Elsevier

Bone

Volume 59, February 2014, Pages 37-43
Bone

Original Full Length Article
Bisphosphonate-osteoclasts: Changes in osteoclast morphology and function induced by antiresorptive nitrogen-containing bisphosphonate treatment in osteoporosis patients

https://doi.org/10.1016/j.bone.2013.10.024Get rights and content

Highlights

  • Osteoclast morphology and resorption parameters were assessed in 23 paired iliac crest bone biopsies from patients on bisphosphonate treatment.

  • After BP treatment resorption indices decreased significantly, while the number of osteoclasts remained unchanged.

  • Characteristic ‘giant-osteoclasts’ weren't found in healthy individuals and untreated osteoporosis patients, but in 39% of patients on bisphosphonate therapy.

  • BP-treated subgroup with ‘giant-osteoclasts’ presented pronounced reduction in resorption indices, but not entirely inhibited resorption capability.

  • Disturbed bisphosphonate-osteoclasts' viability and affinity to bone may suggest them as cells in a prolonged apoptotic process.

Abstract

Osteoclasts are unique cells capable of bone resorption and therefore have become a major target in osteoporosis treatment strategies. Bisphosphonates suppress bone turnover via interference with the internal enzymatic cell system of osteoclasts leading to cytoskeletal disruption. This mechanism found its clinical relevance in reducing bone resorption, stabilizing bone mass and reducing fracture risk in osteoporosis patients. However, knowledge about specific in vivo changes in osteoclast cell morphology and function is still insufficient. We examined osteoclasts in 23 paired bone biopsies from osteoporosis patients (18 males, 5 females; age: 52.6 ± 11.5 yrs) under nitrogen-containing bisphosphonate administration with a mean treatment duration of three years. Formalin-fixed, undecalcified sections were assessed by qualitative and quantitative bone histomorphometry, where the osteoclast morphology, nuclei, distribution, location as well as resorption parameters were investigated to obtain information about cell function and viability. After three years of treatment, resorption parameters decreased significantly while the number of osteoclasts remained unchanged. Out of 23 patients, nine developed previously termed “giant-osteoclasts” with increased size, numerous nuclei (> 10 nuclei/Oc) and oftentimes detachment from the bone surface. These cells frequently had pycnotic nuclei and other morphological signs suggestive of osteoclast apoptosis. Characteristic large-sized osteoclasts were uniquely found in patients treated with nitrogen-containing bisphosphonates, thus being clearly distinguishable from giant-osteoclasts in other bone disorders such as Paget disease, secondary hyperparathyroidism or osteopetrosis. The resorption indices of large-sized osteoclasts, specifically the eroded perimeter and erosion depth, revealed significantly reduced values but not an entirely inhibited resorption capability. Bisphosphonate-osteoclasts' viability and affinity to bone seem significantly disturbed while the apoptotic process may be prolonged for a yet unknown period of time in favor of maintaining a low bone turnover.

Introduction

Bisphosphonates (BP) as a treatment for osteoporosis have been in clinical use for over two decades and proven to be effective in fracture prevention [1], [2], [3]. Following early laboratory experiments by Fleisch et al. in the late 1960s [4], several bone markers and histomorphometry studies have shown the antiresorptive action of BP. Based on their effects on osteoclasts' (Oc) function, BP were reported to cause a general reduction in bone turnover, the degree of which can be perceived as a surrogate parameter for treatment efficacy [5], [6], [7], [8], [9], [10], [11]. BPs are incorporated into bone for an extended period of time and continue to affect bone metabolism long after cessation of intake [11], [12]. The substance is relatively safe, however, possible osteonecrosis of the jaw in oncology patients under high-dose BP administration and atypical femoral fractures are nowadays believed to be caused by oversuppression of bone remodeling — although this is still a matter of debate [11], [13], [14], [15], [16], [17], [18], [19].

BP molecules have a strong affinity to the bone surface [20] where nitrogen-containing BPs (N-BP) once assimilated by osteoclasts inhibit the mevalonate pathway enzyme farnesyldiphosphate (FPP) synthase and disrupt the cytoskeleton which affects the ruffled border and results in a loss of function [21]. Given that the osteoclasts/osteoblasts ratio in histological sections is approximately 1:6 [22] and since osteoclasts display a three- to four-fold shorter lifespan than osteoblasts, the cells capable of bone resorption are rare in histological sections [23], [24]. The short time span of Oc apoptosis makes this brief stage a rarely observed event in histological sections which makes it difficult to acquire large subgroups of this cell type with special findings. Despite extensive research on both the therapeutic benefits and side effects of BP, the true nature of their effects on osteoclast morphology, vitality, life-span and fate remains inconclusive.

Following earlier histological studies that described treatment-induced changes in the Oc phenotype, Weinstein et al. reported data on “giant osteoclast formation after long-term oral BP therapy” [25]. Weinstein's study in a clinical setting frequently showed morphological changes and apoptosis of osteoclasts, as well as an increase in Oc number following BP treatment. Similar morphological cell changes have also been observed by others and ourselves [26], [27], [28], [29]. However, so far a number of questions regarding the affected osteoclasts remain unanswered, such as i) how the bisphosphonates affect Oc function as reflected in erosion surface and resorption depth in osteoporotic patients, and ii) whether there is discordance between osteoclast number and anti-resorptive effects. In addition, the effect of BP in a male cohort without the influence of hormonal changes, the role of intravenous BP administration, as well as the influence of different BP classes all require proper attention [30], [31].

To directly explore the effects of N-BP treatment on osteoclasts' morphology and function in each individual, we conducted a histomorphometric paired-biopsy study in osteoporosis patients before and after the anti-resorptive bisphosphonate treatment.

Section snippets

Material and methods

Bone biopsies from the dorsal iliac crest were acquired by an experienced physician performing the Jamshidi technique from 37 patients at one institution, as described previously [32]. Patients had osteoporosis as defined by the WHO and confirmed by dual X-ray absorptiometry. For each patient, a baseline biopsy and a follow-up biopsy were available. In the treatment group, 23 patients (18 males, 5 females; age: 52.6 ± 11.5 yrs) underwent bone biopsy at two time points: at baseline (before starting

Osteoclast morphology and resorption patterns

Unusual osteoclast morphology or hypernucleation (> 3–4 nuclei) was not observed in the two control groups or at baseline in the treatment group. However, in 39.1% of patients in the N-BP treatment group, there was an obvious increase in osteoclast cell size and shape, as well as in the number of nuclei (> 10 nuclei/Oc) in follow-up biopsies (Fig. 2A). Six out of nine patients who developed bisphosphonate osteoclasts had received oral alendronate treatment, while the remaining three received

Discussion

Osteoclasts play a pivotal role in bone remodeling and are key factors in many bone disorders such as osteoporosis, which is of major interest to the health care community. In this paired-biopsy study of osteoporosis patients, we focused on osteoclast morphology under the influence of antiresorptive treatment with nitrogen-containing bisphosphonates and expand knowledge about Oc vitality, life cycle and implications. Osteoclastogenesis, which includes the development, differentiation and

Conclusion

In paired biopsies from a cohort of osteoporosis patients, we assessed changes in osteoclast morphology induced by nitrogen-containing bisphosphonate treatment. Characteristic large osteoclasts as described here, are uniquely found in patients under bisphosphonate-treatment and were termed ‘Bisphosphonate-Osteoclasts’ to clearly distinguish them from giant osteoclasts in other bone disorders, such as Paget's disease, secondary hyperparathyroidism or osteopetrosis [71], [72], [73]. Our study

Conflict of interest

All authors state that they have no conflicts of interest.

Acknowledgments

Dr. Petar Milovanovic is a fellow of the DAAD (Deutscher Akademischer Austauschdienst — German Academic Exchange Service; A/11/83161), Serbian Ministry of Education and Science (III45005), and acknowledges the support from the European Federation for Experimental Morphology (EFEM Travel Grant 2013). Dr. Björn Busse is a fellow of the DFG — Emmy Noether program (Deutsche Forschungsgemeinschaft — German Research Foundation; BU 2562/2-1). This study was supported by grants from the

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