Bisphosphonate effects on the behaviour of oral epithelial cells and oral fibroblasts

https://doi.org/10.1016/j.archoralbio.2010.11.003Get rights and content

Abstract

Objective

Bisphosphonates (BPs) like Zometa (ZA) are widely used to treat complications of bony metastases in cancer patients. A serious adverse event occurs in 1–12% of patients on BP therapy, osteonecrosis of the jaw (BPONJ). BPONJ develops after oral trauma and is manifested by poor wound healing and soft-tissue breakdown followed by exposure and necrosis of intra-oral bone. Currently, there is no effective clinical treatment for BPONJ.

Design

We evaluated the effect of ZA on the proliferation, apoptosis and migratory capacity of the cell lines CRL-7408, an oral fibroblast culture and OKF/6, an oral epithelial cell line.

Results

In both oral epithelium and fibroblasts, ZA exposure inhibited proliferation and elevated apoptosis; however oral fibroblasts were differentially influenced versus oral epithelial cells. In oral fibroblasts, ZA treatment significantly inhibited motility. Further, quantitative real-time PCR demonstrated that ZA treatment of oral fibroblasts inhibits expression of both the COL1A1 and COL1A2 chains of type-I collagen, consistent with a loss of collagen immunofluorescent staining.

Conclusions

These data support a model wherein ZA treatment impedes oral wound healing by blocking the growth and migratory capacity of oral fibroblasts as well as downregulating the transcription of type-I collagen, functions necessary to deposit the granulation tissue needed for re-epithelization. Therefore, BP released from bone following tooth extraction may delay wound healing of the oral mucosal barrier and contribute to BPONJ pathogenesis.

Introduction

Bisphosphonates (BPs) such as zoledronic acid (ZA: Zometa) are widely used to treat complications of bony metastases in cancer patients, with 3 million patients receiving BP therapy worldwide.1 BPs bind bone with high affinity and mitigate osteoclastic activity, thereby slowing skeletal remodelling and turnover.2 Intravenous (IV) BPs have also shown significant efficacy in reducing bone pain, hypercalcemia and fractures secondary to bony metastases.3 Indeed, monthly IV administration of BP is the standard of care, recommended by the American Society of Clinical Oncology clinical practice guidelines, for treatment of malignancy-associated hypercalcemia and metastatic osteolytic lesions.1, 4, 5 Orally administered, and to a lesser extent IV, BPs are widely used for the management of osteoporosis.2 According to the American Dental Association, more than 190 million prescriptions have been written for BPs worldwide.6

Recent studies have revealed a serious adverse event in patients receiving IV BP therapy, bisphosphonate-associated osteonecrosis of the jaw (BPONJ).1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 BPONJ, defined as exposed nonviable maxillomandibular bone, is a debilitating, painful condition that affects 1–12% of the patients receiving IV BP therapy.6, 9 BPONJ is manifested by poor oral wound healing (particularly following invasive dental procedures), oral soft-tissue breakdown, and exposure of the underlying intra-oral bone, culminating in necrosis of the exposed bone. A working definition of BPONJ has been standardized by the American Association of Oral and Maxillofacial Surgeons and includes prior or current treatment with a BPs followed by persistent (>8 weeks) exposed necrotic bone in the maxillomandibular region, in the absence of radiation therapy to the jaws.1 Early stages of BPONJ are difficult to note via conventional radiography, which will not detect alterations until 30–50% of the bone is demineralized.1, 12 BPONJ patients present with exposed necrotic bone, sites ranging in size from mm to cm, often with surrounding inflammation and pain. Systemic antibiotic treatment and/or an oral antimicrobial rinse are used in patients with asymptomatic exposed bone.10 As debridement is usually unsuccessful and carries the potential for further exposure of healthy bone, a conservative palliative approach is often recommended.10 Currently there is no highly effective clinical treatment for advanced BPONJ, and management may be limited to analgesia and control of disease progression, as complete healing may never occur.10

Presently, as the underlying pathophysiology of BPONJ is uncharacterized, there is a significant gap in our knowledge of the aetiology of BPONJ disease. To evaluate the role of BP therapy in oral wound healing, the current study evaluated the effect of ZA on the proliferation, apoptosis and migratory capacity of CRL-7408, an oral fibroblast cell line and OKF/6, a cell line derived from the oral epithelium. These results suggest a mechanism wherein ZA may affect oral wound healing via the inhibition of interstitial collagen synthesis by oral fibroblasts. In addition to providing an integrative in vitro analysis of the effects of BPs on repair and remodelling of oral soft tissues, ultimately we aim to identify risk factors and novel treatment strategies related to the initiation and progression of BPONJ.

Section snippets

Cell cultures and treatments

Human oral fibroblasts (CRL-7408) were obtained from American Type Culture Collection (ATCC, Manassas, VA) and were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% foetal bovine serum, 100 unit/ml penicillin and 100 μg/ml streptomycin. Human oral epithelial cells (tert-immortalized) were the generous gift of Dr. James Rheinwald of Harvard University (Boston, MA). These OKF/6 cells were cultured in keratinocyte-SFM (Gibco, Carlsbad, CA) supplemented with 25 μg/ml

Effect of ZA on proliferation and apoptosis of oral cells

To examine the potential consequences of bisphosphonate treatment on cell populations in the oral cavity, the effect of ZA treatment on oral epithelial cells (OKF/6) and oral fibroblasts (CRL-7408) was evaluated. Cells were cultured for 24 h in the presence of increasing concentrations of ZA (0–100 μM) followed by analysis of viability/proliferation using a MTS assay. Significant toxicity was observed with ZA concentrations as low as 10 μM (Fig. 1A and B). Oral fibroblasts were more susceptible

Discussion and conclusions

In the current study, we evaluated the effect of ZA on the proliferation, apoptosis and migratory capacity of oral fibroblasts (CRL-7408) and oral epithelial cells (OKF/6). Our results show a differential effect of ZA on these distinct cell populations. Although proliferation of both cell types was inhibited in a ZA concentration-dependent manner, oral fibroblasts were significantly more susceptible to ZA-induced apoptosis relative to oral epithelial cells. This is distinct from a recent study

Funding

University of Missouri Research Board, Department of Pathology and Anatomical Sciences, University of Missouri School of Medicine.

Competing of interest

Not declared.

Ethical approval

Not required.

Acknowledgments

Supported by the Department of Pathology and Anatomical Sciences at the University of Missouri School of Medicine as well as the University of Missouri Research Board (MJR). Dr. James Rheinwald of Harvard University is thanked for access to the OKF/6 cells used in this research. Dr. Robert Zitsch III, Department of Otolaryngology at the University of Missouri School of Medicine, Dr. Thomas Southard, Department of Orthodontics at the University of Iowa School of Dentistry, and two anonymous

References (33)

  • S.L. Ruggiero et al.

    Bisphosphonate-related osteonecrosis of the jaw: diagnosis, prevention, and management

    Annu Rev Med

    (2009)
  • H.A. Fleisch

    Bisphosphonates: preclinical aspects and use in osteoporosis

    Ann Med

    (1997)
  • J. Green et al.

    The molecular basis of bisphosphonate activity: a preclinical perspective

    Semin Oncol

    (2010)
  • M.A. Scheper et al.

    Effect of zoledronic acid on oral fibroblasts and epithelial cells: a potential mechanism of bisphosphonate-associated osteonecrosis

    Br J Haematol

    (2009)
  • C.M. Novince et al.

    Osteonecrosis of the jaw: an update and review of recommendations

    Cells Tissues Organs

    (2009)
  • ...
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