Laboratory methods and biomarkers in the evaluation of bisphosphonate effects on body tissues: a literature reviewby Ana Carolina Uchoa Vasconcelos, Soraya de Azambuja Berti-Couto, Maria Antonia Figueiredo, Fernanda Gonçalves Salum, Tiago Giuliani Lopes, Karen Cherubini

J Oral Pathol Med

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REVIEW

Laboratory methods and biomarkers in the evaluation of bisphosphonate effects on body tissues: a literature review

Ana Carolina Uchoa Vasconcelos1, Soraya de Azambuja Berti-Couto1, Maria Antonia Figueiredo1,

Fernanda Goncalves Salum1, Tiago Giuliani Lopes2, Karen Cherubini1 1Postgraduate Program, Dental College, Pontifical Catholic University of Rio Grande do Sul - PUCRS, Porto Alegre, RS, Brazil; 2Department of Pathology, School of Medicine, Hospital S~ao Lucas, Pontifical Catholic University of Rio Grande do Sul - PUCRS, Porto

Alegre, RS, Brazil

Bisphosphonates are extensively used to treat bone metabolism disorders; meanwhile, these drugs have important side effects such as jaw osteonecrosis and femoral fracture. Therefore, studies have been conducted aimed at better understanding their mechanism of action and determining a course of action to avoid these side effects. We present here a literature review focusing on the laboratory methods available for investigating bisphosphonate effects on body tissues. There are many different methods available for this purpose, but the appropriate application of these methods and knowledge of their limitations are crucial for understanding the effects of bisphosphonates.

J Oral Pathol Med (2013) 42: 577--586

Keywords: biomarkers; bisphosphonates; body tissues; laboratory methods

Introduction

Bisphosphonates are drugs used in the prevention and treatment of bone metabolism diseases with intense resorption activity (1, 2), based on their ability to inhibit both bone resorption (3–5) and angiogenesis (6, 7), even though some effects on non-mineralized tissues have also been reported (8, 9). These compounds are able to interfere with remodeling of bone tissue by acting on different cells (10–12). Their effect on osteoclasts occurs by inhibiting recruitment and differentiation, decreasing life span (4) and promoting apoptosis (10, 13). The mechanism through which bisphosphonates affect osteoblasts is still unclear, but some in vitro studies have demonstrated that high concentrations of nitrogen-containing bisphosphonates inhibit osteoblast proliferation, adhesion, and migration (14, 15), as well as inducing osteoblasts to produce osteoprotegerin (OPG) (16). Studies have shown that zoledronic acid and, to a lesser extent, clodronate have antiangiogenic effects, tending to inhibit the proliferation, migration, and adhesion of endothelial cells (6, 7, 17). Zoledronic acid inhibits their proliferation and migration in vitro in a dose-dependent way (18). Studies have also postulated that bisphosphonates, especially the most potent ones, are able to modulate the secretion of specific growth factors, such as transforming growth factor b1 (TGF-b1) (14).

The adverse effects of these drugs are sporadic and depend on the class of the drug, as well as the route and frequency of administration. Bisphosphonate-related osteonecrosis of the jaws was first described in 2003 (19) and is characterized by an area of exposed bone in the maxillofacial region, which persists for at least 8 weeks in patients undergoing therapy with these drugs and with no history of head and neck radiation therapy (20–22). Several theories have been proposed to explain the pathogenesis of this lesion, whose etiology is multifactorial. It is believed that the impact of the drug on different cell types is capable of contributing to the difficulty of tissue healing after the damage, resulting in clinical manifestation of the disease (23).

Atypical non-traumatic femoral fractures have also been recognized as an important side effect of bisphosphonate use (24–26). These fractures affect the diaphyseal (subtrochanteric or shaft) part of the femur (27) and, although involving a very small number, they are indeed associated with bisphosphonate use and have shown increased incidence over a 12-year period (25), which is directly related to the longer duration of the drug therapy (27). The problem possibly results from the incapacity of bone to heal physiological microcracks, which leads to clinically apparent stress fractures in areas with high mechanical stress loads, such as the outer cortex of the femoral shaft (25).

Considering the risk–benefit relationship, including the low incidence of both atypical femoral fractures and jaw osteonecrosis, and the risk and rate of serious hip and other

Correspondence: Karen Cherubini, Servico de Estomatologia, Hospital S~ao

Lucas PUCRS, Av Ipiranga, 6690, sala 231, Porto Alegre, RS, Brazil. CEP: 90610-000, Tel/Fax: 55(51)33203254, E-mail: karen.cherubini@pucrs.br

Accepted for publication November 17, 2012 doi: 10.1111/jop.12031

J Oral Pathol Med (2013) 42: 577–586 © 2012 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd wileyonlinelibrary.com/journal/jop fractures in elderly osteoporotic patients, it is believed that patients at risk of osteoporotic fractures should not be discouraged from initiating bisphosphonates (27). Nevertheless, these side effects and the challenge of treating osteonecrosis point to the need of better understanding the mechanism of action of these drugs and call for research into their effects on biological tissues (28), to establish criteria to guide the management of patients on bisphosphonates (29). This study reviews the scientific literature with regard to laboratory methods and biomarkers available for the evaluation of biological effects of bisphosphonates. The literature was searched for studies of techniques and markers involved in the main effects of bisphosphonates, that is, bone turnover (3–5) and angiogenesis inhibition (6,7), and they are discussed focusing on their indications, limitations, advantages, and disadvantages. The strategy of the search used structures, cells, and tissues that are the major targets of the drug and also bisphosphonate as key words to achieve the respective markers.

Evaluation of bisphosphonate effects on bone cells

Hematoxylin–eosin evaluation (H&E)

On H&E examination, bisphosphonate-related osteonecrosis shows non-vital bone tissue infected by microorganisms, and inflammatory areas as well (30, 31). In bisphosphonate users without osteonecrosis, bone tissue shows some morphologic changes, with osteoclasts being the focus of attention. In this examination, they appear as acidophilic multinucleated giant cells (Fig. 1a). The active osteoclasts are found intimately associated with the bone surface through their active pole. At this site, the ruffled border and the clear zone are noted, which is a small area outlining the region to be resorbed. The osteoclast–bone interface is poorly stained and is not easily seen. The inactive osteoclasts, in turn, do not stay in contact with bone structure and tend to assume a rounded shape. The H&E technique is easily performed, has low cost, and also permits the visualization of active and inactive osteoclasts, an important feature regarding bisphosphonates. The major limitation lies in the identification of osteoclasts with just one or no nucleus. In these cases, they could be wrongly classified as pre-osteoclasts or not even be identified (32).