Development of oral osteomucosal tissue constructs in vitro and localization of fluorescently-labeled bisphosphonates to hard and soft tissue.

SUSAN BAE,NO-HEE PARK,JU-EUN OH,KI-HYUK SHIN,SOTIRIOS TETRADIS,SHUTING SUN,REUBEN H. KIM,MO K. KANG,CHARLES E. McKENNA,TARA AGHALOO

doi:10.3892/ijmm.2014.1802

Abstract

Bisphosphonates (BPs) are anti-resorptive agents commonly used to treat bone-related diseases; however, soft tissue-related side-effects are frequently reported in some BP users, such as oral or gastrointestinal (GI) ulcerations. BPs are stable analogs of pyrophosphate and have high affinity to hydroxyapatite, allowing them to bind to the bone surfaces and exert suppressive effects on osteoclast functions. However, the underlying mechanisms as to how bone-seeking BPs also exert cytotoxic effects on soft tissue remain unknown. In the present study, we investigated the localization of nitrogen-containing BPs (N-BPs) in hard and soft tissue using fluorescently-labeled N-BPs in vitro. We developed osteomucosal tissue constructs in vitro to recapitulate the hard and soft tissue of the oral cavity. A histological examination of the osteomucosal tissue constructs revealed a differentiated epithelium over the bone containing osteocytes and the periosteum, similar to that observed in the rat palatal tissues. Following treatment with the fluorescently-labeled bisphosphonate, AF647-ZOL, the osteomucosal constructs exhibited fluorescent signals, not only in the bone, but also in the epithelium. No fluorescent signals were observed from the control- or ZOL-treated constructs, as expected. Collectively, the data from the present study suggest that N-BPs localize to epithelial tissue and that such a localization and subsequent toxicity of N-BPs may be associated, at least in part, with soft tissue-related side-effects.

Highlights

Bisphosphonates (BPs) are the most widely used anti-resorptive agents for osteoporosis, a skeletal disorder characterized byKey words: osteomucosal tissue constructs, soft tissue, hard tissue, bisphosphonates, osteonecrosis of the jaw, ulceration, toxicity compromised bone strength with an increased risk of fracture [1,2]
The HOK-16B cells and normal human oral keratinocytes (NHOKs) were grown in keratinocyte growth medium (KGM; Clonetech, San Diego, CA, USA), and the normal human oral fibroblasts (NHOFs) were grown in Dulbecco's modified Eagle's medium (DMEM/199) (4:1) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA)
Such histological findings were comparable to the in vivo oral osteomucosal tissue from the rat palates (Fig. 2A-a, -a1 and -a2), indicating that the in vitro osteomucosal tissue constructs recapitulated the in vivo histological architecture of the soft and hard tissue in the oral cavity

Summary

Introduction

Bisphosphonates (BPs) are the most widely used anti-resorptive agents for osteoporosis, a skeletal disorder characterized by. With frequent use of BPs in multiple clinical settings, a growing body of evidence supports the notion that some N-BPs cause side-effects that are specific to soft tissue [5]. One of the major side-effects of orally administered BPs is inflammation and ulcerations of the gastrointestinal (GI) tract and, to a lesser extent, oral mucosa. GI toxicity is one of the major reasons patients drop out of N-BP clinical trials [6]. Esophageal inflammation and ulcerations are frequently reported in N-BP users [7], and oral ulcerations are observed in patients who suck BP tablets [8]

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