Jaw and long bone marrows have a different osteoclastogenic potential.
ABSTRACT: Osteoclasts, the multinucleated bone-resorbing cells, arise through fusion of precursors from the myeloid lineage. However, not all osteoclasts are alike; osteoclasts at different bone sites appear to differ in numerous respects. We investigated whether bone marrow cells obtained from jaw and long bone differed in their osteoclastogenic potential. Bone marrow cells from murine mandible and tibiae were isolated and cultured for 4 and 6 days on plastic or 6 and 10 days on dentin. Osteoclastogenesis was assessed by counting the number of TRAP(+) multinucleated cells. Bone marrow cell composition was analyzed by FACS. The expression of osteoclast- and osteoclastogenesis-related genes was studied by qPCR. TRAP activity and resorptive activity of osteoclasts were measured by absorbance and morphometric analyses, respectively. At day 4 more osteoclasts were formed in long bone cultures than in jaw cultures. At day 6 the difference in number was no longer observed. The jaw cultures, however, contained more large osteoclasts on plastic and on dentin. Long bone marrow contained more osteoclast precursors, in particular the myeloid blasts, and qPCR revealed that the RANKL:OPG ratio was higher in long bone cultures. TRAP expression was higher for the long bone cultures on dentin. Although jaw osteoclasts were larger than long bone osteoclasts, no differences were found between their resorptive activities. In conclusion, bone marrow cells from different skeletal locations (jaw and long bone) have different dynamics of osteoclastogenesis. We propose that this is primarily due to differences in the cellular composition of the bone site-specific marrow.
Project description:Recently, it was shown that interleukin-1? (IL-1?) has diverse stimulatory effects on different murine long bone marrow osteoclast precursors (OCPs) in vitro. In this study, interleukin-1 receptor antagonist deficient (Il1rn-/-) and wild-type (WT) mice were compared to investigate the effects of enhanced IL-1 signaling on the composition of OCPs in long bone, calvaria, vertebra, and jaw. Bone marrow cells were isolated from these sites and the percentage of early blast (CD31hi Ly-6C-), myeloid blast (CD31+ Ly-6C+), and monocyte (CD31- Ly-6Chi) OCPs was assessed by flow cytometry. At the time-point of cell isolation, Il1rn-/- mice showed no inflammation or bone destruction yet as determined by histology and microcomputed tomography. However, Il1rn-/- mice had an approximately two-fold higher percentage of OCPs in long bone and jaw marrow compared to WT. Conversely, vertebrae and calvaria marrow contained a similar composition of OCPs in both strains. Bone marrow cells were cultured with macrophage colony stimulating factor (M-CSF) and receptor of Nf?B ligand (RANKL) on bone slices to assess osteoclastogenesis and on calcium phosphate-coated plates to analyze mineral dissolution. Deletion of Il1rn increased osteoclastogenesis from long bone, calvaria, and jaw marrows, and all Il1rn-/- cultures showed increased mineral dissolution compared to WT. However, osteoclast markers increased exclusively in Il1rn-/- osteoclasts from long bone and jaw. Collectively, these findings indicate that a lack of IL-1RA increases the numbers of OCPs in vivo, particularly in long bone and jaw, where rheumatoid arthritis and periodontitis develop. Thus, increased bone loss at these sites may be triggered by a larger pool of OCPs due to the disruption of IL-1 inhibitors.
Project description:Osteoprotegerin (OPG) is secreted by stromal and osteoblastic lineage cells and inhibits osteoclastogenesis by preventing the interaction of receptor activator of nuclear factor-?B ligand (RANKL) with receptor activator of nuclear factor-?B (RANK). In this study, the expression of OPG in osteoclasts themselves and its biological functions during osteoclastogenesis were investigated for the first time. OPG expression in vivo in the developing rat maxilla was examined by immunofluorescence analysis. OPG expression in osteoclasts during in vitro osteoclastogenesis was determined by reverse-transcription polymerase chain-reaction (RT-PCR), Western blot, and immunofluorescence staining. We determined the function of OPG produced by osteoclasts during osteoclastogenesis by silencing the OPG gene. The effects of OPG on bone-resorbing activity and apoptosis of mature osteoclasts were examined by the assay of resorptive pit formation on calcium-phosphate-coated plate and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, respectively. In the immunofluorescence findings, strong immunoreactivities were unexpectedly seen in multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts around the growing and erupting tooth germs in the rat alveolar bone. In vitro, OPG expression was significantly increased during the differentiation of osteoclasts from mouse bone-marrow-derived cells treated with a combination of macrophage colony-stimulating factor (M-CSF) and RANKL. Interestingly, it was found that OPG small interfering (si)RNA treatment during osteoclastogenesis enhanced the sizes of osteoclasts, but attenuated their bone-resorbing activity. Also, the increased chromosomal DNA fragmentation and caspase-3 activity in the late phase of osteoclastogenesis were found to be decreased by treatment with OPG siRNA. Furthermore, effects of OPG siRNA treatment on osteoclastogenesis and bone-resorbing activity were recovered by the treatment of exogenous OPG. These results suggest that OPG, expressed by the osteoclasts themselves, may play an auto-regulatory role in the late phase of osteoclastogenesis through the induction of apoptosis.
Project description:Donepezil, an inhibitor of acetylcholinesterase (AChE) targeting the brain, is a common medication for Alzheimer's disease. Interestingly, a recent clinical study found that administration of this agent is associated with lower risk of hip fracture independently of falling, suggesting its direct effect on bone tissues as well. AChE has been reported to be involved in osteoblast function, but the role of AChE on osteoclastogenesis still remains unclear. We analyzed the effect of AChE and donepezil on osteoclastogenesis in vivo and in vitro.Cell-based assays were conducted using osteoclasts generated in cultures of murine bone marrow macrophages (BMMs) with receptor activator of nuclear factor-kappa B ligand (RANKL). The effect of donepezil was also determined in vivo using a mouse model of RANKL-induced bone loss.Recombinant AChE in BMMs cultured with RANKL further promoted RANKL-induced tartrate-resistant acid phosphatase (TRAP)-positive osteoclast differentiation. RANKL also upregulated AChE expression in BMMs. RNA interference-mediated knockdown of AChE significantly inhibited RANKL-induced osteoclast differentiation and suppressed gene expression specific for osteoclasts. AChE upregulated expression of RANK, the receptor of RANKL, in BMMs. Donepezil decreased cathepsin K expression in BMMs and the resorptive function of osteoclasts on dentine slices. Donepezil decreased RANK expression in BMMs, resulting in the inhibition of osteoclast differentiation with downregulation of c-Fos and upregulation of Id2. Moreover, administration of donepezil prevented RANKL-induced bone loss in vivo, which was associated with the inhibition of bone resorption by osteoclasts.AChE promotes osteoclast differentiation in vitro. Donepezil inhibits osteoclast function in vitro and prevents bone loss by suppressing bone resorption in vivo, suggesting the possibility that donepezil reduces fracture risk in patients with Alzheimer's disease.
Project description:BACKGROUND:Platelet-rich fibrin (PRF) membranes can preserve alveolar ridge dimension after tooth extraction. Thus, it can be presumed that PRF suppresses the catabolic events that are caused by osteoclastic bone resorption. METHODS:To address this possibility, we investigated the impact of soluble extracts of PRF membranes on in vitro osteoclastogenesis in murine bone marrow cultures. Osteoclastogenesis was induced by exposing murine bone marrow cultures to receptor activator of nuclear factor kappa B ligand (RANKL), macrophage colony-stimulating factor (M-CSF) and transforming growth factor-beta 1 (TGF-?1) in the presence or absence of PRF. Osteoclastogenesis was evaluated based on histochemical, gene expression, and resorption analysis. Viability was confirmed by formation of formazan crystals, live-dead staining and caspase-3 activity assay. RESULTS:We report here that in vitro osteoclastogenesis is greatly suppressed by soluble extracts of PRF membranes as indicated by tartrate-resistant acid phosphatase (TRAP) staining and pit formation. In support of the histochemical observations, soluble extracts of PRF membranes decreased expression levels of the osteoclast marker genes TRAP, Cathepsin K, dendritic cell-specific transmembrane protein (DCSTAMP), nuclear factor of activated T-cells (NFATc1), and osteoclast-associated receptor (OSCAR). PRF membranes, however, cannot reverse the process once osteoclastogenesis has evolved. CONCLUSION:These in vitro findings indicate that PRF membranes can inhibit the formation of osteoclasts from hematopoietic progenitors in bone marrow cultures. Overall, our results imply that the favorable effects of PRF membranes in alveolar ridge preservation may be attributed, at least in part, by the inhibition of osteoclastogenesis.
Project description:Molecular regulators of osteoclast formation and function are an important area of research due to the central role of osteoclasts in bone resorption. Transcription factors such as MITF are essential for osteoclast generation by regulating expression of the genes required for cellular differentiation and resorptive function. We recently reported that histone deacetylase 7 (HDAC7) binds to and represses the transcriptional activity of MITF in osteoclasts, and that loss of HDAC7 in vitro accelerated osteoclastogenesis. In the current study, we extend this initial observation by showing that conditional deletion of HDAC7 in osteoclasts of mice leads to an in vivo enhancement in osteoclast formation, associated with increased bone resorption and lower bone mass. Expression of multiple MITF target genes is increased in bone marrow derived osteoclast cultures from the HDAC7 knockout mice. Interestingly, multiple regions of the HDAC7 amino-terminus can bind to MITF or exert repressive activity. Moreover, mutation or deletion of the HDAC7 conserved deacetylase catalytic domain had little effect on repressive function. These observations identify HDAC7 in osteoclasts as an important molecular regulator of MITF activity and bone homeostasis, but also highlight a gap in our understanding of exactly how HDAC7 functions as a corepressor.
Project description:Calycosin, an isoflavonoid phytoestrogen, isolated from Radix Astragali, was reported to possess anti-tumor, anti-inflammation, and osteogenic properties, but its impact on osteoclast differentiation remains unclear. In this study, we examined the effects of calycosin on osteoclastogenesis induced by RANKL. The results showed that calycosin significantly inhibited RANKL-induced osteoclast formation from primary bone marrow macrophages (BMMs). Calycosin also dose-dependently suppressed the formation of bone resorption pits by mature osteoclasts. In addition, the expression of osteoclatogenesis-related genes, including cathepsin K (CtsK), tartrate-resistant acid phosphatase (TRAP), and MMP-9, was significantly inhibited by calycosin. Furthermore, the results indicated that calycosin down-regulated the expression levels of NFATc1 and c-Fos through suppressing the activation of NF-?B and MAPKs. Our results indicate that calycosin has an inhibitory role in the bone loss by preventing osteoclast formation, as well as its bone resorptive activity. Therefore, calycosin may be useful as a therapeutic reagent for bone loss-associated diseases.
Project description:Glucocorticoid (GC) therapy decreases bone mass and increases the risk of fractures. We investigated interactions between the GC dexamethasone (DEX) and the bone resorptive agents 1,25(OH)2-vitamin D3 (D3) and parathyroid hormone (PTH) on osteoclastogenesis. We observed a synergistic potentiation of osteoclast progenitor cell differentiation and formation of osteoclasts when DEX was added to either D3- or PTH-treated mouse bone marrow cell (BMC) cultures. Cotreatment of DEX with D3 or PTH increased gene encoding calcitonin receptor (Calcr), acid phosphatase 5, tartrate resistant (Acp5), cathepsin K (Ctsk), and TNF superfamily member 11 (Tnfsf11) mRNA, receptor activator of NF-?B ligand protein (RANKL), numbers of osteoclasts on plastic, and pit formation and release of C-terminal fragment of type I collagen from cells cultured on bone slices. Enhanced RANKL protein expression caused by D3 and DEX was absent in BMC from mice in which the GC receptor (GR) was deleted in stromal cells/osteoblasts. Synergistic interactions between DEX and D3 on RANKL and osteoclast formation were present in BMC from mice with attenuated GR dimerization. These data demonstrate that the GR cooperates with D3 and PTH signaling, causing massive osteoclastogenesis, which may explain the rapid bone loss observed with high dosages of GC treatment.-Conaway, H. H., Henning, P., Lie, A., Tuckermann, J., Lerner, U. H. Glucocorticoids employ the monomeric glucocorticoid receptor to potentiate vitamin D3 and parathyroid hormone-induced osteoclastogenesis.
Project description:<h4>Background</h4>Copper-containing biomaterials are increasingly applied for bone regeneration due to their pro-angiogenetic, pro-osteogenetic and antimicrobial properties. Therefore, the effect of Cu<sup>2+</sup> on osteoclasts, which play a major role in bone remodeling was studied in detail.<h4>Methods</h4>Human primary osteoclasts, differentiated from human monocytes were differentiated or cultivated in the presence of Cu<sup>2+</sup>. Osteoclast formation and activity were analyzed by measurement of osteoclast-specific enzyme activities, gene expression analysis and resorption assays. Furthermore, the glutathione levels of the cells were checked to evaluate oxidative stress induced by Cu<sup>2+</sup>.<h4>Results</h4>Up to 8 µM Cu<sup>2+</sup> did not induce cytotoxic effects. Activity of tartrate-resistant acid phosphatase (TRAP) was significantly increased, while other osteoclast specific enzyme activities were not affected. However, gene expression of TRAP was not upregulated. Resorptive activity of osteoclasts towards dentin was not changed in the presence of 8 µM Cu<sup>2+</sup> but decreased in the presence of extracellular bone matrix. When Cu<sup>2+</sup> was added to mature osteoclasts TRAP activity was not increased and resorption decreased only moderately. The glutathione level of both differentiating and mature osteoclasts was significantly decreased in the presence of Cu<sup>2+</sup>.<h4>Conclusions</h4>Differentiating and mature osteoclasts react differently to Cu<sup>2+</sup>. High TRAP activities are not necessarily related to high resorption.
Project description:Drug-induced osteonecrosis of the jaw (ONJ) is a detrimental intraoral lesion that often occurs after dental-related interventions in patients undergoing treatment with bisphosphonates or denosumab, the neutralizing human anti-receptor activator of NF-?B ligand (RANKL) antibody (Ab). The cause of ONJ by these drugs has been speculated to their direct effects on osteoclasts. However, the extent to which osteoclasts contribute to ONJ pathogenesis remains controversial. Herein, by using a tooth-extraction mouse model with i.v. administration of mouse anti-RANKL Ab or the bisphosphonate zoledronate (ZOL), we show that unresorbed bone due to impaired formation or suppressed functions of osteoclasts, respectively, is associated with ONJ development. After tooth extraction, ONJ-like lesions developed 50% in the anti-RANKL Ab-treated mice and 30% in the ZOL-treated mice. Nonviable and unresorbed bone was found more in anti-RANKL Ab-treated mice compared with mice receiving ZOL. All mice receiving anti-RANKL Ab had an undetectable tartrate-resistant acid phosphatase (TRAP) level in the serum and no TRAP-positive osteoclasts at the extracted sockets, whereas ZOL-treated mice had a decreased TRAP level without altering the numbers of TRAP-positive osteoclasts. Interestingly, the absence of newly formed woven bone in the extracted sockets was evident in ONJ-like lesions from both anti-RANKL Ab- and ZOL-treated mice. Our study suggests that the lack of osteoclasts' bone-resorptive functions by these drugs and suppression of woven bone formation after dental trauma may be associated with ONJ development.