Tartrate-resistant Acid Phosphatase Enzyme Research Articles

C-C Motif Chemokine Ligand 5 (CCL5) Promotes Irradiation-Evoked Osteoclastogenesis.

The imbalance that occurs in bone remodeling induced by irradiation (IR) is the disruption of the balance between bone formation and bone resorption. In this study, primary osteocytes (OCYs) of femoral and tibial origin were cultured and irradiated. It was observed that irradiated OCY showed extensive DNA damage, which led to the initiation of a typical phenotype of cellular senescence, including the secretion of senescence-associated secretory phenotype (SASP), especially the C-C motif chemokine ligand 5 (CCL5). In order to explore the regulation of osteoclastogenic potential by IR-induced senescent OCYs exocytosis factor CCL5, the conditioned medium (CM) of OCYs was co-cultured with RAW264.7 precursor cells. It was observed that in the irradiated OCY co-cultured group, the migration potential increased compared with the vehicle culture group, accompanied by an enhancement of typical mature OCs; the expression of the specific function of enzyme tartrate-resistant acid phosphatase (TRAP) increased; and the bone-destructive function was enhanced. However, a neutralizing antibody to CCL5 could reverse the extra-activation of osteoclastogenesis. Accordingly, the overexpression of p-STAT3 in irradiated OCY was accompanied by CCL5. It was concluded that CCL5 is a potential key molecule and the interventions targeting CCL5 could be a potential strategy for inhibiting osteoclastogenesis and restoring bone remodeling.

Comparison of the Validity of Enzymatic and Immunohistochemical Detection of Tartrate-resistant Acid Phosphatase (TRAP) in the Context of Biocompatibility Analyses of Bone Substitutes.

Macrophages and biomaterial-induced multinucleated giant cells (BMGCs) are central elements in the tissue reaction cascade towards bone substitute materials (BSM). The enzymatic detection of the lytic enzyme tartrate-resistant acid phosphatase (TRAP) has manifoldly been used to examine the so-called "bioactivity" of BSM. The present study aimed to compare the detection validity and expression pattern of the TRAP enzyme using enzymatic and immunohistochemical detection methods in the context of biocompatibility analyses of BSM. Biopsies from 8 patients were analyzed after sinus augmentation with a xenogeneic bone substitute. Analysis of both macrophage and BMGC polarization were performed by histochemical TRAP detection and immunohistochemical detection of TRAP5a. Histomorphometrical analysis was used for comparison of the TRAP detection of BMGCs. The enzymatic TRAP detection method revealed that in 7 out of 8 biopsies only single cells were TRAP-positive, whereas most of the cells and especially the BMGCs were TRAP-negative. The immunohistochemical detection of TRAP5a showed moderate numbers of stained mononuclear cells, while the majority of the BMGCs showed signs of TRAP5a-expression. The enzymatic TRAP detection was comparable to the results obtained via immunohistochemistry only in one case. The histomorphometrical analysis showed that significantly more mononuclear and multinucleated TRAP-positive cells were found using immunohistochemical TRAP5a-staining compared to the enzymatic TRAP detection method. Also, significantly more TRAP-negative BMGCs were found using the enzymatic TRAP detection. The immunohistochemical detection of TRAP is more accurate for examination of the bioactivity and cellular degradability of BSM.

Celecoxib treatment dampens LPS-induced periapical bone resorption in a mouse model.

To evaluate the efficacy of selective and nonselective inhibitors of cyclooxygenase-2 enzymes in the treatment of experimental apical periodontitis induced by bacterial lipopolysaccharide (LPS) in vivo in a mouse model. Thirty-six C57BL/6 mice were used. After access cavity preparation, a solution containing E.coli LPS (1.0µgµL-1 ) was inoculated into the root canals of the mandibular and maxillary right first molars (n=72) After 30days, apical periodontitis was established and the animals were systemically treated with celecoxib, a selective COX-2 inhibitor (15mgkg-1 ), or indomethacin, a nonselective COX-2 inhibitor (5mgkg-1 ), for 7 and 14days. Blocks containing teeth and bone were removed for histopathological and histometric analyses (haematoxylin and eosin), evaluation of osteoclasts numbers (tartrate-resistant acid phosphatase enzyme - TRAP) and immunohistochemistry for RANK, RANKL and OPG. Gene expression was performed using reverse transcription and real-time polymerase chain reaction (qRT-PCR) for RANK, RANKL, OPG, TRAP, MMP-9, cathepsin K and calcitonin receptor. Histopathological, histometric, TRAP, immunohistochemistry and qRT-PCR data were evaluated using Kruskal-Wallis followed by Dunn's test (α=0.05). Systemic administration of celecoxib for 7 and 14days prevented periapical bone resorption (P<0.0001), differently from indomethacin that exacerbated bone resorption at 7days (P<0.0001) or exerted no effect at 14days (P=0.8488). Celecoxib treatment reduced osteoclast formation in apical periodontitis, regardless of the period of treatment (P<0.0001 for 7days and P=0.026 for 14days). Administration of celecoxib or indomethacin differentially modulated the expression of genes involved in bone resorption. At 7days, celecoxib and indomethacin treatment significantly inhibited expression of mRNA for cathepsin K (P=0.0005 and P=0.016, respectively) without changing TRAP, MMP-9 and calcitonin receptor gene expression. At 14days, celecoxib significantly inhibited expression of mRNA for MMP-9 (P<0.0001) and calcitonin receptor (P=0.004), whilst indomethacin exerted no effect on MMP-9 (P=0.216) and calcitonin receptor (P=0.971) but significantly augmented cathepsin K gene expression (P=0.001). The selective COX-2 inhibitor celecoxib reduced osteoclastogenic signalling and activity that dampened bone resorption in LPS-induced apical periodontitis in mice, with greater efficacy than the nonselective inhibitor indomethacin.

Effects of 5-lipoxygenase gene disruption on inflammation, osteoclastogenesis and bone resorption in polymicrobial apical periodontitis

ObjectivesTo investigate the regulation of inflammatory and osteoclastogenic signaling by 5-lipoxygenase (5-LO) in apical periodontitis induced by oral contamination of dental root canals in mice. DesignApical periodontitis was induced in 5-lipoxygenase enzyme knockout (129-Alox5tm1Fun) and 129 wild-type mice (n = 96) by exposure of the dental root canal to the oral cavity. After 7, 14, 21, and 28 days, the animals were euthanized and the tissues removed (n = 12 teeth per period) for histopathological and histometric analyses (hematoxylin and eosin [HE]), evaluation of osteoclastogenic activity (tartrate-resistant acid phosphatase enzyme [TRAP]), and determination of inflammatory and osteoclastogenic signaling (qRT-PCR). ResultsOral contamination of dental root canals induced recruitment of neutrophils and osteoclasts to the periodontal ligament, resulting in bone resorption. Absence of 5-LO did not impair neutrophil recruitment while osteoclastic formation was increased. Nonetheless, early bone resorption progressed similarly to lesions in wild-type animals. Interestingly, in the absence of 5-LO, the synthesis of mRNAs for cytokines, chemokines, and their receptors was significantly reduced while that of regulators of osteoclastogenesis (RANK, RANKL, and OPG) was increased in comparison with the corresponding levels in wild-type animals. ConclusionsThe 5-LO pathway plays a role in the stimulation of inflammatory mediator synthesis and inhibition of osteoclastogenesis in apical periodontitis in mice. However, the paradoxical inflammatory-osteoclastogenic signaling did not impair inflammatory cell recruitment and bone resorption during early development of the disease.

Manipulation of osteoclastogenesis: Bioactive multiphasic silica/collagen composites and their effects of surface and degradation products

The intent of the present study was to demonstrate that multiphasic silica/collagen xerogels are able to manipulate cellular processes. These xerogels were prepared by a sol-gel approach allowing the incorporation of mineral phases. The resulting nanocomposites are designed as biomaterial for bone regeneration. Human osteoclasts derived from peripheral blood mononuclear cells were cultured both indirectly and directly, either in presence of different xerogel types or on their surface, to investigate the factor with the main influence on osteoclastogenesis. To this end, the incorporation of a third phase to silica/collagen xerogels was used to affect osteoclastogenesis. In cell culture, ambient ion conditions controlled by both the degradation products of the xerogel and the bioactivity-dependent ion release and reprecipitation were shown to have the main effect on osteoclast specific enzyme tartrate-resistant acid phosphatase (TRAP) 5b. Late stage of osteoclastogenesis characterized by resorption was strongly dependent on the xerogels composition. Surface chemistry of the xerogels was displayed to play an important role in osteoclast resorption. Biphasic silica/collagen xerogels and triphasic xerogels with calcium carbonate offered widespread resorbed areas, whereas hydroxyapatite containing xerogels showed distinctly reduced resorption. The incorporation of strontium carbonate and phosphate, respectively, as third phase changed TRAP 5b activity dose-dependently and inhibited resorption within 21 days. Quantitative evaluation on osteoclast differentiation was carried out using biochemical methods (TRAP 5b, cathepsin K) and was supported by confocal laser scanning microscopy and scanning electron microscopy (SEM). Qualitative estimation of resorption was carried out by SEM.

A Potent Tartrate Resistant Acid Phosphatase Inhibitor to Study the Function of TRAP in Alveolar Macrophages

The enzyme tartrate resistant acid phosphatase (TRAP, two isoforms 5a and 5b) is highly expressed in alveolar macrophages, but its function there is unclear and potent selective inhibitors of TRAP are required to assess functional aspects of the protein. We found higher TRAP activity/expression in lungs of patients with chronic obstructive pulmonary disease (COPD) and asthma compared to controls and more TRAP activity in lungs of mice with experimental COPD or asthma. Stimuli related to asthma and/or COPD were tested for their capacity to induce TRAP. Receptor activator of NF-κb ligand (RANKL) and Xanthine/Xanthine Oxidase induced TRAP mRNA expression in mouse macrophages, but only RANKL also induced TRAP activity in mouse lung slices. Several Au(III) coordination compounds were tested for their ability to inhibit TRAP activity and [Au(4,4′-dimethoxy-2,2′-bipyridine)Cl2][PF6] (AubipyOMe) was found to be the most potent inhibitor of TRAP5a and 5b activity reported to date (IC50 1.3 and 1.8 μM respectively). AubipyOMe also inhibited TRAP activity in murine macrophage and human lung tissue extracts. In a functional assay with physiological TRAP substrate osteopontin, AubipyOMe inhibited mouse macrophage migration over osteopontin-coated membranes. In conclusion, higher TRAP expression/activity are associated with COPD and asthma and TRAP is involved in regulating macrophage migration.

Osteoclastic differentiation and resorption is modulated by bioactive metal ions Co2+, Cu2+ and Cr3+ incorporated into calcium phosphate bone cements.

Biologically active metal ions in low doses have the potential to accelerate bone defect healing. For successful remodelling the interaction of bone graft materials with both bone-forming osteoblasts and bone resorbing osteoclasts is crucial. In the present study brushite forming calcium phosphate cements (CPC) were doped with Co2+, Cu2+ and Cr3+ and the influence of these materials on osteoclast differentiation and activity was examined. Human osteoclasts were differentiated from human peripheral blood mononuclear cells (PBMC) both on the surface and in indirect contact to the materials on dentin discs. Release of calcium, phosphate and bioactive metal ions was determined using ICP-MS both in the presence and absence of the cells. While Co2+ and Cu2+ showed a burst release, Cr3+ was released steadily at very low concentrations (below 1 μM) and both calcium and phosphate release of the cements was considerably changed in the Cr3+ modified samples. Direct cultivation of PBMC/osteoclasts on Co2+ cements showed lower attached cell number compared to the reference but high activity of osteoclast specific enzymes tartrate resistant acid phosphatase (TRAP), carbonic anhydrase II (CAII) and cathepsin K (CTSK) and significantly increased gene expression of vitronectin receptor. Indirect cultivation with diluted Co2+ cement extracts revealed highest resorbed area compared to all other modifications and the reference. Cu2+ cements had cytotoxic effect on PBMC/osteoclasts during direct cultivation, while indirect cultivation with diluted extracts from Cu2+ cements did not provoke cytotoxic effects but a strictly inhibited resorption. Cr3+ doped cements did not show cytotoxic effects at all. Gene expression and enzyme activity of CTSK was significantly increased in direct culture. Indirect cultivation with Cr3+ doped cements revealed significantly higher resorbed area compared to the reference. In conclusion Cr3+ doped calcium phosphate cements are an innovative cement modification because of their high cytocompatibility and support of active resorption by osteoclasts.

Manipulation of In Vitro Osteoclastogenesis: Bioactive Multiphasic Silica/Collagen Composites and their Effects of Surface and Degradation Products

The intent of the present study was to demonstrate that multiphasic silica/collagen xerogels are able to manipulate cellular processes. These xerogels were prepared by a sol-gel approach allowing the incorporation of mineral phases. The resulting nanocomposites are designed as biomaterial for bone regeneration. Human osteoclasts derived from peripheral blood mononuclear cells were cultured both indirectly and directly, either in presence of different xerogel types or on their surface, to investigate the factor with the main influence on osteoclastogenesis. To this end, the incorporation of a third phase to silica/collagen xerogels was used to affect osteoclastogenesis. In cell culture, ambient ion conditions controlled by both the degradation products of the xerogel and the bioactivity-dependent ion release and reprecipitation were shown to have the main effect on osteoclast specific enzyme tartrate-resistant acid phosphatase (TRAP) 5b. Late stage of osteoclastogenesis characterized by resorption was strongly dependent on the xerogels composition. Surface chemistry of the xerogels was displayed to play an important role in osteoclast resorption. Biphasic silica/collagen xerogels and triphasic xerogels with calcium carbonate offered widespread resorbed areas, whereas hydroxyapatite containing xerogels showed distinctly reduced resorption. The incorporation of strontium carbonate and phosphate, respectively, as third phase changed TRAP 5b activity dose-dependently and inhibited resorption within 21 days. Quantitative evaluation on osteoclast differentiation was carried out using biochemical methods (TRAP 5b, cathepsin K) and was supported by confocal laser scanning microscopy and scanning electron microscopy (SEM). Qualitative estimation of resorption was carried out by SEM.

Retinoic acid stimulates expression of the functional osteoclast integrin alpha v beta 3: transcriptional activation of the beta 3 but not the alpha v gene.

The capacity of osteoclasts to resorb bone depends, in part, on the surface expression of the integrin alpha v beta 3. We have investigated whether the steroid hormone retinoic acid, known to stimulate bone resorption, regulates the appearance of the alpha v beta 3 complex in avian osteoclast presursors. Using surface labeling, followed by immunoprecipitation with a alpha v beta 3-specific antibody, we show that retinoic acid increases surface expression of the heterodimer in a dose- and time-dependent manner. Northern analysis reveals that the high basal steady-state levels of alpha v mRNA do not change, while those for beta 3 rise significantly from their initially low levels. Nuclear run-on studies confirm that steroid treatment stimulates transcription of the beta 3, but not the alpha v gene. Osteoclast precursors treated with retinoic acid exhibit increased multinucleation and expression of the osteoclast marker enzyme tartrate-resistant acid phosphatase. However, the fused cells do not have an increased capacity to resorb bone. In summary, multinucleated cells generated in this study do not represent fully differentiated bone-resorbing polykaryons. These results suggest that treatment of osteoclast precursors with retinoic acid is necessary, but insufficient, for expression of the mature osteoclast phenotype.

The R740S mutation in the V‐ATPase a3 subunit results in osteoclast apoptosis and defective early‐stage autophagy

Vacuolar-type H(+)-ATPases (V-ATPases) are located in lysosomes and at the ruffled border in osteoclasts. We showed previously that the R740S mutation is dominant negative for V-ATPase activity, uncouples proton transport from ATP hydrolysis and causes osteopetrosis in heterozygous mice (+/R740S). Here we show mice homozygous for R740S (R740S/R740S) have more severe osteopetrosis and die by postnatal day 14. Although R740S/R740S osteoclasts express wild-type levels of a3, it is mislocalized. Acridine orange staining of R740S/R740S osteoclasts grown on a Corning resorptive surface reveals no resorption and no acidification of intracellular compartments. Whereas osteoblast and osteocyte apoptosis is normal, R740S/R740S osteoclasts exhibit increased apoptosis compared with wild-type osteoclasts. Localization of the enzyme tartrate-resistant acid phosphatase (TRAP) is also aberrant. Transmission electron microscopy reveals that R740S/R740S osteoclasts do not polarize, lack ruffled borders, and contain fewer autophagosomes. Consistent with an early stage defect in autophagy, expression of LC3II is reduced and expression of p62 is increased in R740S/R740S compared to wild-type osteoclasts. These results indicate the importance of intracellular acidification for the early stages of autophagy as well as for osteoclast survival, maturation, and polarization with appropriate cytoplasmic distribution of key osteoclast enzymes such as TRAP.

Ovariectomy induces osteoporosis in the maxillary alveolar bone: an in vivo micro‐CT and histomorphometric analysis in rats

To investigate whether porotic changes occur in maxillary alveolar bone in ovariectomized rats. Thirty-two 6-week-old female Sprague-Dawley rats were divided into an ovariectomy group (OVX) and a sham-ovariectomy group (sham). Twelve weeks after surgery, maxillae of 16 rats (eight OVX and eight sham) were analyzed by micro-CT. Inter-radicular alveolar bone of the maxillary first molar was reconstructed and analyzed. The remaining 16 maxillae were used for histochemistry, including hematoxylin-eosin staining and tartrate-resistant acid phosphatase enzyme staining. Three-dimensional reconstructed images of the irregular alveolar bone showed an intuitive view of porotic changes in the OVX group. The alveolar bone in OVX rats had a porous microarchitecture including lower bone mass and a looser structure of more widely separated trabeculae. Bone mineral density (BMD), bone volume fraction (BV/TV), and trabecular thickness (Tb.Th.) were significantly lower in the OVX group than those in the sham group (P<0.05). Trabecular separation (Tb.Sp.) increased significantly in the OVX rats compared to the sham rats (P<0.05). Histomorphometric analysis of alveolar bone also revealed porotic changes in the OVX rats. Bone area ratio significantly decreased in the OVX group compared with the sham group (P<0.01). There were also more osteoclasts present in the alveolar bone of OVX rats compared to sham rats (P<0.05). Ovariectomy induces osteoporosis in maxillary alveolar bone in rats, which may be related to the increased number of osteoclasts.

Interleukin‐17A stimulates granulocyte–macrophage colony‐stimulating factor release by murine osteoblasts in the presence of 1,25‐dihydroxyvitamin D3 and inhibits murine osteoclast development in vitro

To investigate the effects of interleukin-17A (IL-17A) on osteoclastogenesis in vitro. Bone marrow cells (BMCs) were isolated from the excised tibia and femora of wild-type C57BL/6J mice, and osteoblasts were obtained by sequential digestion of the calvariae of ddY, C57BL/6J, and granulocyte-macrophage colony-stimulating factor-knockout (GM-CSF(-/-)) mice. Monocultures of BMCs or cocultures of BMCs and osteoblasts were supplemented with or without 1,25-dihydroxyvitamin D(3)(1,25[OH](2)D(3)), recombinant human macrophage colony-stimulating factor (M-CSF), RANKL, and IL-17A. After 5-6 days, the cultures were fixed with 4% paraformaldehyde and subsequently stained for the osteoclast marker enzyme tartrate-resistant acid phosphatase (TRAP). Osteoprotegerin (OPG) and GM-CSF expression were measured by enzyme-linked immunosorbent assay, and transcripts for RANK and RANKL were detected by real-time polymerase chain reaction. In both culture systems, IL-17A alone did not affect the development of osteoclasts. However, the addition of IL-17A plus 1,25(OH)(2)D(3) to cocultures inhibited early osteoclast development within the first 3 days of culture and induced release of GM-CSF into the culture supernatants. Furthermore, in cocultures of GM-CSF(-/-) mouse osteoblasts and wild-type mouse BMCs, IL-17A did not affect osteoclast development, corroborating the role of GM-CSF as the mediator of the observed inhibition of osteoclastogenesis by IL-17A. These findings suggest that IL-17A interferes with the differentiation of osteoclast precursors by inducing the release of GM-CSF from osteoblasts.

Role of solasodine hydrochloride in AS2O3 induced HeLa cells apoptosis as well as its effect on cell telomerase activity in vitro

Objective To study whether solasodine hydrochloride (SBHL) could enhance the effect of arsenic trioxide in inducing apoptosis and affecting telomerase activity in cervical cancer HeLa cells. Methods Using cell culture methods, cervical cancer HeLa cells were cultured in vitro. The optimal concentration of SBHL was determined by MTT method from 0, 10, 20, 40, 80, 160, to 320 μmol/L. HeLa cells were grown in improved RPMI1640 supplemented respectively with arsenic trioxide(5 μmol/L As2O3), As2O3(5 μmol/L)+ SBHL( 40 μmol/L) and none (control group). The growth morphology of HeLa cells was observed under phase contrast microscopy after culture for 24, 48, and 72 h. Apoptosis of HeLa cells was determined under transmission electronic microscopy. The method of MTT was used to study the cell survival percentage. The technique of flow cytometry was used to measure cell cycle and cell apoptosis percentage. The method of tartrate-resistant acid phosphatase-enzyme linked immunosorbent assay (TRAP-ELISA) was used to determine telomerase activity of HeLa cells. Results Under phase contrast microscopy, in control group HeLa cells were round, densely packed; in As2O3 group the numbers of the cells were less, cell spacing increased; in As2O3 + SBHL group the cells shrinked significantly, nuclear fragmented as a petal-like, gap became larger. Under transmission electronic microscopy, there were rich microvillus on the cell surface in control group, cell intervals clear, immature connections, and the intervals did not close. The structure of the mitochondria in the cytoplasm was integrated. Most of the chromatin in the nucleus were, euchromatin and characteristics of apoptosis with heterochromatin increased and the chromatin condensed into masses, on the boundary of nuclear membrane. The microvillud on the cell surface were ruptured and decreased in As2O3 + SBHL group. The chromatin condensed into masses. The formation of apoptotic bodies was observed. The difference was statistically significant between groups in cell survival percentage at 24, 48, 72h(x2 = 10.39 , 13.88 , 17.21,respectively, all P < 0.05). Cell survival percentage in SBHL + As2O3 group (52.80%) was significantly less than that of As2O3 group(77.51%, x2 = 9.29, P < 0.05) at 72 h. In cell cycles, the difference was statistically significant between groups in C1 phase and S phase(F = 7.46,22.14, all P < 0.05), respectively. Compared with , control group[ (41.57 ± 1.56)%, (50.45 ± 2.37)%], cell percentages in S phase in As2O3 + SBHL group[(20.06 ± 4.98)%] and As2O3 group[(27.10 ± 5.32)%] were decreased(P< 0.05 or < 0.01), while cell percentage in C1 phase was increased[(58.70 ± 5.18)%, (69.67 ± 4.17)%, P< 0.05 or < 0.01]. The difference was statistically significant between groups in apoptotic percentage of HeLa cells (F = 4.01, P < 0.05). Compared with control group[ (1.18 ± 1.40)%], apoptosis percentage was significantly increased in As2O3 + SBHL group and As2O3 group [(21.08± 1.22)%, (6.04±2.53)%, P< 0.05 or < 0.01], respectively, and As2O3 + SBHL group was higher than As2O3 group(P < 0.01). The difference was statistically significant between groups in telomerase activity (F = 21.28, P< 0.05). Telomerase activity was inhibited in As2O3 group(1.214 ± 0.621) and As2O3A + SBHL group(0.865 ± 0.284) compared to control group (2.107 ± 0.057, all P < 0.05), and telomerase activity in As2O3 + SBHL group was lower than that of As2O3 group (P < 0.05). Conclusions SBHL enhances the effect of As2O3 in inducing apoptosis in HeLa cells, which is related to its inhibiting telomerase activity in HeLa cells. Key words: Solasodine hydrochloride; As2O3; HeLa cells; Apoptosis; Telomerase

Long bone osteoclasts display an augmented osteoclast phenotype compared to calvarial osteoclasts

Osteoclasts are multinucleated cells specialized in degrading bone and characterized by high expression of the enzymes tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CtsK). Recent studies show that osteoclasts exhibit phenotypic differences depending on their anatomical site of action. Using immunohistochemistry, RT-qPCR, FPLC chromatography and immunoblotting, we compared TRAP expression in calvaria and long bone. TRAP protein and enzyme activity levels were higher in long bones compared to calvaria. In addition, proteolytic processing of TRAP was more extensive in long bones than calvaria which correlated with higher cysteine proteinase activity and protein expression of CtsK. These two types of bones also exhibited a differential expression of monomeric TRAP and CtsK isoforms. Analysis of CtsK −/− mice revealed that CtsK is involved in proteolytic processing of TRAP in calvaria. Moreover, long bone osteoclasts exhibited higher expression of not only TRAP and CtsK but also of the membrane markers CD68 and CD163. The results suggest that long bone osteoclasts display an augmented osteoclastic phenotype with stronger expression of both membranous and secreted osteoclast proteins.

Interface membrane fibroblasts around aseptically loosened endoprostheses express MMP‐13

The objective of this article was to assess whether matrix metalloproteinase-13 (MMP-13) is produced by cells of the peri-implant interface tissues and to further characterize these cells. Tissue specimens were collected from the bone-prosthesis interface at the time of revision surgery of clinically loosened hip and knee arthroplasties (n = 27). Synovial tissues from osteoarthritic patients and young patients with mild joint deformity were used as controls (n = 6). Tissue samples were fixed in 4% PFA, decalcified with EDTA, and embedded in paraffin. Sections (4 microm) were stained with hematoxylin/eosin and for the osteoclastic marker enzyme tartrate resistant acid phosphatase. Monocytes/macrophages were characterized with a monoclonal antibody against CD68 and mRNAs encoding MMP-13 and alpha(1) collagen I (COL1A1) were detected by in situ hybridization. Cells expressing transcripts encoding MMP-13 were found in 70% of the interface tissues. These cells colocalized with a cell population expressing COL1A1 mRNA, and were fibroblastic in appearance. MMP-13 expressing cells were found in the close vicinity of osteoclasts and multinuclear giant cells. No signals for transcripts encoding MMP-13 were detected in multinuclear giant cells or in osteoclasts. Control tissues were negative for transcripts encoding MMP-13 mRNA. Fibroblasts of the interface from aseptically loosened endoprostheses selectively express MMP-13. By the expression and the release of MMP-13, these fibroblastic cells may contribute to the local degradation of the extracellular matrix and to bone resorption.

In vitro studies on the effect of delaminated a-C:H film fragments on bone marrow cell cultures

Amorphous hydrogenated carbon (a-C:H) films have many outstanding properties required for a protective coating material on load bearing medical implants. Recently, titanium doped a-C:H films have been evaluated regarding their effects on bone marrow cell cultures. But many materials that are well-tolerated in bulk form are able to induce toxic reaction if present particulate form. In order to further assess biocompatibility aspects of these two coatings, film delamination has been mimicked in exposure to fluids. In the present study, particles from a-C:H, a-C:H/Ti and a-C:H–a-C:H/Ti bilayer films were added to bone marrow cell cultures in vitro. The results showed that plain a-C:H and to a certain extent a-CH/Ti particles were inert. Both kinds of particles did not significantly stimulate the osteoclast-related enzyme tartrate resistant acid phosphatase (TRAP). A slight increase in cell proliferation and total culture TRAP was found in cultures treated by a-C:H–a-C:H/Ti bilayer films. Latter effect can probably be traced back by the relative high percentage of small particles of a size of around 2 μm. However, if corrected by the cell number also no differences between particle-treated and untreated control cultures could be found, indicating the absence of a toxic effect from delaminated a-C:H coatings.

ATP downregulates P2X7 and inhibits osteoclast formation in RAW cells.

Multinucleated giant cells derive from fusion of precursor cells of the macrophage lineage. It has been proposed that the purinoreceptor P2X(7) is involved in this fusion process. Prolonged exposure of macrophages to ATP, the ligand for P2X(7), induces the formation of plasma membrane pores and eventual cell death. We took advantage of this cytolytic property to select RAW 264.7 (RAW) cells that lacked P2X(7) function by maintaining them in ATP (RAW ATP-R cells). RAW ATP-R cells failed to fuse to form multinucleated osteoclasts in response to receptor activator nuclear factor-kappaB ligand, although they did become positive for the osteoclast marker enzyme tartrate-resistant acid phosphatase, and upregulated expression of other osteoclast marker genes. RAW ATP-R cells and wild-type RAW cells expressed similar amounts of P2X(7) protein, but little P2X(7) was present on the surface of RAW ATP-R cells. After ATP was removed from the medium of RAW ATP-R cells, the cells reexpressed P2X(7) on the cell surface, regained sensitivity to ATP, and formed multinucleated osteoclasts. These results suggest that P2X(7) or another protein that is downregulated in concert with P2X(7) is involved either in the mechanics of cell fusion to form osteoclasts or in a signaling pathway proximal to this event. These results also suggest that P2X(7) may be regulated by ligand-mediated internalization and that extracellular ATP may regulate the formation of osteoclasts and other multinucleated giant cells.

The effect of particle size and electrical charge on macrophage-osteoclast differentiation and bone resorption.

In aseptic loosening, there is commonly periprosthetic bone loss and a heavy macrophage infiltrate in response to biomaterial wear particles generated from the implant materials. Macrophages which have phagocytosed wear particles are known to be capable of differentiation into bone resorbing osteoclasts. In this investigation we determine the role of particle size and particle charge on this process. Mouse monocytes and macrophages were co-cultured with osteoblast-like UMR106 cells and 1,25 dihydroxyvitamin D3 in the presence or the absence of (i) various sizes of latex beads (0.1, 1, 10 and 100 microm) and (ii) uncharged, positively- or negatively-charged sephadex beads of uniform shape and composition. The extent of osteoclast differentiation by monocytes or foreign body macrophages was determined by the expression of the osteoclast-associated enzyme tartrate-resistant acid phosphatase and lacunar bone resorption. No significant difference in the extent of osteoclast formation and bone resorption was noted in response to particle size. Osteoclast formation was also not significantly different in the presence of positively/negatively charged and uncharged particles. These findings indicate that osteoclast formation is not significantly influenced by particle characteristics, such as particle size. They also add support to the hypothesis that macrophage involvement in periprosthetic osteolysis is not dependent on particle phagocytosis and that it may be induced by particle contact.

Features of mono- and multinucleated bone resorbing cells of the zebrafish Danio rerio and their contribution to skeletal development, remodeling, and growth.

To provide basic data about bone resorbing cells in the skeleton during the life cycle of Danio rerio, larvae, juveniles, and adults (divided into six age groups) were studied by histological procedures and by demonstration of the osteoclast marker enzyme tartrate-resistant acid phosphatase (TRAP). Special attention was paid to the lower jaw, which is a standard element for fish bone studies. The presence of osteoclasts at endosteal surfaces of growing bones of all animals older than 20 days reveals that resorption is an important part of zebrafish skeletal development. The first bone-resorbing cells to form are mononucleated. They appear in 20-day-old animals concurrently in the craniofacial skeleton and vertebral column. Mononucleated osteoclasts are predominant in juveniles. Regional differences characterize the appearance of osteoclasts; at thin skeletal elements (neural arches, nasal) mononucleated osteoclasts are predominant even in adults. Multinucleated bone-resorbing cells were first observed in 40-day-old animals and are the predominant osteoclast type of adults. Both mono- and multinucleated osteoclasts contribute to allometric bone growth but multinucleated osteoclasts are also involved in lacunar bone resorption and repeated bone remodeling. Resorption of the dentary follows the pattern described above (mononucleated osteoclasts precede multinucleated cells) and includes the partial removal of Meckel's cartilage. Bone marrow spaces created by resorption are usually filled with adipose tissue. In conclusion, bone resorption is primarily subjected to the demands of growth, the appearance of mono- and multinucleated osteoclasts is site- and age-related, and bone remodeling occurs. The results are discussed in relation to findings in other teleosts and in mammals.

Particle-induced Phagocytic Cell Responses are Material Dependent: Foreign Body Giant Cells Vs. Osteoclasts from a Chick Chorioallantoic Membrane Particle-implantation Model

There is increasing concern about particles generated from wear-prone implants that are placed in body tissues, including artificial hip, knee, and jaw joints. Although phagocytes and foreign body giant cells are associated with inhaled or embedded particulate debris, some particles also induce bone digestion by eliciting the differentiation and proliferation of highly specialized osteoclastic cells. This report describes the differential phagocytic cellular responses to four implant-related types of ground, model wear particles in a live-egg cell-response model, as implants to the chick chorioallantoic membrane (CAM): polymethylmethacrylate (PMMA), a main constituent of some temporomandibular joint (TMJ) implants and orthopedic cements used to retain artificial hips and knees; Proplast-HA, an implantable composite of polytetrafluoroethylene (PTFE) and degradable mineral (hydroxyapatite) that has been associated with bone erosion around failed TMJ implants; talc, a nondegradable mineral sometimes found in tissues as a contaminant from talc-coated surgical gloves; and authentic bone, known to induce the formation of osteoclastic cells. Light and electron microscopy of CAM tissues harvested, sectioned and stained with special reagents for the enzymes tartrate-resistant acid phosphatase (TRAP) and tartrate-resistant adenosine triphosphatase (TrATPase), and for the osteoclast-specific antigen 121F, showed that only authentic bone and the degradable HA-rich particles induced osteoclast formation. From these results, and supporting data with polypropylene particles, it is concluded that nonbiodegradable polymer particles, alone, do not induce bone dissolution. Inert polymers do induce foreign body giant cells without the external mineral digestion qualities unique to osteoclasts, however. The chick embryo model system allows quick and affordable examination of material-dependent differences in phagocytic cellular responses to implant wear debris and particles from various occupational environments.