Localized,tumor-associated osteolysis involves the recruitment and activation of osteoclasts

The cellular and biochemical mechanisms that direct destruction of bone at the site of tumor osteolysis are unknown. In order to understand this process better, a murine model designed for the study of tumor osteolysis was developed and the influence of osteolytic and nonosteolytic tumors on bone was investigated. Tumors developed following femoral intramedullary injection of sarcoma (2472) and melanoma (G3.26) cell lines: however, only tumors from the 2472 cell line caused osteolysis. It was determined that 2472 tumor-induced osteolysis commenced 6 days after the femora had been inoculated with 2472 cells. There were more osteoclasts per millimeter of bone surface in 2472 tumor-bearing limbs (16.7 ± 5.0) than in sham-injected limbs (3.8 ± 0.9) (p < 0.015). In addition, an increase in the osteoclast size (area) was detected in 2472 tumor-bearing limbs: 412 ± 65 μm² compared with 187 ± 17 μm² (p < 0.01). In vitro bone resorption experiments indicated that 2472 tumor cells had a limited ability to destroy bone in comparison with macrophages and osteoclasts. Taken in total, these findings define a model that is useful for the study of tumor osteolysis, and the data from analyses of the model demonstrate that the cellular mechanisms responsible for 2472 tumor-induced osteolysis include both an increase in the number of osteoclasts and activation of mature osteoclasts.     

Osteoclasts are required for bone tumors to grow and destroy bone

It has been hypothesized that bone resorption during tumor osteolysis is performed by osteoclasts. Data supporting this hypothesis have been provided from analysis of human biopsy specimens obtained from sites of tumor osteolysis, as well as from experimentation with in vivo animal models. Experiments in this report take this concept one step further by testing the hypothesis that osteoclasts are required for bone tumors to grow and destroy bone. To test this hypothesis, the influence of an osteolytic sarcoma tumor, NCTC clone 2472 (2472). on bone was studied in animals that are osteoclast deficient (microphthalmic, strain B6C3Fe-a/a-Mitf^mi) but whose osteoclast deficiency can be reversed following bone marrow transplantation. Femora of these mice and unaffected wild-type siblings were injected with 10⁵ 2472 cells, and after 14 days the femora were analyzed by radiographic and histomorphometric analysis. Macroscopic tumor, tumor-induced osteolysis, and increased osteoclast number were noted in femora of normal mice but not in femora of osteoclast-deficient mice (p < 0.001). Bone marrow transplantation converted osteoclast-deficient mice to mice with femora that contained osteoclasts in 4 weeks. Femora of these mice were then injected with 10⁵ 2472 tumor cells; after 14 days, in contrast to the findings in the original osteoclast-deficient mice, macroscopic tumor was present, tumor-induced osteolysis was noted on roentgenograms, and osteoclast number was increased when tumor-bearing limbs were compared with sham-injected limbs (p < 0.001). These data prove the hypothesis that osteoclasts are required for 2472 tumor-induced osteolysis, and they introduce the exciting possibility that osteoclasts are also required for tumors to grow in bone.     

Osteoclast formation during tumor osteolysis does not require proliferating osteoclast precursor cells

The cellular mechanism or mechanisms through which tumors induce osteoclast formation at sites of tumor osteolysis is unknown. To test the hypothesis that osteoclast formation at sites of tumor osteolysis reflects influences that tumors have on proliferating osteoclast precursor cells, a novel in vivo experimental model was developed that produced mice that were deficient in osteoclasts (op/op) and were depleted (by way of total body irradiation) of proliferating osteoclast precursor cells. The femora of irradiated op/op mice were injected with tumor cells (2472 clone) that had been previously shown to form osteolytic tumors and to induce focal osteoclastogenesis, and the influence of these tumor cells on osteoclast formation was determined in op/op mice that were depleted of proliferating osteoelast precursor cells. The results indicated that 2472 tumor cells induced osteoclast formation in op/op mice despite the absence of proliferating osteoclast precursor cells. This finding disproved the hypothesis under investigation and suggests that osteoclast formation at sites of tumor osteolysis reflects influences of tumors on postmitotic, not proliferating, osteoclast precursor cells.     

Human breast cancer induces osteoclast activation and increases the number of osteoclasts at sites of tumor osteolysis

The cellular mechanism through which osseous breast cancer metastases induce the focal destruction of bone (tumor osteolysis) is unknown. An athymic mouse model designed for the study of tumor osteolysis was developed and the influence of two human breast cancer tumors on bone was studied. Tumor-induced osteolysis occurred between 7 and 10 weeks after inoculation of mouse femora with MDA-MB-231 or MDA-MB-435s breast cancer cells. An increase in osteoclast number and an increase in osteoclast size (area) were detected when tumor-bearing and sham-injected limbs were compared. In vitro analysis of the influence of the tumor-conditioned medium on osteoclast-mediated bone resorption revealed that this conditioned medium stimulated the resorption by increasing both the number of osteoclasts bound to bone and the number of bone resorption pits formed per osteoclast. In addition, in vitro analysis of the influence of breast cancer tumor cells on osteoclast formation or survival, or both, demonstrated that breast cancer cells induced a dramatic increase in the number of osteoclasts detected in culture. Taken in total, these findings suggest that human breast cancer tumors induce osteolysis by enhancing osteoclast adherence to bone, stimulating osteoclast-mediated bone resorption, and either prolonging the survival of osteoclasts or increasing osteoclast formation.     

Insertion Mutation in Tnfrsf11a Causes a Paget's Disease–Like Phenotype in Heterozygous Mice and Osteopetrosis in Homozygous Mice

Early onset familial Paget's disease of bone (EoPDB), familial expansile osteolysis, and expansile skeletal hyperphosphatasia are related disorders caused by insertion mutations in exon 1 of the TNFRSF11A gene, which encodes receptor activator of nuclear factor κB (RANK) protein. To understand the mechanisms underlying these disorders, we developed a mouse model carrying the 75dup27 mutation which causes EoPDB. Mice heterozygous for the mutation (Tnfrsf11a^75dup27/−) developed a PDB-like disorder with focal osteolytic lesions in the hind limbs with increasing age. Treatment of these mice with zoledronic acid completely prevented the development of lesions. Studies in vitro showed that RANK ligand (RANKL)-induced osteoclast formation and signaling was impaired in bone marrow cells from Tnfrsf11a^75dup27/− animals, but that osteoclast survival was increased independent of RANKL stimulation. Surprisingly, Tnfrsf11a^{75dup27/75dup27} homozygotes had osteopetrosis at birth, with complete absence of osteoclasts. Bone marrow cells from these mice failed to form osteoclasts in response to RANKL and macrophage colony-stimulating factor (M-CSF) stimulation. This intriguing study has shown that in heterozygous form, the 75dup27 mutation causes focal osteolytic lesions in vivo reminiscent of the human disorder and extends osteoclast survival independently of RANKL signaling. In homozygous form, however, the mutation causes osteopetrosis due to failure of osteoclast formation and insensitivity to RANKL stimulation. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..     

Review of cellular mechanisms of tumor osteolysis

The cellular and biochemical mechanisms that direct the destruction of bone at sites of tumor osteolysis are unknown. To better understand the mechanisms through which tumors direct bone resorption, research has focused on developing in vivo and in vitro experimental models that are useful for studying this process. In vivo experimental systems have been developed that permit study of tumor osteolysis from human and murine tumors, and that permit the study of tumors that arise from (sarcoma) or can metastasize (breast cancer) to bone. Recent research has focused on three questions: (1) Are osteoclasts or tumor cells responsible for bone resorption during tumor osteolysis? (2) What are the cellular mechanisms that are responsible for bone resorption during tumor osteolysis, and (3) what are the tumor cell products that regulate the cellular mechanisms that are responsible for tumor osteolysis? It has been determined that osteoclasts are responsible for bone resorption at sites of tumor osteolysis by enhancing the binding of osteoclast to bone, by inducing osteoclastic bone resorption, and by stimulating osteoclast formation. Attempts to identify tumor cell products that regulate these cellular mechanisms are in progress, and findings suggest that production of macrophage colony stimulating factor may be required for tumor osteolysis to occur with some tumors.     

IKKβ activation is sufficient for RANK‐independent osteoclast differentiation and osteolysis

Monocytes differentiate into osteoclasts through stimulation of receptor activator of NF‐κB (RANK). Many downstream effectors of RANK play a positive role in osteoclastogenesis, but their relative importance in osteoclast differentiation is unclear. We report the discovery that activation of a single pathway downstream of RANK is sufficient for osteoclast differentiation. In this regard, introduction of constitutively activated IKKβ (IKKβ^SSEE) but not wild‐type IKKβ into monocytes stimulates differentiation of bona fide osteoclasts in the absence of RANK ligand (RANKL). This phenomenon is independent of upstream signals because IKKβ^SSEE induced the development of bone‐resorbing osteoclasts from RANK and IKKα knockout monocytes and in conditions in which NEMO‐IKKβ association was inhibited. NF‐κB p100 and p105, but not RelB, were critical mediators of this effect. Inflammatory autocrine signaling by tumor necrosis factor α (TNF‐α) and interleukin 1 (IL‐1) were dispensable for the spontaneous osteoclastogenesis driven by IKKβ^SSEE. More important, adenoviral gene transfer of IKKβ^SSEE induced osteoclasts and osteolysis in calvariae and knees of mice. Our data establish the sufficiency of IKKβ activation for osteolysis and suggest that IKKβ hyperactivation may play a role in conditions of pathologic bone destruction refractory to RANK/RANKL proximal therapeutic interventions. © 2010 American Society for Bone and Mineral Research     

Stimulation of Expression of the Intestinal Glutamine Transporter ATB0 in Tumor-Bearing Rats

Background Glutamine supplementation ameliorates host catabolic response in tumor bearing states. The purpose of this in vivo study was to investigate intestinal glutamine transport and expression of glutamine transporter ATB⁰ in methyl-cholanthrene (MCA)-sarcoma bearing rats.Methods Fisher-344 rats underwent subcutaneous flank implantation of MCA-sarcoma cells (saline as control) and were pair-fed an equal quantity of chow as controls, to account for tumor-induced anorexia, until tumors reached 10 or 20% body weight. Intestinal mucosal brush border membrane [3H]-Glutamine transport was measured. Glutamine transporter ATB⁰ mRNA and protein levels were measured by real-time PCR and western blot techniques, respectively.Results Glutamine transport activity across the intestinal brush border membrane (BBM) was 3.7-fold higher in tumor-bearing rats (TBR) than in controls (TBR 153 ± 22.6 vs. Control 41.9 ± 9.7 pmol/mg protein/10s, P < .01). Transporter ATB⁰ mRNA levels were 1.4-fold higher in tumor-bearing rats (Relative value TBR .61 ± .12 vs. Control .43 ± .1, P < .05). A 1.4-fold increase in transporter ATB⁰ protein levels was observed in the tumor-bearing rats (Relative value TBR .52 ± .07 vs. Control .37 ± .04, P < .05). Circulating aortic plasma glutamine levels were 1.3-fold higher in tumor bearing rats ([Glutamine] = .63 ± .02 Control vs. [Glutamine] = .74 ± .01 mmol/l TBR, P < .0001). Portal venous plasma glutamine levels were also higher in tumor bearing rats ([Glutamine] = .47 ± .01 Control vs. [Glutamine] = .60 ± .02 mmol/l TBR, P < .0001).Conclusion Intestinal brush border membrane glutamine transport activity, transporter ATB⁰ mRNA and protein levels are up-regulate in tumor-bearing rats.Presented at the 59th Society of Surgical Oncology Annual Meeting, San Diego, CA, March 23–26, 2006.     

Utilizing 808 nm laser for sensitizing of melanoma tumors to megavoltage radiation therapy

Melanotic melanoma has high content of melanin and laser can destroy melanin-containing cells through thermal effect. In this study, the therapeutic effect of 808 nm laser therapy was investigated on B16-F10 melanoma tumor growth and tumor-bearing mice survival time. In addition, as laser can destroy melanin as the main cause of melanoma radioresistance, the effect of laser administration to enhance radiation therapy efficacy at B16-F10 cancer cells was evaluated in vitro and in vivo. Laser therapy (1 W/cm²?×?4 min) could cause significant (P?<?0.05) inhibition of melanoma tumors’ growth (~?61%) and about three times increase of the tumor-bearing mice survival time in comparison with no-treatment group. In addition, the mice which were treated with 1 W/cm²?×?4 min laser administration plus 6 Gy megavoltage radiation therapy exhibited ~?68% lesser tumors’ volume and 27 days increase of survival time in comparison with 6 Gy irradiated tumor-bearing mice. Also, significantly higher (P?<?0.05) tumor necrosis percentage was observed at the histopathological slides of 1 W/cm²?×?4 min laser + RT treated mice tumors (57?±?12%) in comparison with radiation therapy group (31?±?10%). Therefore, not only laser therapy can inhibit melanoma tumors’ growth per se but also its combination with radiation therapy can cause a significant enhancement of radiation therapy efficacy. The laser administration can be used as a radiosensitizing method for melanotic melanoma radiation therapy.

     

Pamidronate decreases tumor-induced osteoclastogenesis in osteopetrotic mice.

Recent studies indicate that the bisphosphonate pamidronate reduces skeletal complications caused by tumor osteolysis. In this investigation, the cellular mechanism through which pamidronate affects tumor-induced osteoclastogenesis is studied in osteopetrotic mice. A unique animal model is employed which studies the effect of pamidronate on a tumor (2472 sarcoma) which induces osteoclastogenesis in osteoclast-deficient mice (oplop). This model provides opportunity to specifically study effects on osteoclast formation and findings suggest that pamidronate decreases the number of osteoclasts at sites of 2472 tumor by decreasing the number of osteoclast precursor cells at the level of myeloid precursors.     

Differential effects of biologic versus bisphosphonate inhibition of wear debris‐induced osteolysis assessed by longitudinal micro‐CT

Aseptic loosening of total joint replacements is caused by wear debris‐induced osteoclastic bone resorption, for which bisphosphonates (BPs) and RANK antagonists have been developed. Although BPs are effective in preventing metabolic bone loss, they are less effective for inflammatory bone loss. Because this difference has been attributed to the antiapoptotic inflammatory signals that protect osteoclasts from BP‐induced apoptosis, but not RANK antagonists, we tested the hypothesis that osteoprotegerin (OPG) is more effective in preventing wear debris‐induced osteolysis than zoledronic acid (ZA) or alendronate (Aln) in the murine calvaria model using in vivo micro‐CT and traditional histology. Although micro‐CT proved to be incompatible with titanium (Ti) particles, we were able to demonstrate a 3.2‐fold increase in osteolytic volume over 10 days induced by polyethylene (PE) particles versus sham controls (0.49 ± 0.23mm³ versus 0.15 ± 0.067mm³; p < 0.01). Although OPG and high‐dose ZA completely inhibited this PE‐induced osteolysis (p < 0.001), pharmacological doses of ZA and Aln were less effective but still reached statistical significance (p < 0.05). Traditional histomorphometry of the sagital suture area of calvaria from both Ti and PE‐treated mice confirmed the remarkable suppression of resorption by OPG (p < 0.001) versus the lack of effect by physiological BPs. The differences in drug effects on osteolysis were largely explained by the significant difference in osteoclast numbers observed between OPG versus BPs in both Ti‐ and PE‐treated calvaria; and linear regression analyses that demonstrated a highly significant correlation between osteolysis volume and sagittal suture area versus osteoclast numbers (p < 0.001). © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1340–1346, 2008     

Low dose metal particles can induce monocyte/macrophage survival

Aseptic loosening results in pain, loss of function, and ultimately prosthetic joint failure and revision surgery. The generation of wear particles from the prosthesis is a major factor in local osteolysis. We investigated the effects of such wear particles on the survival of monocytes and macrophages, populations implicated in wear particle‐driven pathology. Particles from titanium aluminum vanadium (TiAlV) and cobalt chromium (CoCr) alloys were generated in‐house and were equivalent in size (0.5–3 µm) to those seen in patients. Human CD14⁺ monocytes and murine bone marrow‐derived macrophages (BMM) were treated with TiAlV and CoCr particles in vitro, and cell survival was assayed. Both particles increased monocyte and macrophage survival in a dose‐dependent manner, with an optimal concentration of around 10⁷ particles/mL. Conditioned media from particle‐treated BMM also increased macrophage survival. Studies with antibody blockade and gene‐deficient mice suggest that particle‐induced BMM survival is independent of endogenous CSF‐1 (M‐CSF), GM‐CSF, and TNFα. These data indicate that wear particles can promote monocyte/macrophage survival in vitro possibly via an endogenous mediator. If this phenomenon occurs in vivo, it could mean that increased numbers of macrophages (and osteoclasts) would be found at a site of joint implant failure, which could contribute to the local inflammatory reaction and osteolysis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1481–1486, 2009     

IL-34 and CSF-1: similarities and differences

Colony-stimulating factor-1 (CSF-1) is widely expressed and considered to regulate the development, maintenance, and function of mononuclear phagocyte lineage cells such as monocytes, macrophages, dendritic cells (DCs), Langerhans cells (LCs), microglia, and osteoclasts. Interleukin-34 (IL-34) was recently identified as an alternative ligand for the CSF-1 receptor (CSF-1R) through functional proteomics experiments. It is well established that the phenotype of CSF-1R-deficient (CSF-1R^?/?) mice is more severe than that of mice bearing a spontaneous null mutation in CSF-1 (CSF-1^op/op). CSF-1R^?/? mice are severely depleted of macrophages and completely lack LCs, microglia, and osteoclasts during their lifetime. In contrast, CSF-1^op/op mice exhibit late-onset macrophage development and osteoclastogenesis, whereas they show modestly reduced numbers of microglia and a relatively normal LC development. In contrast, IL-34-deficient (IL-34^?/?) mice show a marked reduction of LCs and a decrease in microglia. IL-34 and CSF-1 display different spatiotemporal expression patterns and have distinct biological functions. In this review, we focus on the functional similarities and differences between IL-34 and CSF-1 in vivo.     

Inhibiting wear particles-induced osteolysis with naringin

Purpose

The purpose of this study was to determine the effects of naringin on osteoclastogenesis and osteolysis both in vitro and in vivo.

Methods

In this research osteoclasts were generated from mouse bone marrow monocytes with the receptor activator of NF-КB ligand and the macrophage colony stimulating factor. Naringin, at a concentration of 1, 10, 50, and 100 μg/mL, was respectively added to the medium. Seven days later, the osteoclasts were determined through tartrate-resistant acid phosphatase (TRAP) staining. Mature osteoclasts were isolated from newborn rabbits and cultured for three days on bone slices. Naringin at a concentration of 1, 10, 50, and 100 μg/mL was respectively added to the medium. The resorption bone slices were quantified, and the area was calculated after toluidine blue and Mayer-hematoxylin staining. Polymethyl methacrylate (PMMA) particles were implanted on the calvariae of C57BL/J6 mice. Naringin, at a dose of 50 μg/kg and 100 μg/kg, was respectively given intraperitoneally for seven. Seven days later, the calvariae were removed and processed for pathological analysis.

Results

The result indicated that naringin treatment effectively inhibited in vitro osteoclastogenesis and inhibited mature osteoclasts. In vivo data indicated that naringin strongly inhibited PMMA-induced osteolysis.

Conclusion

Naringin can effectively inhibit osteoclastogenesis and suppress wear particles-induced osteolysis and might be useful in the treatment or prevention of wear particles-induced osteolysis and aseptic loosening for its effect on osteoclast generation and function.     

Antisense oligonucleotide targeting TNF‐α can suppress co‐cr‐mo particle‐induced osteolysis

The most common cause of implant failure in joint replacement is aseptic loosening due to particle‐induced osteolysis. TNF‐α has been shown to be one of the key factors in the process of osteoclastogenesis. Anti‐TNF agents are useful in the treatment of joint inflammation related to osteolysis. This study investigated the effect of a single subcutaneous dose of an antisense oligonucleotide (ASO) on particle‐induced osteolysis. We utilized the murine calvaria osteolysis model in C57BL/J6 mice. Bone resorption was measured by the toluidine blue staining. Osteoclasts were detected by tartrate resistant acid phosphatase (TRAP) staining assay and were quantified by a TRAP quantification kit. Results show that bone resorption is 0.347 ± 0.09 mm² in mice with particle implantation, and decreased to 0.123 ± 0.05 mm² and 0.052 ± 0.02 mm² after ASO treatment with low and high doses, respectively. The number of osteoclasts in animal calvaria treated with ASO is reduced compared with that of untreated animals, and the quantification results indicate that about 90% of osteoclastogenesis is suppressed by the ASO. In addition, the osteoclastogenesis can be reestablished by the addition of TNF‐α. In conclusion, we demonstrate that the antisense oligonucleotide targeting to TNF‐α can suppress osteolysis induced by metal particles in a murine calvaria model. This new finding may be of value in the search for novel therapeutic methods for implant loosening. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1114–1120, 2008     

Transforming growth factor-beta induces osteoclast ruffling and chemotaxis: potential role in osteoclast recruitment.

Transforming growth factor-beta (TGF-beta) is released from the matrix during bone resorption and has been implicated in the pathogenesis of giant cell tumors of bone and the expansion of breast cancer metastases in bone. Because osteoclasts mediate tumor-induced osteolysis, we investigated whether TGF-beta stimulates osteoclast recruitment. Osteoclasts were isolated from rat long bones and time-lapse video microscopy was used to monitor their morphology and motility. Within 5 minutes, TGF-beta (0.1 nM) induced dynamic ruffling, with 65% of osteoclasts displaying membrane ruffles compared with 35% in untreated controls. Over a 2-h period, osteoclasts exhibited significant directed migration toward a source of TGF-beta, indicating chemotaxis. echistatin, an alphavbeta3 integrin blocker that inhibits macrophage colony-stimulating factor (M-CSF)-induced osteoclast migration, did not prevent the migration of osteoclasts toward TGF-beta. In contrast, a beta1 integrin blocking antibody inhibited osteoclast chemotaxis toward TGF-beta but not M-CSF. These data indicate the selective use of integrins by osteoclasts migrating in response to different chemotaxins. In addition, wortmannin and U0126 inhibited TGF-beta-induced chemotaxis, suggesting involvement of the phosphatidylinositol 3 (PI 3) kinase and mitogen-activated protein (MAP) kinase signaling pathways. Physiologically, TGF-beta, may coordinate osteoclast activity by recruiting osteoclasts to existing sites of resorption. Pathologically, TGF-beta-induced osteoclast recruitment may be critical for expansion of primary and metastatic tumors in bone.     

Temporal changes in bone mass and mechanical properties in a murine model of tumor osteolysis

Pathological fracture is a devastating complication of osteolytic bone metastases. The progression of osteolysis and its effect on bone fracture risk are poorly understood. The goal of this study was to determine the temporal changes in bone strength following tumor inoculation in a preclinical model of tumor osteolysis. In addition, a predictive model was developed between non-invasive radiographic measures and bone strength. The right femora of female nude mice were injected with breast cancer cells; the left limb served as a sham-operated control. Radiographs and DEXA scans were obtained at the time of surgery and at 3, 6, and 9 weeks. Groups of mice were euthanized at each time point for mechanical assessment. Micro-CT analysis was performed on a sub-set of mice with advanced state disease to quantify bone loss. Radiographs documented an increase in tumor osteolysis over time, with 58% of the mice showing signs of osteolysis at 3 weeks, 75% at 6 weeks, and 81% at 9 weeks. BMD measurements revealed a 21.6% increase from baseline in the controls whereas tumor-injected femora failed to increase in BMD over the same time course. Tumor-bearing limbs exhibited statistically significant decreases in torque at failure (86%), energy to failure (88%), and initial stiffness (94%) compared to the controls. Both lysis scores and BMD measurements proved to be modest predictors of mechanical strength, accounting for approximately 73% and 41% of variation in torque at failure, respectively. Micro-CT analysis revealed decreases in both total bone volume in the distal femur (31%) and metaphyseal fractional trabecular bone (89%). We have shown that non-invasive radiographic techniques provide a useful tool for monitoring the progression of tumor osteolysis and for predicting the mechanical strength of tumor-bearing bones in this model. By integrating non-invasive measures of tumor osteolysis and fracture risk, we have validated a clinically relevant platform for evaluating new therapeutic approaches for preserving and/or restoring bone affected by metastatic disease.     

In Vitro Generation of Mature Human Osteoclasts

Mononuclear precursors of human osteoclasts are found in the CD14⁺ monocyte fraction of circulating peripheral blood mononuclear cells (PBMCs). It is possible to generate osteoclasts in vitro from PBMCs cultured with macrophage colony-stimulating factor and receptor activator for nuclear factor κB ligand. In these cultures, however, it is not possible to distinguish the effect of a specific agent on osteoclast resorption activity as opposed to osteoclast differentiation. To produce a population of mature human osteoclasts to study osteoclast lacunar resorption specifically, we cultured CD14⁺ human monocytes on hydrophobic dishes in order to generate and maintain osteoclasts in suspension prior to culturing them on coverslips and dentine slices. Multinucleated cells formed in these cultures expressed vitronectin receptor, tartrate-resistant acid phosphatase, and cathepsin K. These cells also produced F-actin rings and were capable of extensive lacunar resorption on dentine slices after 24 h in culture. Lacunar resorption was inhibited by calcitonin and zoledronate but not by osteoprotegerin. This method of generating a highly enriched population of mature human osteoclasts should provide a valuable means of specifically assessing the effect of molecular factors (e.g., cytokines, growth factors, hormones) and therapeutic agents on osteoclast resorption activity.     

PDGF Receptor Signaling in Osteoblast Lineage Cells Controls Bone Resorption Through Upregulation of Csf1 Expression

The physiological functions of platelet-derived growth factor receptors (PDGFRs) α and β in osteoblast biology and bone metabolism remain to be established. Here, we show that PDGFRA and PDGFRB genes are expressed by osteoblast-lineage canopy and reversal cells in close proximity to PDGFB-expressing osteoclasts within human trabecular bone remodeling units. We also report that, although removal of only one of the two PDGFRs in Osterix-positive cells does not affect bone phenotype, suppression of both PDGFRs in those osteoblast lineage cells increases trabecular bone volume in male mice as well as in female gonad-intact and ovariectomized mice. Furthermore, osteoblast lineage-specific suppression of PDGFRs reduces Csf1 expression, bone marrow level of macrophage colony-stimulating factor (M-CSF), number of osteoclasts, and, therefore, bone resorption, but does not change bone formation. Finally, abrogation of PDGFR signaling in osteoblasts blocks PDGF-induced ERK1/2-mediated Csf1 expression and M-CSF secretion in osteoblast cultures and calcitriol-mediated osteoclastogenesis in co-cultures. In conclusion, our results indicate that PDGFR signaling in osteoblast lineage cells controls bone resorption through ERK1/2-mediated Csf1 expression. © 2020 American Society for Bone and Mineral Research (ASBMR).     

Bone modeling in gallium nitrate-treated rats

Summary Gallium nitrate (GaN) reduces cancer-related hypercalcemia and inhibits bone resorptionin vitro. This study investigated the effects of chronic GaN administration on bone, kidney, and parathyroid gland activity of growing rats. Experimental animals received GaN (1.75 mg elemental gallium i.p. QOD×8, Ga⁺), and controls received the solvent (Ga⁻). In the bone of Ga⁺ rats the number of osteoclasts was increased (Ga⁺: 70.4±2.31 osteoclasts/mm²; Ga⁻: 46.5±1.61 osteoclasts/mm²,P<0.001), and apposition rate and osteoid width were unchanged. Ga was concentrated in bone (2.4 μmol/g cortical bone) and detected by electron microprobe on the surface of a few trabeculae. Alkaline (Alp) and acid (Acp) phosphatase activities were higher in Ga⁺ than in Ga⁻ calvaria (Ga⁺: Alp 223±23.4 U/mg prot, Ga⁻: Alp 145±13.3 U/mg prot,P<0.02; Ga⁺: Acp 69.5±4.7 U/mg prot, Ga⁻: 57.5±2.8 U/mg prot,P<0.05). Serum iPTH was increased (Ga⁺: 112.9±17.6 pg/ml, Ga⁻: 41.4±7.4 pg/ml,P<0.01), serum calcium was reduced (Ga⁺: 2.4±0.02 mmol/l, Ga⁻: 2.6±0.03 mmol/l,P<0.001); calciuria remained comparable to controls. Relative to the hypocalcemia this suggests renal loss of Ca. The calcemic response to hPTH 1-34 (i.v. 50 μ/kg) was decreased 2 hours after injection of the hormone (ΔCa: TPTX Ga⁺: 0.11±0.04 mmol/l, Ga⁻: 0.33 ±0.03 mmol/lP<0.01). In conclusion, Ga, at the dosage used, does not inhibit the activity of osteoblasts in rats and does not interfere with mineralization but increases the number of osteoclasts through stimulation of parathyroid gland activity, induced by a fall in serum calcium. The hypocalcemia seems to be related to skeletal resistance to PTH and to increased renal calcium loss.