Elsevier

Bone

Volume 51, Issue 3, September 2012, Pages 353-361
Bone

Original Full Length Article
A specific subtype of osteoclasts secretes factors inducing nodule formation by osteoblasts

https://doi.org/10.1016/j.bone.2012.06.007Get rights and content

Abstract

Osteoclasts are known to be important for the coupling process between bone resorption and formation.

The aim of this study was to address when osteoclasts are anabolically active.

Human monocytes were differentiated into mature osteoclasts by treatment with M-CSF and RANKL. Conditioned medium was collected from macrophages, pre-osteoclasts, and mature functional or non-resorbing osteopetrotic osteoclasts on either bone, plastic, decalcified bone or dentine with or without diphyllin, E64 or GM6001. Osteoclasts numbers were measured by TRACP activity. Bone resorption was evaluated by CTX-I and calcium release. The osteoblastic cell line 2 T3 was treated with 50% of CM or non-CM for 12 days. Bone formation was assessed by Alizarin Red extraction.

CM from mature osteoclasts induced bone formation, while CM from macrophages did not. Non-resorbing osteoclasts generated from osteopetrosis patients showed little resorption, but still an induction of bone formation by osteoblasts. Mimicking the reduction in bone resorption using the V-ATPase inhibitor Diphyllin, the cysteine proteinase inhibitor E64 and the MMP-inhibitor GM6001 showed that CM from diphyllin and E64 treated osteoclasts showed reduced ability to induce bone formation compared to CM from vehicle treated osteoclasts, while CM from GM6001 treated osteoclasts equaled vehicle CM. Osteoclasts on either dentine or decalcified bone showed strongly attenuated anabolic capacities.

In conclusion, we present evidence that osteoclasts, both dependent and independent of their resorptive activity, secrete factors stimulating osteoblastic bone formation.

Highlights

► Only mature osteoclasts are sources of anabolic stimuli for osteoblasts. ► Osteopetrotic osteoclasts secrete anabolic factors for osteoblasts. ► Enhanced osteoclast survival compensates for the lack of resorption derived factors. ► Osteoclasts on non-natural substrates fail to secrete anabolic molecules. ► Interaction with matrix strongly modulates anabolic potential of the osteoclasts.

Introduction

Bone remodeling is an essential process for maintaining bone strength. Bone remodeling is a complex process most likely initiated by the osteocytes sensing stress, which then activate the osteoclasts leading to bone resorption [1], [2]. After bone resorption bone formation by the osteoblasts is initiated and the removed bone is completely replaced, a process originally identified and termed coupling by Harold Frost and co-workers [1], [3], [4], [5], [6]. Further evidence that coupling was to some extent controlled locally in bone was published by the Baylink group, who showed that bone organ cultures with activated resorption, secreted a bone anabolic molecule [7].

Alterations in the balance between bone resorption and bone formation result in pathologies, of which the most common is osteoporosis, where the osteoblasts are unable to counter the excessive bone resorption taking place [8]. Another situation, where the balance is destroyed, is the much rarer cases of osteoclast-rich forms of osteopetrosis [9]. In these cases bone resorption is strongly reduced, often due to defective acid secretion by the osteoclasts, while osteoclast numbers are increased [9]. Importantly, bone formation by the osteoblasts is often increased [8]. Furthermore, it was shown that the number of mature bone forming osteoblasts in these patients correlated to the number of non-resorbing osteoclasts [10], [11]. These findings highlight that anabolic signaling from the osteoclasts is not strictly mediated by bone resorption, but most likely also involves the presence of osteoclasts [8]. Additionally, a study conducted in adult mice indicated that when the mature non-resorbing osteoclasts are present, this leads to increased bone formation resulting in stronger bones [12]. Furthermore, studies conducted in c-fos deficient mice, which have macrophages but no osteoclasts, showed reduced bone formation, as well as failure to provide an anabolic response to PTH, while c-src deficient mice, which have non-resorbing osteoclasts, showed a full anabolic response to PTH treatment [13], [14], [15]. Finally, these data are supported by early findings in humans with no osteoclasts, where the bone phenotype is milder than that observed in osteoclast-rich osteopetrosis, despite both phenotypes having no bone resorption [16], [17].

Within recent years, several studies have highlighted that an osteoclasts are different depending on the circumstances, a phenomenon termed osteoclast subtypes [8]. These studies have indicated that resorptive activity, acid transport and proteolytic machinery and more parameters are highly influenced by the origin of the osteoclast as well as the interaction with the matrix [18], [19], [20], [21], [22], [23], [24], [25], [26], [27]. However, how the differences in osteoclast subtype influence the anabolic activity of the osteoclasts is presently unclear.

Following the paradigm shift in the understanding of the coupling between osteoclasts and osteoblasts, several studies have studied local communication between osteoclasts and osteoblasts, and how different parameters, such as bone resorption and osteoclast subtype, regulate this process [8]. Several studies have indicated different molecular candidates, including TGF-β, the IGFs, Wnt10b, BMP-6, S1P, TRACP, Cardiotrophin-1 and bidirectional signaling through the ephrin-eph system, and several of these are independent of bone resorption [6], [28], [29], [30].

When it comes to how much of the coupling signal that is derived from osteoclasts and how much that originates from bone resorption, this is still unclear. However, it was shown that cathepsin K inhibitors lead to increased secretion of bone matrix derived anabolic molecules, such as IGF-1, as the inhibitors prevented degradation of these factors [31], although it presently is unclear whether this has any relevance in vivo [32], [33].

With these things in mind, this study focused on providing a thorough investigation of when the osteoclasts secrete anabolic molecules. Importantly, we utilized naturally non-resorbing osteoclasts, as well resorption inhibitors to investigate the role resorption in this process. Furthermore, we investigated whether seeding the osteoclasts on different matrices, including the non-remodeled matrix dentine and the “artificial” matrix decalcified bone, modulated the ability of the osteoclasts to secrete anabolic molecules.

Section snippets

Chemicals and other reagents

Diphyllin was from Bioduro, Beijing, China, GM6001 and E64 from Calbiochem. All other materials were from Sigma-Aldrich unless otherwise specified.

CD34+ Cell isolation

Samples of peripheral blood from an Infantile Malignant Osteopetrosis patient or umbilical cord blood from normal deliveries were obtained after informed consent under protocols approved by institutional ethical boards. Mononuclear cells from these cell sources were isolated on Ficoll gradient, and subsequently CD34+ cells were separated from the

The appearance of the bone anabolic signal correlates with the appearance of osteoclasts

Previous studies have shown that osteoclasts secrete anabolic signals independent of bone resorption [29], [30]; however, at what time of development and whether macrophages possess this ability when cultured long term is not known. To shed light on the time profile of secretion of anabolic molecules by osteoclasts and the corresponding macrophages, we cultured CD14+ monocytes on bone slices in the presence or absence of RANKL, while we collected the conditioned media (CM) and the corresponding

Discussion

In this study we investigated the subtype of osteoclast needed from communication from osteoclasts to osteoblasts in detail, and we here show that osteoclasts, both dependently and independently of bone resorption produce bone anabolic signals to osteoblasts. Furthermore, our data show that the interaction between osteoclasts and culture matrix is highly important, as matrices such as dentine and decalcified bone attenuated the ability of the osteoclasts to produce anabolic signals.

These

Author contributions

KH and AVN devised most of the experiments and assisted in data analysis. KH wrote the manuscript. KVA and CECH performed the majority of the experiments and analyzed data. CST and NSG performed the CD34+ osteoclast experiments. JH, IM, AS provided and expanded the CD34+ cells from both IMO and control. MHD provided human blood. MAK participated in data analysis.

All authors have read, commented and approved the manuscript.

Conflicts of Interest

Morten A. Karsdal owns stock options in Nordic Bioscience A/S, all other authors have no conflicts of interest.

Acknowledgments

We thank the Danish Research Foundation (Den Danske Forskningsfond) for support of Kim Henriksen and Kim V. Andreassen. The support from the Ministry of Science, Technology and Innovation for the PhD education of the PhD education of Anita V. Neutzsky-Wulff and Catherine E. Crüger-Hansen is acknowledged. Christian S. Thudium is on a PhD Scholarship partially funded by Nordforsk. IM received funding by Blanceflor. JR was supported by grants from The Swedish Childhood Cancer Foundation, a

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