophys Res Commun

    (2007)
  • A. Lipton

    New therapeutic agents for the treatment of bone diseases

    Expert Opin Biol Ther

    (2005)
  • J.R. Berenson et al.

    American Society of Clinical Oncology clinical practice guidelines: the role of bisphosphonates in multiple myeloma

    J Clin Oncol

    (2002)
  • A. Lipton et al.

    Pamidronate prevents skeletal complications and is effective palliative treatment in women with breast carcinoma and osteolytic bone metastases: long-term follow-up of two randomized, placebo-controlled trials

    Cancer

    (2000)
  • J.R. Berenson et al.

    Zoledronic acid reduces skeletal-related events in patients with osteolytic metastases

    Cancer

    (2001)
  • S.L. Silverman et al.

    Osteonecrosis of the jaw and the role of bisphosphonates: a critical review

    Am J Med

    (2009)
  • M. Sato et al.

    Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure

    J Clin Invest

    (1991)
  • H. Fujita et al.

    Effect of risedronate on osteoblast differentiation, expression of receptor activator of NF-kappa B ligand and apoptosis in mesenchymal stem cells

    Basic Clin Pharmacol Toxicol

    (2011)
  • S. Patntirapong et al.

    Zoledronic acid suppresses mineralization through direct cytotoxicity and osteoblast differentiation inhibition

    J Oral Pathol Med

    (2012)
  • S.J. Wimalawansa

    Insight into bisphosphonate-associated osteomyelitis of the jaw: pathophysiology, mechanisms and clinical management

    Expert Opin Drug Saf

    (2008)
  • J.R. Ross et al.

    Systematic review of role of bisphosphonates on skeletal morbidity in metastatic cancer

    BMJ

    (2003)
  • B.M. Kuehn

    Reports of adverse events from bone drugs prompt caution

    JAMA

    (2006)
  • G.S. Wilkinson et al.

    Intravenous bisphosphonate therapy and inflammatory conditions or surgery of the jaw: a population-based analysis

    J Natl Cancer Inst

    (2007)
  • S.B. Woo et al.

    Systematic review: bisphosphonates and osteonecrosis of the jaws

    Ann Intern Med

    (2006)
  • Cited by (74)

    • Preclinical models of medication-related osteonecrosis of the jaw (MRONJ)

      2021, Bone
      Citation 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.

    • Conditioned medium from mesenchymal stem cells improves condylar resorption induced by mandibular distraction osteogenesis in a rat model

      2021, Heliyon
      Citation Excerpt :

      Serum-free conditioned medium from human MSCs (MSC-CM) has been reported to serve multiple positive functions [13, 14]. In particular, studies conducted till date have supported the theory that the paracrine factors included in MSC-CM are important for turnover of local bone status [15, 16]. We have reported that MSC-CM contains numerous growth factors such as insulin growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), and transforming growth factor-β1 (TGF-β1), which accelerate osteoblastic differentiation and bone regeneration [13, 14].

    • PDGF-BB exhibited therapeutic effects on rat model of bisphosphonate-related osteonecrosis of the jaw by enhancing angiogenesis and osteogenesis

      2021, Bone
      Citation Excerpt :

      BPs, especially nitrogen-containing bisphosphonates was widely reported to inhibit endothelial cells via RhoA and MAPK signaling and block the activity of farnesyl pyrophosphate synthase (FPPS) [24,25]. And lower VEGF concentrations in serums were found in BRONJ patients and animal models [7,13] However, anti-angiogenic effect of BPs was generally observed in a high concentration in vitro [25]. Another study suggested that anti-angiogenesis alone was not the main cause of osteonecrosis of jaw (ONJ) but the combination of anti-angiogenesis and suppressed osteoclasts or other risk factors [26].

    View all citing articles on Scopus
    View full text