Showing papers on "Tartrate-resistant acid phosphatase published in 2010"

The Microphthalmia Transcription Factor Regulates Expression of the Tartrate‐Resistant Acid Phosphatase Gene During Terminal Differentiation of Osteoclasts

Abstract: The defective terminal differentiation of osteoclasts in mice homozygous for the mi allele of the microphthalmia transcription factor (MITF) gene implies that MITF plays a critical role in regulating gene expression during osteoclast ontogeny. To begin addressing the role of this transcription factor in the osteoclast, target genes need to be identified. In the present work, several lines of evidence show that the gene encoding the enzyme tartrate-resistant acid phosphatase (TRAP) is a target of MITF. Analysis of osteoclasts in vivo in the embryonic forelimb showed that MITF and TRAP RNA were coexpressed in a dynamic pattern during the process of endochondral ossification of long bone. Primary osteoclast-like cells (OCLs) produced from mi/mi mutant mice expressed TRAP messenger RNA (mRNA) at 8-fold lower levels than in OCLs derived from normal mice, indicating a direct link between MITF function and TRAP expression. The activity of mouse TRAP promoter-reporter genes was assayed in the primary OCLs by DNA-mediated transfection, and this activity was shown to depend on a conserved sequence (GGTCATGTGAG) located in the proximal promoter. Recombinant MITF protein recognized specifically this conserved sequence element. Expression of a TRAP promoter-green fluorescent protein (GFP) transgene mimicked the expression of the endogenous TRAP gene during differentiation of osteoclast-like cells, and the expression of the transgene was decreased 8-fold when placed into the mutant mi/mi background. These results are consistent with a role for MITF in gene expression during terminal differentiation of the osteoclast and will allow osteoclast-specific mechanisms of gene regulation to be studied in greater detail.

Diet-induced obesity alters bone remodeling leading to decreased femoral trabecular bone mass in mice

Abstract: Obesity-derived body mass may be detrimental to bone health through not well-defined mechanisms. In this study we determined changes in bone structure and serum cytokines related to bone metabolism in diet-induced obese mice. Mice fed a high-fat diet (HFD) had higher serum tartrate-resistant acid phosphatase (TRAP) and leptin but lower osteocalcin concentrations than those fed the normal-fat diet. The HFD increased multinucleated TRAP-positive osteoclasts in bone marrow compared to the control diet. Despite being much heavier, mice fed the HFD had lower femoral bone volume, trabecular number, and connectivity density and higher trabecular separation than mice on the control diet. These findings suggest that obesity induced by a HFD increases bone resorption that may blunt any positive effects of increased body weight on bone.

Direct and indirect actions of fibroblast growth factor 2 on osteoclastic bone resorption in cultures.

Abstract: Fibroblast growth factor 2 (FGF-2 or basic FGF) is known to show variable actions on bone formation and bone resorption This study was undertaken to elucidate the mechanisms whereby FGF-2 affects bone metabolism, especially bone resorption, using three different culture systems FGF-2 at 10(-9) M and higher concentrations induced osteoclastic cell formation in the coculture system of mouse osteoblastic cells and bone marrow cells, and this induction was abrogated by nonsteroidal anti-inflammatory drugs (NSAIDs) 45Ca release from prelabeled cultured mouse calvariae stimulated by FGF-2 (10(-8) M) was also inhibited by NSAIDs, and the inhibition was stronger by NSAIDs, which are more selective for inhibition of cyclooxygenase 2 (COX-2) than COX-1, suggesting the mediation of COX-2 induction COX-2 was highly expressed and its messenger RNA (mRNA) level was stimulated by FGF-2 in osteoblastic cells whereas it was undetectable or not stimulated by FGF-2 in cells of osteoclast lineage To further investigate the direct actions of FGF-2 on osteoclasts, resorbed pit formation was compared between cultures of purified osteoclasts and unfractionated bone cells from rabbit long bones FGF-2 (> or = 10(-12) M) stimulated resorbed pit formation by purified osteoclasts with a maximum effect of 20-fold at 10(-11) M, and no further stimulation was observed at higher concentrations However, FGF-2 at 10(-9) M - 10(-8) M stimulated resorbed pit formation by unfractionated bone cells up to 97-fold NS-398, a specific COX-2 inhibitor, did not affect the FGF-2 stimulation on purified osteoclasts but inhibited that on unfractionated bone cells We conclude that FGF-2 at low concentrations (> or =10(-12) M) acts directly on mature osteoclasts to resorb bone moderately, whereas at high concentrations (> or = 10(-9) M) it acts on osteoblastic cells to induce COX-2 and stimulates bone resorption potently

Decreased nitric oxide levels stimulate osteoclastogenesis and bone resorption both in vitro and in vivo on the chick chorioallantoic membrane in association with neoangiogenesis.

Abstract: High nitric oxide (NO) levels inhibit osteoclast (OC)-mediated bone resorption in vivo and in vitro, and nitrate donors protect against estrogen-deficient bone loss in postmenopausal women. Conversely, decreased NO production potentiates OC bone resorption in vitro and is associated with in vivo bone loss in rats and humans. Previously, we reported that bone sections from rats administered aminoguanidine (AG), a selective inhibitor of NO production via inducible NO synthase, exhibited both increased OC resorptive activity as well as greater numbers of OC. Here, we investigated further whether AG promoted osteoclastogenesis, in addition to stimulating mature OC function, using a modified in vivo chick chorioallantoic membrane (CAM) system and an in vitro chick bone marrow OC-like cell developmental model. AG, focally administered in small agarose plugs placed directly adjacent to a bone chip implanted on the CAM, dose-dependently elicited neoangiogenesis while stimulating the number, size, and bone pit resorptive activity of individual OC ectopically formed in vivo. In addition to enhancing OC precursor recruitment via neoangiogenesis, AG also exerted other vascular-independent effects on osteoclastogenesis. Thus, AG promoted the in vitro fusion and formation from bone marrow precursor cells of larger OC-like cells that contained more nuclei per cell and exhibited multiple OC differentiation markers. AG stimulated development was inversely correlated with declining medium nitrite levels. In contrast, three different NO donors each dose-dependently inhibited in vitro OC-like cell development while raising medium nitrite levels. Therefore, NO sensitively regulates OC-mediated bone resorption through affecting OC recruitment (angiogenesis), formation (fusion and differentiation), and bone resorptive activity in vitro and in vivo. Possibly, the stimulation of neoangiogenesis and OC-mediated bone remodeling via AG or other pro-angiogenic agents may find clinical applications in reconstructive surgery, fracture repair, or the treatment of avascular necrosis.

Gallium modulates osteoclastic bone resorption in vitro without affecting osteoblasts

Abstract: Background and purpose: Gallium (Ga) has been shown to be effective in the treatment of disorders associated with accelerated bone loss, including cancer-related hypercalcemia and Paget's disease. These clinical applications suggest that Ga could reduce bone resorption. However, few studies have studied the effects of Ga on osteoclastic resorption. Here, we have explored the effects of Ga on bone cells in vitro. Experimental approach: In different osteoclastic models [osteoclasts isolated from long bones of neonatal rabbits (RBC), murine RAW 264.7 cells and human CD14-positive cells], we have performed resorption activity tests, staining for tartrate resistant acid phosphatase (TRAP), real-time polymerase chain reaction analysis, viability and apoptotic assays. We also evaluated the effect of Ga on osteoblasts in terms of proliferation, viability and activity by using an osteoblastic cell line (MC3T3-E1) and primary mouse osteoblasts. Key results: Gallium dose-dependently (0–100 µM) inhibited the in vitro resorption activity of RBC and induced a significant decrease in the expression level of transcripts coding for osteoclastic markers in RAW 264.7 cells. Ga also dramatically reduced the formation of TRAP-positive multinucleated cells. Ga down-regulated in a dose-dependant manner the expression of the transcription factor NFATc1. However, Ga did not affect the viability or activity of primary and MC3T3-E1 osteoblasts. Conclusions and implications: Gallium exhibits a dose-dependent anti-osteoclastic effect by reducing in vitro osteoclastic resorption, differentiation and formation without negatively affecting osteoblasts. We provide evidence that this inhibitory mechanism involves down-regulation of NFATc1 expression, a master regulator of RANK-induced osteoclastic differentiation.

Loss of Osteoclasts Contributes to Development of Osteosarcoma Pulmonary Metastases

Abstract: We conducted a transcriptomic screen of osteosarcoma (OS) biopsies and found that expression of osteoclast-specific tartrate-resistant acid phosphatase 5 (ACP5/TRAP) is significantly downregulated in OS compared with nonmalignant bone (P < 0.0001). Moreover, lesions from OS patients with pulmonary metastases had 2-fold less ACP5/TRAP expression (P < 0.018) than lesions from patients without metastases. In addition, we found a direct correlation (P = 0.0166) between ACP5/TRAP expression and time to metastasis. Therefore, we examined whether metastasis-competent (MC) OS cells could induce loss of ACP5(+) osteoclasts and contribute to metastasis. We found that MC OS cell lines can inhibit osteoclastogenesis in vitro and in vivo. In addition, osteoclasts can inhibit the migration of MC OS cells in vitro. Finally, ablation of osteoclasts with zoledronic acid increases the number of metastatic lung lesions in an orthotopic OS model, whereas fulvestrant treatment increases osteoclast numbers and reduces metastatic lesions. These data indicate that the metastatic potential of OS is determined early in tumor development and that loss of osteoclasts in the primary lesion enhances OS metastasis.

Crosstalk of osteoblast and osteoclast precursors on mineralized collagen—towards an in vitro model for bone remodeling

Abstract: Bone remodeling and, therefore, integration of implant materials require the coordinated regulation of osteoblast and osteoclast activity. This is why the in vitro evaluation of biomaterials for bone regeneration should involve not only the analysis of osteoblast differentiation but also the formation and differentiation of osteoclasts. In the present study, we applied a material made of mineralized collagen I that mimics extracellular bone matrix to establish a culture system, which allows the cocultivation of human monocytes and human mesenchymal stem cells (hMSCs), which were differentiated into osteoclast-like cells and osteoblasts, respectively. Both cell types were cultivated on membrane-like structures from mineralized collagen. Transwell inserts were used to spatially separate the cell types but allowed exchange of soluble factors. The osteoclastogenesis and osteogenic differentiation were evaluated by analysis of gene expression, determination of alkaline phosphatase (ALP), and tartrate-resistant acidic phosphatase (TRAP) activity. Furthermore, cell morphology was studied using scanning electron and transmission electron microscopy. Osteogenically induced hMSC showed an increased specific ALP activity as well as increased gene expression of gene coding for alkaline phosphatase (ALPL), when cocultivated with differentiating osteoclasts. Adipogenic differentiation of hMSCs was suppressed by the presence of osteoclasts as indicated by a major decrease in adipocyte cell number and a decrease in gene expression of adipogenic markers. The formation of multinucleated osteoclasts seems to be decreased in the presence of osteogenically induced hMSC as indicated by electron microscopic evaluation and determination of TRAP activity. However, gene expression of osteoclast markers was not decreased in coculture with osteogenically induced hMSC.

Retinoic Acid Increases Proliferation of Human Osteoclast Progenitors and Inhibits RANKL-Stimulated Osteoclast Differentiation by Suppressing RANK

Abstract: It has been shown that high vitamin A intake is associated with bone fragility and fractures in both animals and humans. However, the mechanism by which vitamin A affects bones is unclear. In the present study, the direct effects of retinoic acid (RA) on human and murine osteoclastogenesis were evaluated using cultured peripheral blood CD14(+) monocytes and RAW264.7 cells. Both the activity of the osteoclast marker tartrate resistant acid phosphatase (TRAP) in culture supernatant and the expression of the genes involved in osteoclast differentiation together with bone resorption were measured. To our knowledge, this is the first time that the effects of RA on human osteoclast progenitors and mature osteoclasts have been studied in vitro. RA stimulated proliferation of osteoclast progenitors both from humans and mice. In contrast, RA inhibited differentiation of the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis of human and murine osteoclast progenitors via retinoic acid receptors (RARs). We also show that the mRNA levels of receptor activator of nuclear factor κB (RANK), the key initiating factor and osteoclast associated receptor for RANKL, were potently suppressed by RA in osteoclast progenitors. More importantly, RA abolished the RANK protein in osteoclast progenitors. This inhibition could be partially reversed by a RAR pan-antagonist. Furthermore, RA treatment suppressed the expression of the transcription factor nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and increased the expression of interferon regulatory factor-8 (IRF-8) in osteoclast progenitors via RARs. Also, RA demonstrated differential effects depending on the material supporting the cell culture. RA did not affect TRAP activity in the culture supernatant in the bone slice culture system, but inhibited the release of TRAP activity if cells were cultured on plastic. In conclusion, our results suggest that retinoic acid increases proliferation of human osteoclast progenitors and that it inhibits RANK-stimulated osteoclast differentiation by suppressing RANK.

Generation of Novel Bone Forming Cells (Monoosteophils) from the Cathelicidin-Derived Peptide LL-37 Treated Monocytes

Abstract: Background Bone generation and maintenance involve osteoblasts, osteoclasts, and osteocytes which originate from unique precursors and rely on key growth factors for differentiation. However, an incomplete understanding of bone forming cells during wound healing has led to an unfilled clinical need such as nonunion of bone fractures. Since circulating monocytes are often recruited to sites of injury and may differentiate into various cell types including osteoclasts, we investigated the possibility that circulating monocytes in the context of tissue injury may also contribute to bone repair. In particular, we hypothesized that LL-37 (produced from hCAP-18, cathelicidin), which recruits circulating monocytes during injury, may play a role in bone repair. Methods and Findings Treatment of monocytes from blood with LL-37 for 6 days resulted in their differentiation to large adherent cells. Growth of LL-37-differentiated monocytes on osteologic discs reveals bone-like nodule formation by scanning electron microscopy (SEM). In vivo transplantation studies in NOD/SCID mice show that LL-37-differentiated monocytes form bone-like structures similar to endochondral bone formation. Importantly, LL-37-differentiated monocytes are distinct from conventional monocyte-derived osteoclasts, macrophages, and dendritic cells and do not express markers of the mesenchymal stem cells (MSC) lineage, distinguishing them from the conventional precursors of osteoblasts. Furthermore, LL-37 differentiated monocytes express intracellular proteins of both the osteoblast and osteoclast lineage including osteocalcin (OC), osteonectin (ON), bone sialoprotein II (BSP II), osteopontin (OP), RANK, RANKL, MMP-9, tartrate resistant acid phosphatase (TRAP), and cathepsin K (CK). Conclusion Blood derived monocytes treated with LL-37 can be differentiated into a novel bone forming cell that functions both in vitro and in vivo. We propose the name monoosteophil to indicate their monocyte derived lineage and their bone forming phenotype. These cells may have wide ranging implications in the clinic including repair of broken bones and treatment of osteoporosis.

RESEARCH PAPER Gallium modulates osteoclastic bone resorption in vitro without affecting osteoblasts

Abstract: Background and purpose: Gallium (Ga) has been shown to be effective in the treatment of disorders associated with accelerated bone loss, including cancer-related hypercalcemia and Paget’s disease. These clinical applications suggest that Ga could reduce bone resorption. However, few studies have studied the effects of Ga on osteoclastic resorption. Here, we have explored the effects of Ga on bone cells in vitro. Experimental approach: In different osteoclastic models [osteoclasts isolated from long bones of neonatal rabbits (RBC), murine RAW 264.7 cells and human CD14-positive cells], we have performed resorption activity tests, staining for tartrate resistant acid phosphatase (TRAP), real-time polymerase chain reaction analysis, viability and apoptotic assays. We also evaluated the effect of Ga on osteoblasts in terms of proliferation, viability and activity by using an osteoblastic cell line (MC3T3-E1) and primary mouse osteoblasts. Key results: Gallium dose-dependently (0–100 mM) inhibited the in vitro resorption activity of RBC and induced a significant decrease in the expression level of transcripts coding for osteoclastic markers in RAW 264.7 cells. Ga also dramatically reduced the formation of TRAP-positive multinucleated cells. Ga down-regulated in a dose-dependant manner the expression of the transcription factor NFATc1. However, Ga did not affect the viability or activity of primary and MC3T3-E1 osteoblasts. Conclusions and implications: Gallium exhibits a dose-dependent anti-osteoclastic effect by reducing in vitro osteoclastic resorption, differentiation and formation without negatively affecting osteoblasts. We provide evidence that this inhibitory mechanism involves down-regulation of NFATc1 expression, a master regulator of RANK-induced osteoclastic differentiation. British Journal of Pharmacology (2010) 159, 1681–1692; doi:10.1111/j.1476-5381.2010.00665.x; published online 23 March 2010

In vitro osteoclastogenesis on textile chitosan scaffold.

Abstract: Textile chitosan fibre scaffolds were evaluated in terms of interaction with osteoclast-like cells, derived from human primary monocytes. Part of the scaffolds was further modified by coating with fibrillar collagen type I in order to make the surface biocompatible. Monocytes were cultured directly on the scaffolds in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL) for up to 18 days. Confocal laser scanning microscopy (CLSM) as well as scanning electron microscopy (SEM) revealed the formation of multinuclear osteoclast-like cells on both the raw chitosan fibres and the collagen-coated scaffolds. The modified surface supported the osteoclastogenesis. Differentiation towards the osteoclastic lineage was confirmed by the microscopic detection of cathepsin K, tartrate resistant acid phosphatase (TRAP), acidic compartments using 3-(2,4-dinitroanillino)3’-amino-N-methyldipropylamine (DAMP), immunological detection of TRAP isoform 5b, and analysis of gene expression of the osteoclastic markers TRAP, cathepsin K, vitronectin receptor, and calcitonin receptor using reverse transcription-polymerase chain reaction (RT-PCR). The feature of the collagen-coated but also of the raw chitosan fibre scaffolds to support attachment and differentiation of human monocytes facilitates cell-induced material resorption – one main requirement for successful bone tissue engineering.

Structural and cellular features in metaphyseal and diaphyseal periosteum of osteoporotic rats

Abstract: Despite the important physiological role of periosteum in the pathogenesis and treatment of osteoporosis, little is known about the structural and cellular characteristics of periosteum in osteoporosis. To study the structural and cellular differences in both diaphyseal and metaphyseal periosteum of osteoporotic rats, samples from the right femur of osteoporotic and normal female Lewis rats were collected and tissue sections were stained with hematoxylin and eosin, antibodies or staining kit against tartrate resistant acid phosphatase (TRAP), alkaline phosphatase (ALP), vascular endothelial growth factor (VEGF), von Willebrand (vWF), tyrosine hydroxylase (TH) and calcitonin gene-related peptide (CGRP). The results showed that the osteoporotic rats had much thicker and more cellular cambial layer of metaphyseal periosteum compared with other periosteal areas and normal rats (P < 0.001). The number of TRAP+ osteoclasts in bone resorption pits, VEGF+ cells and the degree of vascularization were found to be greater in the cambial layer of metaphyseal periosteum of osteoporotic rats (P < 0.05), while no significant difference was detected in the number of ALP+ cells between the two groups. Sympathetic nerve fibers identified by TH staining were predominantly located in the cambial layer of metaphyseal periosteum of osteoporotic rats. No obvious difference in the expression of CGRP between the two groups was found. In conclusion, periosteum may play an important role in the cortical bone resorption in osteoporotic rats and this pathological process may be regulated by the sympathetic nervous system.

A Quick Whole-Mount Staining Protocol for Bone Deposition and Resorption

Abstract: Osteoblasts and osteoclasts, two cell types important in bone development, are associated with enzymes capable of hydrolyzing phosphate groups. These enzymes are important for their function of bone deposition and resorption. Alkaline phosphatase (AP) activity is associated with the cell surface of osteoblasts, while osteoclasts secrete tartrate-resistant acid phosphatase (TRAP). Due to their shared enzymatic properties, we were able to develop a simple whole-mount staining protocol to stain for both enzymes (TRAP and AP) within the same teleostean tissue sample. In addition, we were able to perform each reaction individually. Further, AP and TRAP stains were maintained through decalcification, embedding, and sectioning procedures. Staining can also be conducted after sectioning depending on the question under investigation. These rapid staining protocols can thus be used to observe the processes involved in bone remodeling in whole teleost specimens, and/or the location of the stain can be determined through sectioning. The ability to observe bone deposition and resorption in such a capacity will significantly advance our understanding of bone remodeling throughout the life history of organisms and also within particular skeletal elements.

Systemic RANK‐Fc protein therapy is efficacious against primary osteosarcoma growth in a murine model via activity against osteoclasts

Abstract: Objectives Osteosarcoma (OS) is the most common primary malignant bone tumour, and mainly affects adolescents and young adults. Although there has been substantial improvement in management of OS with surgery and chemotherapy, further survival increase has not been achieved over the past two decades. Methods We focused on the receptor activator of nuclear factor κB ligand (RANKL)–osteoclast (OCL) system as a biological target for OS. RANKL is a critical factor for OCL formation and bone resorption activity. The primary lesion in bone and ensuing metastasis in OS both require the induction of OCLs. RANK-Fc is a potent RANKL antagonist and inhibitor of OCL formation and activity. Key findings In an orthotopic model in Balb/c nu/nu mice, a twice weekly dosing regimen of 350 μg of RANK-Fc per mouse subcutaneously (n= 5) reduced lung metastasis (P > 0.05), preserved bone structure and reduced tartrate-resistant acid phosphatase (TRAP)+ OCLs (P < 0.005) in OS-bearing bone. In vitro, RANK-Fc suppressed OCL formation (P < 0.005), bone resorption activity (P < 0.005) and RANKL-induced anti-apoptosis (P < 0.5) of OCLs.

Osteoclast‐specific inactivation of the integrin‐linked kinase (ILK) inhibits bone resorption

Abstract: Bone resorption requires the adhesion of osteoclasts to extracellular matrix (ECM) components, a process mediated by the alpha(v)beta(3) integrin. Following engagement with the ECM, integrin receptors signal via multiple downstream effectors, including the integrin-linked kinase (ILK). In order to characterize the physiological role of ILK in bone resorption, we generated mice with an osteoclast-specific Ilk gene ablation by mating mice with a floxed Ilk allele with TRAP-Cre transgenic mice. The TRAP-Cre mice specifically excised floxed alleles in osteoclasts, as revealed by crossing them with the ROSA26R reporter strain. Osteoclast-specific Ilk mutant mice appeared phenotypically normal, but histomorphometric analysis of the proximal tibia revealed an increase in bone volume and trabecular thickness. Osteoclast-specific Ilk ablation was associated with an increase in osteoclastogenesis both in vitro and in vivo. However, the mutant osteoclasts displayed a decrease in resorption activity as assessed by reduced pit formation on dentin slices in vitro and decreased serum concentrations of the C-terminal telopeptide of collagen in vivo. Interestingly, compound heterozygous mice in which one allele of Ilk and one allele of the beta(3) integrin gene were inactivated (ILK(+/-); beta(3) (+/-)) also had increased trabecular thickness, confirming that beta(3) integrin and Ilk form part of the same genetic cascade. Our results show that ILK is important for the function, but not the differentiation, of osteoclasts.

Regulation of osteoblast and osteoclast functions by FGF-6.

Abstract: Fibroblast growth factor-6 (FGF-6) is known to be the key ligand for fibroblast growth factor receptor 4 (FGFR4) during muscle regeneration but its role in bone has yet to be verified. FGFR signaling is known to be important in the initiation and regulation of osteogenesis, so in this study the actions of FGF-6 on human osteoblasts and osteoclasts were investigated. Human primary osteoblasts (hOB) were used to study the effect of FGF-6 on proliferation (by ATP quantification), signal transduction (by ERK and AKT phosphorylation), differentiation (by alkaline phosphatase activity, APA), and mineralization (by calcein staining). To study FGF-6 activity on osteoclast differentiation, human bone marrow cells were used and tartrate-resistant acid phosphatase (TRAP) multinucleated cells together with actin filaments arrangements were quantified. Human primary mature osteoclasts were used to evaluate the effect of FGF-6 on osteoclast reabsorbing activity by reabsorbed pit measurements. FGF-6 >10(-9) M as FGF-2 10(-7) M induced hOB proliferation mediated by pERK together with a reduction in APA and reduced mineralization of the treated cells. Moreover FGF-6 increased the formation of TRAP-positive multinucleated cells in a dose-dependent manner (maximal effect at 10(-8) M). FGF-6-treated cells showed also a greater percentage of cells that formed typical osteoclast sealing zones. Mature osteoclasts cultured on dentine slice increased the area of reabsorption with a maximal effect of FGF-6 at 10(-12) M. FGF-6 may be considered a regulator of bone metabolism as shown by its activity on both osteoblasts and osteoclasts.

Dlx homeobox gene family expression in osteoclasts

Abstract: Skeletal growth and homeostasis require the finely orchestrated secretion of mineralized tissue matrices by highly specialized cells, balanced with their degradation by osteoclasts. Time- and site-specific expression of Dlx and Msx homeobox genes in the cells secreting these matrices have been identified as important elements in the regulation of skeletal morphology. Such specific expression patterns have also been reported in osteoclasts for Msx genes. The aim of the present study was to establish the expression patterns of Dlx genes in osteoclasts and identify their function in regulating skeletal morphology. The expression patterns of all Dlx genes were examined during the whole osteoclastogenesis using different in vitro models. The results revealed that Dlx1 and Dlx2 are the only Dlx family members with a possible function in osteoclastogenesis as well as in mature osteoclasts. Dlx5 and Dlx6 were detected in the cultures but appear to be markers of monocytes and their derivatives. In vivo, Dlx2 expression in osteoclasts was examined using a Dlx2/LacZ transgenic mouse. Dlx2 is expressed in a subpopulation of osteoclasts in association with tooth, brain, nerve, and bone marrow volumetric growths. Altogether the present data suggest a role for Dlx2 in regulation of skeletal morphogenesis via functions within osteoclasts.

Osteoblast activity in the goldfish scale responds sensitively to mechanical stress.

Abstract: The adaptive response of bone to mechanical loading in teleosts is not well understood. We recently developed a new assay system using teleost scales, which consists of osteoblasts, osteoclasts, and bone matrix protein. In this system, alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) were used as markers of osteoblasts and osteoclasts, respectively. Using this assay system, we examined the effects of mechanical loading on ALP and TRAP activity in goldfish scales. ALP activity in the scales was significantly elevated (p<0.01) by ultrasound stimuli (1 MHz, 50% duty factor, 0.5 Hz pulse repetition frequency, 60 mW/cm(2) [I(SATA)] and 6 min) after both 18 h and 24h of incubation while TRAP activity remained unchanged. In addition, mRNA expression of both insulin-like growth factor-I (IGF-I) and estrogen receptors (ER) increased significantly, as did ALP activity. After the goldfish had been swimming for 3 days (speed: 2 body lengths/second, duration: 3h/day), the scales' ALP activity increased significantly (p<0.01) but TRAP activity did not change. These in vitro and in vivo results strongly suggest that osteoblasts in the goldfish scale respond sensitively to mechanical stress and may be important in promoting bone formation.

Bone resorptive activity of osteoclast-like cells generated in vitro by PEG-induced macrophage fusion

Abstract: Normal bone remodeling is maintained by a balance between osteoclast and osteoblast activity, whereas defects in osteoclast activity affecting such balance result in metabolic bone disease. Macrophage-macrophage fusion leading to multinucleated osteoclasts being formed is still not well understood. Here we present PEG-induced fusion of macrophages from both U937/A and J774 cell lines and the induced differentiation and activation of osteoclast-like cells according to the expression of osteoclast markers such as tartrate resistant acid phosphatase (TRAP) and bone resorptive activity. PEG-induced macrophage fusion, during the non-confluent stage, significantly increased the osteoclastogenic activity of macrophages from cell lines compared to that of spontaneous cell fusion in the absence of PEG (polyethylene glycol). The results shown in this work provide evidence that cell fusion per se induces osteoclast-like activity. PEG-fused macrophage differential response to pretreatment with osteoclastogenic factors was also examined in terms of its ability to form TRAP positive multinucleated cells (TPMNC) and its resorptive activity on bovine cortical bone slices. Our work has also led to a relatively simple method regarding those previously reported involving cell co-cultures. Multinucleated osteoclast-like cells obtained by PEG-induced fusion of macrophages from cell lines could represent a suitable system for conducting biochemical studies related to basic macrophage fusion mechanisms, bone-resorption activity and the experimental search for bone disease therapeutic alternatives.

Differentiation analyses of adult suspension mononucleated peripheral blood cells of Mus musculus

Abstract: Background: The purpose of this study is to determine whether isolated suspension mouse peripheral mononucleated blood cells have the potential to differentiate into two distinct types of cells, i.e., osteoblasts and osteoclasts. Results: Differentiation into osteoblast cells was concomitant with the activation of the Opn gene, increment of alkaline phosphatase (ALP) activity and the existence of bone nodules, whereas osteoclast cells activated the Catk gene, increment of tartrate resistant acid phosphatase (TRAP) activity and showed resorption activities via resorption pits. Morphology analyses showed the morphology of osteoblast and osteoclast cells after von Kossa and May-Grunwald-Giemsa staining respectively. Conclusions: In conclusion, suspension mononucleated cells have the potentiality to differentiate into mature osteoblasts and osteoclasts, and hence can be categorized as multipotent stem cells.

Potential differentiation of three types of primitive cells originated from different proliferation terms of mouse blood

Abstract: The aim of this study was to differentiate central blood system mononucleated cells in vitro into osteoblast and osteoclast cells for three different proliferation terms of cells. The mononucleated cells were cultured in a selective proliferation medium for three different proliferation terms, i.e., short (5 days), medium (15 days) and long term (30 days) prior to analysis of osteoblast and osteoclast cells’ differentiation potentialities. The proliferation medium was then supplemented with differentiation factors, i.e., ascorbic acid and β-glycerophosphate to differentiate mononucleated cells into osteoblast cells. For osteoclast assay, RANKL and M-CSF were added into proliferation medium. For control, the same cells were used without supplementation of respective differentiation factors. The viability of differentiated cells from short, medium and long types of cells showed that they were able to survive until 10 to 14 days in the presence of respective differentiation factors without significant increased in the specific differentiation medium. Biochemical analyses on both alkaline phosphatase (ALP) and tartrate resistant acid phosphatase (TRAP) activities were significantly increased (p<0.05) once cultured in their respective differentiation medium. In conclusion, the three types of primitive cells have the same potentiality to differentiate into mature osteoblast and osteoclast cells even though the proliferation rates are different, i.e. 0.37, 0.55 and 0.72 division/day for short, medium and long term cells respectively. Mononucleated cells isolated from peripheral blood are primitive enough to differentiate into two distinct types of mature cells which originated from two different stem cells lineage hence can be categorized as multipotent stem cells.

Profiles of lactate dehydrogenase, tartrate resistant acid phosphatase and alkaline photophatase in saliva during orthodontic treatment

Abstract: During orthodontic treatment, the progress of tooth movements can be classified into four stages, i.e. activation (related to tissue inflammation or cells’ death), resorption, reversal and restructuring of new bones. Tooth movement is related to metabolic changes around the mouth. The objective of this study was to determine enzymes profiles from patient’s saliva during orthodontic tooth movement, i.e., lactate dehydrogenase (LDH) for inflammation, tartrate resistant acid phosphatase (TRAP) for bone resorption and alkaline phosphatase (ALP) for bone formation. Saliva samples were collected from 6 orthodontic patients. Activities of all enzymes were measured before placement of orthodontic fixed appliances (normal activity) followed with day 3, 7, 10, 14, 17, 21, 24, 28 and 31 after the braces were activated. All enzymes (LDH, TRAP and ALP) showed significantly (p≤0.05) higher specific activities as compared to normal. LDH showed the increment at the early phase of treatment (day 3,7 and 10), TRAP at day 14 and 17 followed by ALP at day 17, 21 and 24. As a conclusion, the profiles of enzymes activities showed that inflammation occured during early phase of treatment followed by bone resorption and bone formation. In addition, the inflammations, resorption and bone formation phases in orthodontic treatment were found to be completed within 24 days.