acid-phosphatase and Osteolysis

Topics: Acid Phosphatase; Adult; Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Female; Humans; Isoenzymes; Laryngeal Diseases; Laryngeal Neoplasms; Male; Middle Aged; Osteoclasts; Osteolysis; Tartrates

We examined the effects of triptolide on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and on titanium (Ti) particle-induced osteolysis.. To examine the effect of triptolide on osteoclast differentiation, bone marrow macrophages (BMMs) were treated with 100 ng/mL of RANKL and 30 ng/mL of macrophage-colony stimulating factor, or co-cultured with osteoblasts stimulated with 10 nM vitamin D3 and 1 μM prostaglandin E2 in the presence or absence of triptolide (2.8-14 nM). Osteoclast differentiation and activation were assessed using tartrate-resistant acid phosphatase staining, reverse transcriptase-polymerase chain reaction analysis to determine differentiation marker gene expression and pit formation assays. To examine the effect of triptolide on wear debris-induced osteolysis, titanium (Ti) particles were injected into the calvaria of ICR mice. Then, the mice were divided into three groups and were orally administered vehicle, or 16 or 32 μg/kg/day triptolide for ten days, followed by histomorphometric analysis.. Triptolide suppressed RANKL-mediated osteoclast differentiation of BMMs in a dose-dependent manner. In a co-culture system, osteoblasts treated with triptolide could not induce osteoclast differentiation of BMMs, which was accompanied by down-regulation of RANKL and up-regulation of osteoprotegrin. Moreover, triptolide significantly inhibited bone resorption, and expression of the bone resorption marker genes. RANKL-induced activation of p38, ERK, and JNK was substantially inhibited by triptolide. Further, in a Ti-induced mouse calvarial erosion model, mice perorally administrated with triptolide showed significant attenuation of Ti-mediated osteolysis.. Our data indicated that triptolide had an anti-osteoclastic effect and significantly suppressed wear debris-induced osteolysis in mice.

Topics: Acid Phosphatase; Animals; Blotting, Western; Cell Differentiation; Cell Survival; Diterpenes; Epoxy Compounds; Isoenzymes; Macrophages; Male; Mice; Mice, Inbred ICR; Osteoclasts; Osteolysis; Phenanthrenes; Receptor Activator of Nuclear Factor-kappa B; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Tartrate-Resistant Acid Phosphatase; Titanium

Receptor activator of nuclear factor kappa-B (RANK) and RANK-ligand are relevant targets for the treatment of polyethylene particle-induced osteolysis. This study assessed the local administration of siRNA, targeting both human RANK and mouse Rank transcripts in a mouse model. Four groups of mice were implanted with polyethylene (PE) particles in the calvaria and treated locally with 2.5, 5 and 10 μg of RANK siRNA or a control siRNA delivered by the cationic liposome DMAPAP/DOPE. The tissues were harvested at day 9 after surgery and evaluated by micro-computed tomography, tartrate-resistant acid phosphatase (TRAP) immunohistochemistry for macrophages and osteoblasts, and gene relative expression of inflammatory and osteolytic markers. 10 μg of RANK siRNA exerted a protective effect against PE particle-induced osteolysis, decreasing the bone loss and the osteoclastogenesis, demonstrated by the significant increase in the bone volume (P<0.001) and by the reduction in both the number of TRAP(+) cells and osteoclast activity (P<0.01). A bone anabolic effect demonstrated by the formation of new trabecular bone was confirmed by the increased immunopositive staining for osteoblast-specific proteins. In addition, 5 and 10 μg of RANK siRNA downregulated the expression of pro-inflammatory cytokines (P<0.01) without depletion of macrophages. Our findings show that RANK siRNA delivered locally by a synthetic vector may be an effective approach for reducing osteolysis and may even stimulate bone formation in aseptic loosening of prosthetic implants.

Topics: Acid Phosphatase; Animals; Disease Models, Animal; Gene Expression Regulation; Genetic Vectors; HEK293 Cells; Humans; Isoenzymes; Liposomes; Mice; Osteoblasts; Osteolysis; Polyethylene; Receptor Activator of Nuclear Factor-kappa B; RNA, Small Interfering; Tartrate-Resistant Acid Phosphatase

Aseptic loosening associated with wear particle-induced inflammation is a major cause of joint implant failure. Recent studies have demonstrated the therapeutic effects of p38 mitogen-activated protein kinase (MAPK)-based therapies on chronic inflammatory diseases. The purpose of this study was to investigate whether SB203580, a p38 MAPK inhibitor, inhibits wear debris-induced inflammatory osteolysis in mice through downregulation of receptor activator of nuclear factor kβ (RANK)/RANK ligand (RANKL). We used a murine osteolysis model to study the effect of SB203580 on RANKL/RANK signaling and titanium particle-induced osteolysis in vivo. Pouch membranes with intact bone implants were analyzed using histological analysis and transmission electron microscopy, and the levels of RANK and RANKL protein and mRNA were evaluated by immunohistological staining and real-time reverse transcriptase-polymerase chain reaction. SB203580 had less of an effect on RANK and RANKL expression under wear debris-induced conditions. The number of TRAP-positive cells was remarkably reduced in Ti-particle-induced pouch tissues. These effects were confirmed through the transmission electron microscopy results. These results suggest that p38 MAPK-based therapies are beneficial in preventing aseptic loosening associated with total joint replacement by modulating RANK-RANKL signaling.

Topics: Acid Phosphatase; Animals; Bone and Bones; Colonic Pouches; Disease Models, Animal; Down-Regulation; Female; Immunohistochemistry; Inflammation; Isoenzymes; Mice, Inbred BALB C; Osteolysis; p38 Mitogen-Activated Protein Kinases; Prostheses and Implants; Protective Agents; Protein Kinase Inhibitors; RANK Ligand; Real-Time Polymerase Chain Reaction; Receptor Activator of Nuclear Factor-kappa B; Tartrate-Resistant Acid Phosphatase; Titanium

We examined the effects of vitamin E supplementation (VES) on osteoclast (OC) resorbing activity and cytomorphometry in Walker 256/B tumor osteolytic rats. Twenty-four aged male rats were randomized into 3 groups: 6 were sham operated; 9 were injected in the right hind limb with Walker 256/B cells (W256 group); and 9 were injected as above and supplemented with VE (45mg/kg BW) (W256VE group). Twenty days later, bone mass (BV/TV) and some microarchitectural parameters were assessed. Some histodynamic parameters, cellular and nuclear form factors (FFC and FFN), and nuclear-cytoplasmic ratio (N/C) of OC were measured for each group. W256 group exhibited osteolytic lesions in the operated femora. Walker 256/B induced trabecular perforation and decreased BV/TV associated with significant increases in OC numbering (N.Oc/B.Ar and Oc.N/B.Pm) and activity (ES/BS and Oc.S/BS). While FFN remain unchanged, the FFC and N/C ratio increased in the W256 group. W256VE showed less osteolytic lesions. Moreover, disruption of bone microarchitecture and OC activity in W256VE group decreased. VES reduced the malignant Walker 256/B-induced enhanced OC resorbing activity with cytoinhibition rate reaching 41%. The protective effect of VE may be due to its modulation of OC cytomorphometry and subsequently their activity.

Topics: Acid Phosphatase; alpha-Tocopherol; Animals; Biomarkers; Bone Neoplasms; Bone Remodeling; Breast Neoplasms, Male; Dietary Supplements; Femur; Isoenzymes; Male; Osteoclasts; Osteolysis; Rats; Rats, Sprague-Dawley; Tartrate-Resistant Acid Phosphatase; Time Factors; Vitamins

Wear debris associated periprosthetic osteolysis represents a major pathological process associated with the aseptic loosening of joint prostheses. Naringin is a major flavonoid identified in grapefruit. Studies have shown that naringin possesses many pharmacological properties including effects on bone metabolism. The current study evaluated the influence of naringin on wear debris induced osteoclastic bone resorption both in vitro and in vivo. The osteoclast precursor cell line RAW 264.7 was cultured and stimulated with polymethylmethacrylate (PMMA) particles followed by treatment with naringin at several doses. Tartrate resistant acid phosphatase (TRAP), calcium release, and gene expression profiles of TRAP, cathepsin K, and receptor activator of nuclear factor-kappa B were sequentially evaluated. PMMA challenged murine air pouch and the load bearing tibia titanium pin-implantation mouse models were used to evaluate the effects of naringin in controlling PMMA induced bone resorption. Histological analyses and biomechanical pullout tests were performed following the animal experimentation. The in vitro data clearly demonstrated the inhibitory effects of naringin in PMMA induced osteoclastogenesis. The naringin dose of 10 μg/mL exhibited the most significant influence on the suppression of TRAP activities. Naringin treatment also markedly decreased calcium release in the stimulated cell culture medium. The short-term air pouch mouse study revealed that local injection of naringin ameliorated the PMMA induced inflammatory tissue response and subsequent bone resorption. The long-term tibia pin-implantation mouse model study suggested that daily oral gavage of naringin at 300 mg/kg dosage for 30 days significantly alleviated the periprosthetic bone resorption. A significant increase of periprosthetic bone volume and regaining of the pin stability were found in naringin treated mice. Overall, this study suggests that naringin may serve as a potential therapeutic agent to treat wear debris associated osteolysis.

Topics: Acid Phosphatase; Animals; Bone Resorption; Calcium; Cells, Cultured; Female; Flavanones; Isoenzymes; Mice; Mice, Inbred BALB C; Osteoclasts; Osteogenesis; Osteolysis; Polymethyl Methacrylate; Tartrate-Resistant Acid Phosphatase

Connexin 43 (Cx43) is the predominant gap junction protein in bone. Mice with a bone-specific deletion of Cx43 (cKO) have an osteopenic cortical phenotype. In a recent study, we demonstrated that cKO mice are resistant to bone loss induced by hindlimb suspension (HLS), an animal model of skeletal unloading. This protective effect occurred primarily as a result of lower osteoclast-mediated bone resorption. Interestingly, we also documented a significant increase in cortical osteocyte apoptosis and reduced sclerostin production. In the present study, we investigated whether osteocytic osteolysis - bone resorption by osteocytes within lacunae - is induced by HLS and the potential effect of Cx43 deficiency on this process during unloading.. 6-month-old male Cx43 cKO or wild-type (WT) mice were subjected to three weeks of HLS (Suspended) or normal loading conditions (Control) (n = 5/group). Lacunar morphology and tartrate-resistant acid phosphatase (TRACP) staining were assessed on sections of femur cortical bone. Experimental groups were compared via two-way ANOVA.. Empty lacunae were 26% larger in cKO-Control vs. WT-Control (p < 0.05), while there was no difference in the size of empty lacunae between Control and Suspended within WT or cKO (p > 0.05). Similarly, there was a trend (p = 0.06) for 11% larger lacunae containing viable osteocytes for cKO-Control vs. WT-Control, with no apparent effect of loading condition. There was no difference in the proportion of TRACP + cells between WT-Control and cKO-Control (p > 0.05); however, WT-Suspended mice had 246% more TRACP + osteocytes compared WT-Control mice (p < 0.05). There was no difference in TRACP staining between cKO-Control and cKO-Suspended (p > 0.05).. Prior to undergoing apoptosis, osteocytes in cKO mice may be actively resorbing their respective lacunae via the process of osteocytic osteolysis. Interestingly, the proportion of TRACP + osteocytes increased dramatically following unloading of WT mice, an effect that was not observed in cKO mice subjected to HLS. The results of the present study provide initial evidence that osteocytic osteolysis is occurring in cortical bone in response to mechanical unloading. Furthermore, Cx43 deficiency appears to protect against osteocytic osteolysis in a manner similar to the inhibition of unloading-induced osteoclast activation that we have documented previously.

Topics: Acid Phosphatase; Animals; Biomarkers; Cell Communication; Connexin 43; Femur; Genotype; Hindlimb Suspension; Isoenzymes; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Osteocytes; Osteolysis; Phenotype; Porosity; Signal Transduction; Tartrate-Resistant Acid Phosphatase; Time Factors

Wear-particle-induced osteolysis is considered to be the main reason for revision after arthroplasty. Although the exact mechanism remains unclear, inflammatory osteoclastogenesis plays an important role in this process. Strontium ranelate (SR) was found to have a therapeutic effect on osteoporosis in postmenopausal women. Based on prior studies, the present authors hypothesized that SR prevents wear-particle-induced osteolysis through restraining inflammatory osteoclastogenesis. The present study used 80 male C57BL/J6 mice to test this hypothesis in a murine osteolysis model. All experimental animals were randomly divided into four groups: a control group; a SR group; a titanium group; and a titanium+SR group. Once titanium particles had been implanted in mice, the mice were administered SR (900 mg kg(-1) day(-1)) by gavage for 14 days. After 14 days, the calvaria were collected for micro-computed tomography (μCT), histological and molecular analysis. The results of μCT and histomorphometric analysis demonstrated that SR markedly inhibited bone resorption and the generation of tartrate-resistant acid-phosphatase-positive cells in vivo, compared with titanium-stimulated calvaria. Reverse transcription polymerase chain reaction and ELISAs showed that SR stimulated the mRNA and protein expression of osteoprotegerin, and inhibited gene and protein expression of receptor activators of nuclear factor-kappa B ligand in titanium-particle-charged calvaria. In addition, SR obviously reduced the secretion of tumor necrosis factor-α and interleukin-1β in the calvaria of the titanium group. It was concluded that SR inhibits titanium-induced osteolysis by restraining inflammatory osteoclastogenesis, and that it could be developed as a new drug to prevent and treat aseptic loosening.

Topics: Acid Phosphatase; Animals; Inflammation; Interleukin-1beta; Isoenzymes; Male; Mice, Inbred C57BL; Osteoclasts; Osteogenesis; Osteolysis; Osteoprotegerin; RANK Ligand; RNA, Messenger; Skull; Staining and Labeling; Tartrate-Resistant Acid Phosphatase; Thiophenes; Titanium; Tumor Necrosis Factor-alpha; X-Ray Microtomography

Wear-particle-induced osteolysis leads to prosthesis loosening, which is one of the most common causes of joint-implant failure, a problem that must be fixed using revision surgery. Thus, a potential treatment for prosthetic loosening is focused on inhibiting osteoclastic bone resorption, which prevents wear-particle-induced osteolysis. In this study, we synthesized a compound named OA-14 (N-(3- (dodecylcarbamoyl)phenyl)-1H-indole-2-carboxamide) and examined how OA-14 affects titanium (Ti)-particle-induced osteolysis and osteoclastogenesis. We report that OA-14 treatment protected against Ti-particle-induced osteolysis in a mouse calvarial model. Interestingly, the number of tartrate-resistant acid phosphatase-positive osteoclasts decreased after treatment with OA-14 in vivo, which suggested that OA-14 inhibits osteoclast formation. To test this hypothesis, we conducted in vitro studies, and our results revealed that OA-14 markedly diminished osteoclast differentiation and osteoclast-specific gene expression in a dose- and time-dependent manner. Moreover, OA-14 suppressed osteoclastic bone resorption and F-actin ring formation. Furthermore, we determined that OA-14 inhibited osteoclastogenesis by specifically blocking the p38-Mitf-c-fos-NFATc1 signaling cascade induced by RANKL (ligand of receptor activator of nuclear factor κB). Collectively, our results suggest that the compound OA-14 can be safely used for treating particle-induced peri-implant osteolysis and other diseases caused by excessive osteoclast formation and function.

Topics: Acid Phosphatase; Actins; Animals; Benzamides; Bone Resorption; Cell Survival; Cells, Cultured; Indoles; Isoenzymes; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Osteoclasts; Osteolysis; RANK Ligand; Tartrate-Resistant Acid Phosphatase; Titanium

The effects of pyrroloquinoline quinine (PQQ) on RANKL-induced osteoclast differentiation and on wear particle-induced osteolysis were examined in this study. PQQ inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages (BMMs) in a dose-dependent manner without any evidence of cytotoxicity. The mRNA expression of c-Fos, NFATc1, and TRAP in RANKL-treated BMMs was inhibited by PQQ treatment. Moreover, RANKL-induced c-Fos and NFATc1 protein expression was suppressed by PQQ. PQQ additionally inhibited the bone resorptive activity of differentiated osteoclasts. Further a UHMWPE-induced murine calvaria erosion model study was performed to assess the effects of PQQ on wear particle-induced osteolysis in vivo. Mice treated with PQQ demonstrated marked attenuation of bone erosion based on Micro-CT and histologic analysis of calvaria. These results collectively suggested that PQQ demonstrated inhibitory effects on osteoclast differentiation in vitro and may suppress wear particle-induced osteolysis in vivo, indicating that PQQ may therefore serve as a useful drug in the prevention of bone loss.

Topics: Acid Phosphatase; Animals; Bone Marrow Cells; Cell Death; Cell Differentiation; Gene Expression Regulation; Isoenzymes; Mice; Mice, Inbred C57BL; NFATC Transcription Factors; Osteoclasts; Osteogenesis; Osteolysis; Polyethylenes; Proto-Oncogene Proteins c-fos; Pyrroles; Quinine; Quinolines; RANK Ligand; RNA, Messenger; Skull; Tartrate-Resistant Acid Phosphatase; X-Ray Microtomography

We administered recombinant interleukin (IL)-4 and recombinant IL-13 locally into the air pouch of mice to improve bone resorption induced by ultra-high-molecular-weight polyethylene (UHMWPE) particles.. Air pouches were established on the back of BALB/c mice, followed by the surgical introduction of a section of calvaria from a syngeneic mouse donor. We stimulated the bone-implanted pouches with the UHMWPE suspension. We divided UHMWPE-containing mice into four study groups to receive injections of phosphate-buffered saline (control), IL-4 alone, IL-13 alone, or IL-4 and IL-13 into the pouches. We harvested the tissues at 14 d after treatment for molecular and histological analyses.. The inhibitory effect of IL-4 was stronger than that of IL-13 toward osteoclast differentiation and osteoblast for the induction of osteoprotegerin production and down-regulation of receptor for activation of nuclear factor-κB ligand production. Furthermore, the combined treatment with both IL-4 and 1L-13 had a more important role in inhibiting bone resorption in these pouches with UHMWPE stimulation, compared with IL-4 or IL-13 treatment alone.. Local administration of recombinant IL-4 and IL-13 may be a feasible and effective therapeutic candidate to treat or prevent wear debris-associated osteolysis.

Topics: Acid Phosphatase; Animals; Cell Differentiation; Disease Models, Animal; Female; Interleukin-13; Interleukin-4; Isoenzymes; Mice; Mice, Inbred BALB C; Osteoclasts; Osteolysis; Polyethylenes; Receptor Activator of Nuclear Factor-kappa B; Recombinant Proteins; Tartrate-Resistant Acid Phosphatase

Wear particles generated with use of total joint replacements incite a chronic macrophage-mediated inflammatory reaction, which leads to implant failure. Macrophage activation may be polarized into two states, with an M1 proinflammatory state dominating an alternatively activated M2 anti-inflammatory state. We hypothesized that IL-4, an activator of M2 macrophages, could modulate polyethylene (PE) particle-induced osteolysis in an experimental murine model. Four animal groups included (a) calvarial saline injection with harvest at 14 days (b) single calvarial injection of PE particles subcutaneously (SC) without IL-4 (c) PE particles placed as in (b), then IL-4 given SC for 14 consecutive days and (d) PE particles as in (b) then IL-4 beginning 7 days after particle injection for 7 days. The calvarial bone volume to total tissue volume was measured using microCT and histomorphometry. Calvaria were cultured for 24 h to assess release of RANKL, OPG, TNF-α, and IL-1ra and isolation and identification of M1 and M2 specific proteins. MicroCT and histomorphometric analysis showed that bone loss was significantly decreased following IL-4 administration to PE treated calvaria for both 7 and 14 days. Western blot analysis showed an increased M1/M2 ratio in the PE treated calvaria, which decreased with addition of IL-4. Cytokine analysis showed that the RANKL/OPG ratio and TNF-α/IL-1ra ratio decreased in PE-treated calvaria following IL-4 addition for 14 days. IL-4 delivery mitigated PE particle-induced osteolysis through macrophage polarization. Modulation of macrophage polarization is a potential treatment strategy for wear particle induced periprosthetic osteolysis.

Topics: Acid Phosphatase; Animals; beta-N-Acetylhexosaminidases; Biomarkers; Blotting, Western; Bone Remodeling; Cytokines; Drug Delivery Systems; Enzyme-Linked Immunosorbent Assay; Interleukin-4; Isoenzymes; Lectins; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase Type II; Osteoclasts; Osteolysis; Polyethylenes; RANK Ligand; Skull; Tartrate-Resistant Acid Phosphatase; Tomography, X-Ray Computed; Tumor Necrosis Factor-alpha

Skeletal homeostasis relies upon a fine tuning of osteoclast (OCL)-mediated bone resorption and osteoblast (OBL)-dependent bone formation. This balance is unsettled by multiple myeloma (MM) cells, which impair OBL function and stimulate OCLs to generate lytic lesions. Emerging experimental evidence is disclosing a key regulatory role of microRNAs (miRNAs) in the regulation of bone homeostasis suggesting the miRNA network as potential novel target for the treatment of MM-related bone disease (BD). Here, we report that miR-29b expression decreases progressively during human OCL differentiation in vitro. We found that lentiviral transduction of miR-29b into OCLs, even in the presence of MM cells, significantly impairs tartrate acid phosphatase (TRAcP) expression, lacunae generation, and collagen degradation, which are relevant hallmarks of OCL activity. Accordingly, expression of cathepsin K and metalloproteinase 9 (MMP9) as well as actin ring rearrangement were impaired in the presence of miR-29b. Moreover, we found that canonical targets C-FOS and metalloproteinase 2 are suppressed by constitutive miR-29b expression which also downregulated the master OCL transcription factor, NAFTc-1. Overall, these data indicate that enforced expression of miR-29b impairs OCL differentiation and overcomes OCL activation triggered by MM cells, providing a rationale for miR-29b-based treatment of MM-related BD.

Topics: Acid Phosphatase; Actins; Bone Resorption; Cathepsin K; Cell Differentiation; Cell Line, Tumor; Cells, Cultured; Collagen Type I; Gene Expression; Genes, fos; Humans; Isoenzymes; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; MicroRNAs; Multiple Myeloma; NFATC Transcription Factors; Osteoblasts; Osteoclasts; Osteolysis; Receptor Activator of Nuclear Factor-kappa B; Tartrate-Resistant Acid Phosphatase

To explore the potential roles of serum Dickkopf-1 (DKK-1) and tartrate-resistant acid phosphatase 5b (TRAP5b) in gouty arthritis.. Blood serum DKK-1 and TRAP5b were assessed by ELISA method. The serum samples were collected from 150 patients with gouty arthritis, 100 with hyperuricemia and 100 healthy controls. Of the 150 gouty arthritis patients, 40 were diagnosed as acute gout (joint rash and pain), and the other 110 as chronic gout. At the time of serum sampling, various clinical and laboratory parameters were assessed. The correlations of DKK-1 or TRAP5b and clinical/laboratory parameters were analyzed.. (1) The serum levels of DKK-1 were elevated in the patients with gouty arthritis [(2 574.8± 997.9) ng/L] and with hyperuricemia [(2 009.4±756.9) ng/L] compared with the healthy controls [(981.8±770.7) ng/L],F=49.59, P<0.001. (2) The serum levels of TRAP5b in the three groups were (3.2±1.4)U/L, (2.5±1.4)U/L and (0.2±0.2)U/L, respectively. F=103.039, P<0.001. (3) A significant difference of DKK-1 was observed between the patients with gouty arthritis and with hyperuricemia (t=3.998, P<0.001). Similarly, a significant difference of TRAP5b was observed between the patients with gouty arthritis and with hyperuricemia(t=3.391, P=0.004). (4) In the patients with gouty arthritis, there was a positive correlation between DKK-1 and TRAP5b(r=0.47, P<0.001), while the levels of DKK-1 were not related with age, disease duration, body mass index or serum uric acid(r=-0.153, -0.123, 0.158, 0.00,P=0.126, 0.509, 0.381, 0.926). (5) In the patients with gouty arthritis, the serum levels of TRAP5b were elevated in the patients with tophi compared with the patients without tophi[(8.4±6.4)U/L vs. (4.0±1.6)U/L,t=-2.938,P=0.007]. The level of TRAP5b was associated with the disease duration(r=0.455,P=0.01), while there was no correlation between TRAP5b and age, body mass index or serum uric acid (r=0.135, 0.278, 0.144,P=0.595, 0.117,0.132).. In the patients with gouty arthritis, the levels of DKK-1 were remarkably elevated, and there was a positive correlation between DKK-1 and TRAP5b. Our results demonstrate that DKK-1 is involved in bone destruction in gouty arthritis.

Topics: Acid Phosphatase; Adolescent; Adult; Aged; Aged, 80 and over; Arthritis, Gouty; Female; Humans; Hyperuricemia; Intercellular Signaling Peptides and Proteins; Isoenzymes; Male; Middle Aged; Osteolysis; Tartrate-Resistant Acid Phosphatase; Young Adult

The p38 mitogen-activated protein kinase (p38 MAPK) pathway is involved in the osteoclast differentiation. The aim of the study was to investigate whether SB203580, a p38 MAPK inhibitor, inhibits wear-debris-induced inflammatory osteolysis in mice. Forty-five mice were implanted with calvaria bone from syngeneic littermates; then, titanium (Ti) particles were injected into established air pouches to provoke inflammatory osteolysis. At 14 days after bone/Ti implantation, pouch membranes with intact bone implants underwent histological and molecular analysis. SB203580 had less effect on MMP-9 and TNF-α expression under wear-debris-induced conditions. SB203580, by inhibiting the expression of p38 MAPK and phospho-p38 MAPK, inhibited Ti particle wear-debris-induced inflammatory osteolysis. It also remarkably decreased the number of tartrate-resistant acid phosphatase positive cells in Ti-particle-induced pouch tissues. Results suggest that p38 MAPK may be critical in a murine osteolysis model. SB203580 may notably inhibit wear-debris-induced inflammatory osteolysis by down-regulating expression of MMP-9 and TNF-α via the p38 MAPK pathway.

Topics: Acid Phosphatase; Animals; Bone and Bones; Down-Regulation; Enzyme Inhibitors; Female; Imidazoles; Inflammation; Isoenzymes; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Osteoclasts; Osteolysis; p38 Mitogen-Activated Protein Kinases; Pyridines; RNA, Messenger; Tartrate-Resistant Acid Phosphatase; Titanium; Tumor Necrosis Factor-alpha

Osteoclasts and osteoblasts regulate bone resorption and formation to allow bone remodeling and homeostasis. The balance between bone resorption and formation is disturbed by abnormal recruitment of osteoclasts. Osteoclast differentiation is dependent on the receptor activator of nuclear factor NF-kappa B (RANK) ligand (RANKL) as well as the macrophage colony-stimulating factor (M-CSF). The RANKL/RANK system and RANK signaling induce osteoclast formation mediated by various cytokines. The RANK/RANKL pathway has been primarily implicated in metabolic, degenerative and neoplastic bone disorders or osteolysis. The central role of RANK/RANKL interaction in osteoclastogenesis makes RANK an attractive target for potential therapies in treatment of osteolysis. The purpose of this study was to assess the effect of inhibition of RANK expression in mouse bone marrow macrophages on osteoclast differentiation and bone resorption.. Three pairs of short hairpin RNAs (shRNA) targeting RANK were designed and synthesized. The optimal shRNA was selected among three pairs of shRNAs by RANK expression analyzed by Western blot and Real-time PCR. We investigated suppression of osteoclastogenesis of mouse bone marrow macrophages (BMMs) using the optimal shRNA by targeting RANK.. Among the three shRANKs examined, shRANK-3 significantly suppressed [88.3%] the RANK expression (p < 0.01). shRANK-3 also brought about a marked inhibition of osteoclast formation and bone resorption as demonstrated by tartrate-resistant acid phosphatase (TRAP) staining and osteoclast resorption assay. The results of our study show that retrovirus-mediated shRANK-3 suppresses osteoclast differentiation and osteolysis of BMMs.. These findings suggest that retrovirus-mediated shRNA targeting RANK inhibits osteoclast differentiation and osteolysis. It may appear an attractive target for preventing osteolysis in humans with a potential clinical application.

Topics: Acid Phosphatase; Animals; Biomarkers; Blotting, Western; Cell Differentiation; Cells, Cultured; Genetic Vectors; Isoenzymes; Macrophages; Mice; Mice, Inbred BALB C; Osteoclasts; Osteolysis; Real-Time Polymerase Chain Reaction; Receptor Activator of Nuclear Factor-kappa B; Retroviridae; RNA Interference; RNA, Small Interfering; Tartrate-Resistant Acid Phosphatase; Transfection

Particle-wear-induced inflammatory osteolysis remains a major problem for the long-term success of total joint arthroplasty. Previous studies have demonstrated that cyclooxygenase-2 (COX-2) is expressed abundantly in the tissue around a failed implant. However, the role of COX-2 in the development of particle-wear-induced osteoclastogenesis remains unclear. The aim of the study was to test the hypothesis that Dynastat, a COX-2 inhibitor, ameliorates particle-wear-induced inflammatory osteoclastogenesis through the down-regulation of the receptor activators of nuclear factor-κB (RANK) and nuclear factor-κB ligand (RANKL) expression in a murine osteolysis model. Titanium (Ti) particles were introduced into established air pouches in BALB/c mice, followed by the implantation of calvaria bone from syngeneic littermates. Dynastat was given to mice intraperitoneally 2 days before the introduction of Ti particles and maintained until the mice were sacrificed. Pouch tissues were collected 14 days after Ti inoculation for molecular and histological analysis. The results showed that Dynastat has more impact on Ti-particle-induced prostaglandin E(2) expression and less on the expression of interleukin-1β and tumor necrosis factor-α. Dynastat inhibited Ti-particle-induced osteoclastogenesis by reducing the gene activation of RANK and RANKL, and diminishing the RANKL expression in Ti-particle-charged pouches. Dynastat markedly reduced the number of tartrate-resistant acid-phosphatase-positive cells in pouch tissues stimulated by Ti particles. In conclusion, this study provides evidence that Dynastat can markedly inhibit Ti-particle-induced osteoclastogenesis by the down-regulation of RANK/RANKL in a murine air pouch model, and is a promising therapeutic candidate for the treatment of inflammatory osteolysis induced by wear particles.

Topics: Acid Phosphatase; Animals; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dinoprostone; Down-Regulation; Immunohistochemistry; Interleukin-1beta; Isoenzymes; Isoxazoles; Membranes; Mice; Mice, Inbred BALB C; Osteoclasts; Osteogenesis; Osteolysis; Particulate Matter; Protective Agents; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Tartrate-Resistant Acid Phosphatase; Titanium; Tumor Necrosis Factor-alpha

In a cohort study, the role of the active tartrate-resistant acid phosphatase (TRACP 5b), a marker of bone-resorbing osteoclasts, for the assessment of loosening after total hip arthroplasty (THA), was analyzed, as well as its correlation with osteolysis and multinucleated cell appearance in the retrievals. Eighty THA patients, who went consecutively to the orthopedic department, were asked to participate, and 54 accepted and were enrolled in the study. Finally, 46 subjects were analyzed, clinical-radiographic evaluation was considered the gold standard, serum TRACP 5b was blindly measured, and a cut-off was obtained, by performing a ROC Curve. Based on the gold standard, patients were split by 19 stable and 27 loosened subjects, and results were matched. TRACP 5b was significantly higher in loosened patients than in stable ones (p < 0.001). A good specificity (89.5%), positive predictive value (90.0%), and likelihood ratio (6.33) were calculated, that provided strong evidence of loosening with TRACP 5b levels higher than the cut-off. Moreover, TRACP 5b and osteolysis (Fisher's exact test, p = 0.03) were found significantly correlated. TRACP 5b has been proven a reliable marker, specifically related to resorbing-multinucleated cells, to ascertain late loosening in THA, and could support standard procedures, if confirmed by performing prospective studies.

Topics: Acid Phosphatase; Adult; Aged; Aged, 80 and over; Arthroplasty, Replacement, Hip; Biomarkers; Cohort Studies; Female; Follow-Up Studies; Giant Cells; Humans; Isoenzymes; Macrophages; Male; Middle Aged; Osteoclasts; Osteolysis; Predictive Value of Tests; Prosthesis Failure; Radiography; Sensitivity and Specificity; Tartrate-Resistant Acid Phosphatase

Exogenous osteoprotegerin (OPG) gene modification appears a therapeutic strategy for osteolytic aseptic loosening. The feasibility and efficacy of a cell-based OPG gene delivery approach were investigated using a murine model of knee prosthesis failure. A titanium pin was implanted into mouse proximal tibia to mimic a weight-bearing knee arthroplasty, followed by titanium particles challenge to induce periprosthetic osteolysis. Mouse fibroblast-like synoviocytes were transduced in vitro with either AAV-OPG or AAV-LacZ before transfused into the osteolytic prosthetic joint 3 weeks post surgery. Successful transgene expression at the local site was confirmed 4 weeks later after killing. Biomechanical pullout test indicated a significant restoration of implant stability after the cell-based OPG gene therapy. Histology revealed that inflammatory pseudo-membranes existed ubiquitously at bone-implant interface in control groups, whereas only observed sporadically in OPG gene-modified groups. Tartrate-resistant acid phosphatase+osteoclasts and tumor necrosis factor α, interleukin-1β, CD68+ expressing cells were significantly reduced in periprosthetic tissues of OPG gene-modified mice. No transgene dissemination or tumorigenesis was detected in remote organs and tissues. Data suggest that cell-based ex vivo OPG gene therapy was comparable in efficacy with in vivo local gene transfer technique to deliver functional therapeutic OPG activities, effectively halted the debris-induced osteolysis and regained the implant stability in this model.

Topics: Acid Phosphatase; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Genetic Therapy; Genetic Vectors; Isoenzymes; Knee Prosthesis; Mice; Mice, Inbred BALB C; Osteolysis; Osteoprotegerin; Prosthesis Failure; Tartrate-Resistant Acid Phosphatase; Weight-Bearing

Bone metastases contribute to morbidity in patients with common cancers, and conventional therapy provides only palliation and can induce systemic side effects. The development of nanostructured delivery systems that combine carriers with bone-targeting molecules can potentially overcome the drawbacks presented by conventional approaches. We have recently developed biodegradable, biocompatible nanoparticles (NP) made of a conjugate between poly (D,L-lactide-co-glycolic) acid and alendronate, suitable for systemic administration, and directly targeting the site of tumor-induced osteolysis. Here, we loaded NP with doxorubicin (DXR), and analyzed the in vitro and in vivo activity of the drug encapsulated in the carrier system. After confirming the intracellular uptake of DXR-loaded NP, we evaluated the anti-tumor effects in a panel of human cell lines, representative for primary or metastatic bone tumors, and in an orthotopic mouse model of breast cancer bone metastases. In vitro, both free DXR and DXR-loaded NP, (58-580 ng/mL) determined a significant dose-dependent growth inhibition of all cell lines. Similarly, both DXR-loaded NP and free DXR reduced the incidence of metastases in mice. Unloaded NP were ineffective, although both DXR-loaded and unloaded NP significantly reduced the osteoclast number at the tumor site (P = 0.014, P = 0.040, respectively), possibly as a consequence of alendronate activity. In summary, NP may act effectively as a delivery system of anticancer drugs to the bone, and deserve further evaluation for the treatment of bone tumors.

Topics: Acid Phosphatase; Alendronate; Animals; Antineoplastic Agents; Biological Transport; Bone Density Conservation Agents; Bone Neoplasms; Carcinoma; Cell Count; Cell Line, Tumor; Cell Proliferation; Doxorubicin; Female; Humans; Isoenzymes; Mice; Mice, Nude; Nanocapsules; Osteoclasts; Osteolysis; Radiography; Tartrate-Resistant Acid Phosphatase; Xenograft Model Antitumor Assays

Breast cancer metastases to bone are common in advanced stage disease. We have recently demonstrated that vitamin D deficiency enhances breast cancer growth in an osteolytic mouse model of breast cancer metastasis. In this study, we examined the effects of vitamin D deficiency on tumor growth in an osteosclerotic model of intra-skeletal breast cancer in mice.. The effects of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] on proliferation and apoptosis of MCF-7 breast cancer cells, and changes in the expression of genes within the vitamin D metabolic pathway (VDR, 1α- and 24-hydroxylase) were examined in vitro. MCF-7 breast cancer cells were injected intra-tibially into vitamin D deficient and vitamin D sufficient mice co-treated with and without osteoprotegerin (OPG). The development of tumor-related lesions was monitored via serial X-ray analysis. Tumor burden and indices of proliferation and apoptosis were determined by histology along with markers of bone turnover and serum intact PTH levels.. In vitro, MCF-7 cells expressed critical genes for vitamin D signalling and metabolism. Treatment with 1,25(OH)(2)D(3) inhibited cell growth and proliferation, and increased apoptosis. In vivo, osteosclerotic lesions developed faster and were larger at endpoint in the tibiae of vitamin D deficient mice compared to vitamin D sufficient mice (1.49±0.08 mm(2) versus 1.68±0.15 mm(2), P<0.05). Tumor area was increased by 55.8% in vitamin D deficient mice (0.81±0.13 mm(2) versus 0.52±0.11 mm(2) in vitamin D sufficient mice). OPG treatment inhibited bone turnover and caused an increase in PTH levels, while tumor burden was reduced by 90.4% in vitamin D sufficient mice and by 92.6% in vitamin D deficient mice. Tumor mitotic activity was increased in the tibiae of vitamin D deficient mice and apoptosis was decreased, consistent with faster growth.. Vitamin D deficiency enhances both the growth of tumors and the tumor-induced osteosclerotic changes in the tibiae of mice following intratibial implantation of MCF-7 cells. Enhancement of tumor growth appears dependent on increased bone resorption rather than increased bone formation induced by these tumors.

Topics: Acid Phosphatase; Adipose Tissue; Animals; Apoptosis; Bone and Bones; Bone Neoplasms; Bone Remodeling; Breast Neoplasms; Calcitriol; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Isoenzymes; Mice; Osteolysis; Osteosclerosis; Radiography; Tartrate-Resistant Acid Phosphatase; Tumor Burden; Vitamin D Deficiency; Xenograft Model Antitumor Assays

Squamous cell carcinoma (SCC) is the most common form of oral cancer. Destruction and invasion of mandibular and maxillary bone frequently occurs and contributes to morbidity and mortality. We hypothesized that the bisphosphonate drug zoledronic acid (ZOL) would inhibit tumor-induced osteolysis and reduce tumor growth and invasion in a murine xenograft model of bone-invasive oral SCC (OSCC) derived from an osteolytic feline OSCC. Luciferase-expressing OSCC cells (SCCF2Luc) were injected into the perimaxillary subgingiva of nude mice, which were then treated with 100 μg/kg ZOL or vehicle. ZOL treatment reduced tumor growth and prevented loss of bone volume and surface area but had no effect on tumor invasion. Effects on bone were associated with reduced osteolysis and increased periosteal new bone formation. ZOL-mediated inhibition of tumor-induced osteolysis was characterized by reduced numbers of tartrate-resistant acid phosphatase-positive osteoclasts at the tumor-bone interface, where it was associated with osteoclast vacuolar degeneration. The ratio of eroded to total bone surface was not affected by treatment, arguing that ZOL-mediated inhibition of osteolysis was independent of effects on osteoclast activation or initiation of bone resorption. In summary, our results establish that ZOL can reduce OSCC-induced osteolysis and may be valuable as an adjuvant therapy in OSCC to preserve mandibular and maxillary bone volume and function.

Topics: Acid Phosphatase; Animals; Bone Density Conservation Agents; Bone Resorption; Calcium; Carcinoma, Squamous Cell; Cats; Diphosphonates; Disease Models, Animal; Imidazoles; Isoenzymes; Luciferases; Male; Mice; Mice, Nude; Mouth Neoplasms; Osteoclasts; Osteolysis; Tartrate-Resistant Acid Phosphatase; Transplantation, Heterologous; X-Ray Microtomography; Zoledronic Acid

Metastasis to bone is the leading cause of morbidity and mortality in advanced prostate cancer patients. Considering the complex reciprocal interactions between the tumor cells and the bone microenvironment, there is increasing interest in developing combination therapies targeting both the tumor growth and the bone microenvironment. In this study, we investigated the effect of simultaneous blockade of BMP pathway and RANK/RANKL axis in an osteolytic prostate cancer lesion in bone. We used a retroviral vector encoding noggin (RetroNoggin) to antagonize the effect of BMPs and RANK:Fc, which is a recombinant RANKL antagonist was used to inhibit RANK/RANKL axis. The tumor growth and bone loss were evaluated using plain radiographs, hind limb tumor measurements, micro PET/CT ((18)FDG and (18)F-fluoride tracer), and histology. Tibias implanted with PC-3 cells developed pure osteolytic lesions at 2-weeks with progressive increase in cortical bone destruction at successive time points. Tibias implanted with PC-3 cells over expressing noggin (RetroNoggin) resulted in reduced tumor size and decreased bone loss compared to the implanted tibias in untreated control animals. RANK:Fc administration inhibited the formation of osteoclasts, delayed the development of osteolytic lesions, decreased bone loss and reduced tumor size in tibias implanted with PC-3 cells. The combination therapy with RANK:Fc and noggin over expression effectively delayed the radiographic development of osteolytic lesions, and decreased the bone loss and tumor burden compared to implanted tibias treated with noggin over expression alone. Furthermore, the animals treated with the combination strategy exhibited decreased bone loss (micro CT) and lower tumor burden (FDG micro PET) compared to animals treated with RANK:Fc alone. Combined blockade of RANK/RANKL axis and BMP pathway resulted in reduced tumor burden and decreased bone loss compared to inhibition of either individual pathway alone in osteolytic prostate cancer lesion in bone. These results suggest that simultaneous targeting of tumor cells and osteoclasts may be the most effective method of inhibiting the progression of established osteolytic metastatic lesions in vivo.

Topics: Acid Phosphatase; Animals; Bone Morphogenetic Proteins; Cell Line, Tumor; Fluorine Radioisotopes; Fluorodeoxyglucose F18; Hindlimb; Humans; Isoenzymes; Male; Mice; Mice, SCID; Neoplasm Transplantation; Osteolysis; Positron-Emission Tomography; Prostatic Neoplasms; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Tartrate-Resistant Acid Phosphatase; Tibia; Tomography, X-Ray Computed

Breast cancer frequently metastasizes to bone, where tumor cells induce osteoclasts to locally destroy bone. During bone resorption, growth factors are locally released that may support bone metastatic growth. Differently from most other tissues, drugs that can limit local turnover, such as bisphosphonates and osteoprotegerin (OPG), are available for bone. We examined the hypothesis that inhibition of bone resorption by two different mechanisms may also affect the growth of cancer cells in bone. For this, we tested the effects of high doses of OPG and zoledronic acid (ZOL) on progression of MDA-231-B/Luc+ breast cancer cells in the bone microenvironment using whole body bioluminescent reporter imaging (BLI). Both treatments significantly inhibited the development of radiographically detectable osteolytic lesions. Histologic examination corroborated the radiographic findings, showing that both treatments preserved the integrity of bone trabeculae and prevented bone destruction (significantly higher trabecular bone volumes vs. vehicle). However, whereas practically no TRAcP-positive osteoclasts were observed in tibiae preparations of animals treated with Fc-OPG, TRAcP-positive osteoclasts were still present in the animals treated with ZOL. Intra-bone tumor burden was reduced with ZOL and Fc-OPG treatment. Although there appeared to be a trend for less overall total tumor burden upon treatment with both compounds, this was not significant as assessed by BLI and histomorphometric analysis due to the extramedullary growth of cancer cells which was not affected by these treatments. Collectively, anti-resorptive agents with different mechanisms of action - ZOL and OPG - significantly reduced cancer-induced osteolysis and intra-osseous tumor burden, but failed to restrain local tumor growth. However, interference with the bone micro-environmental growth support could still be of therapeutic relevance when given to patients early in the course of bone metastatic disease.

Topics: Acid Phosphatase; Animals; Bone and Bones; Bone Resorption; Breast Neoplasms; Cell Count; Cell Line, Tumor; Cell Proliferation; Diphosphonates; Female; Humans; Imidazoles; Isoenzymes; Luminescent Measurements; Mice; Mice, Inbred BALB C; Mice, Nude; Osteoclasts; Osteolysis; Osteoprotegerin; Radiography; Tartrate-Resistant Acid Phosphatase; Whole Body Imaging; Zoledronic Acid

Particle-induced osteolysis is a major cause of aseptic loosening after total joint replacement. Earlier studies demonstrated apoptotic macrophages, giant cells, fibroblasts and T-lymphocytes in capsules and interface membranes of patients with aseptic hip implant loosening. The aim of the current study was to determine in a murine calvarial model of wear particle-induced osteolysis whether inhibition of apoptosis using the pan-caspase inhibitor BOC-D-FMK reduces aseptic loosening. Healthy 12-week-old male C57BL/6J mice were treated with UHMWPE particles and received a daily peritoneal injection of BOK-D-FMK, respectively only buffer at a dose of 3 mg/kg of body weight for 12 days until sacrifice. Bone resorption was measured by histomorphometry, micro CT (computed tomography) and TRAP-5b serum analysis. Apoptosis was measured using caspase-3 cleaved staining. The results demonstrated that UHMWPE particles induced stronger apoptotic reactions in macrophages and osteoblasts and increased bone resorption in non-specifically treated mice, whereas peritoneal application of BOC-D-FMK significantly counteracted these adverse particle-related effects. We think that in particle-induced osteolysis apoptosis is pathologically increased, and that failure to reduce the quantity of apoptotic bodies leads to an up-regulation of proinflammatory cytokines, which may be responsible for the induction of osteolysis. We showed for the first time in vivo that a reduction in apoptosis leads to a significant reduction in particle-induced osteolysis. Clinically, the apoptotic cascade could become an interesting novel therapeutic target to modulate particle-induced osteolysis.

Topics: Acid Phosphatase; Animals; Benzyl Compounds; Caspase 3; Caspase Inhibitors; Hydrocarbons, Fluorinated; Immunohistochemistry; Isoenzymes; Male; Mice; Mice, Inbred C57BL; Organ Size; Osteoclasts; Osteolysis; Polyethylenes; Prosthesis Implantation; Skull; Tartrate-Resistant Acid Phosphatase; X-Ray Microtomography

The skeleton is the most common site of breast cancer metastasis, which can occur in up to 85% of patients during their lifetime. The morbidity associated with bone metastases in patients with breast cancer includes pathological fractures, bone pain, hypercalcaemia, and spinal cord compression. When breast cancer metastasizes to bone, the balance of bone resorption (mediated by osteoclasts) and bone formation (mediated by osteoblasts) favors bone resorption, which leads to net bone destruction (i.e., osteolysis). Anti-resorptive agents such as bisphosphonates are commonly used to treat bone resorption in osteoporosis or osteolytic cancer patients. However, bisphosphonates by themselves are unable to rebuild lost bone tissue, and can cause severe side effects. In this study, we developed a bovine bone explant culture system and have observed that murine osteoblasts can modulate the activity of osteotropic human breast cancer cells on this substrate. Using markers of bone metabolism, we observe diminished bone turnover in organ culture following the addition of exogenous osteoblasts. The data presented in this study supports further investigation into the use of cytotherapies to limit breast cancer mediated osteolysis.

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Bone Neoplasms; Breast Neoplasms; Cattle; Cell Line, Tumor; Coculture Techniques; Humans; Mice; Organ Culture Techniques; Osteoblasts; Osteolysis

Bone metastases have a considerable impact on quality of life in patients with breast and other cancers. Tumors produce osteoclast-activating factors, whereas bone resorption promotes the growth of tumor cells, thus leading to a "vicious cycle" of bone metastasis. Sagopilone, a novel, fully synthetic epothilone, inhibits the growth of breast cancer cells in vitro and in vivo, and here we report its activity in the MDA-MB-231(SA) breast cancer bone metastasis mouse model.. The potency of sagopilone was determined in treatment models simulating the adjuvant (preventive) and metastatic (therapeutic) settings in the clinic.. We showed that sagopilone inhibited tumor burden and bone destruction, in addition to reducing tumor-induced cachexia and paraplegia. The reduction in osteolytic lesions, tumor growth in bone, and weight loss was statistically significant in the preventive model compared with the vehicle group. In the therapeutic model, sagopilone treatment significantly lowered the number of activated osteoclasts and significantly reduced the osteolytic lesion area, bone volume loss, and bone resorption compared with vehicle treatment while simultaneously inhibiting tumor burden. An in vitro assay confirmed that sagopilone inhibited osteoclast activation without cytotoxic effects, whereas paclitaxel resulted in lower inhibition and high levels of cytotoxicity.. Sagopilone seems to inhibit the vicious cycle at both the tumor growth and bone resorption stages, suggesting the possibility for substantial benefit in the treatment of patients with breast cancer at risk from bone metastases or with bone lesions already present. Phase II clinical trials with sagopilone in patients with breast cancer are ongoing.

Topics: Acid Phosphatase; Animals; Antineoplastic Agents, Phytogenic; Benzothiazoles; Bone and Bones; Bone Neoplasms; Bone Resorption; Cachexia; Cell Line, Tumor; Dose-Response Relationship, Drug; Epothilones; Female; Humans; Isoenzymes; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Osteoclasts; Osteolysis; Paclitaxel; Tartrate-Resistant Acid Phosphatase; Tumor Burden; Xenograft Model Antitumor Assays

Nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) plays an important role in host defense, as well as in inflammation-induced tissue lesions. Here we evaluated the role of NO in bone loss in bacterial infection-induced apical periodontitis by using iNOS-deficient mice (iNOS(-/-)). The iNOS(-/-) mice developed greater inflammatory cell recruitment and osteolytic lesions than WT mice. Moreover, tartrate-resistant acid-phosphatase-positive (TRAP(+)) osteoclasts were significantly more numerous in iNOS(-/-) mice. Furthermore, the increased bone resorption in iNOS(-/-) mice also correlated with the increased expression of receptor activator NF-kappaB (RANK), stromal-cell-derived factor-1 alpha (SDF-1 alpha/CXCL12), and reduced expression of osteoprotegerin (OPG). These results show that NO deficiency was associated with an imbalance of bone-resorption-modulating factors, leading to severe infection-stimulated bone loss.

Topics: Acid Phosphatase; Actinomycosis; Alveolar Bone Loss; Animals; Bacterial Infections; Bacteroidaceae Infections; Biomarkers; Cell Count; Cell Movement; Chemokine CXCL12; Dental Pulp Exposure; Isoenzymes; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Nitric Oxide; Nitric Oxide Synthase Type II; Osteoclasts; Osteolysis; Osteoprotegerin; Periapical Periodontitis; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Tartrate-Resistant Acid Phosphatase

Total hip replacement can be complicated by periprosthetic osteolysis. Monocytes/macrophages play a major role in the formation of the foreign body granulomas induced by wear debris. We hypothesized that periprosthetic monocytes/macrophages do not only accelerate inflammatory and osteoclast-mediated osteolytic processes, but also resorb periprosthetic bone directly by themselves. This study was designed to evaluate the osteolytic potential in vitro of monocytes/macrophages derived from bone marrow.. Monocytes/macrophages were produced by filtration of rat bone marrow cells, followed by culture in the presence of macrophage-colony stimulating factor (M-CSF). Monocyte/macrophage properties were ascertained using immunocytochemistry and phagocytic activity. Osteolytic cytokines and extracellular matrix degrading proteinases were quantified at the mRNA level.. Adherent cell fraction was immunoreactive for the monocyte/macrophage specific marker CD68 and active in the phagocytosis of carbon particles up to 72 h. They also showed immunoreactivity to cathepsin K, IL-1beta, IL-6, and M-CSF, but mostly did not react to TRAP. mRNA levels of osteolytic cytokines and extracellular matrix degrading proteinases were enhanced, but that of RANKL were not. Monocytes/macrophages resorbed dentine discs and carbonated calcium phosphate was very actively resorbed after stimulation with titanium particles.. Harvested bone marrow cells expressed monocyte/macrophage phenotype, but not osteoclastic markers. The capacity of these cathepsin-K-positive phagocytic cells to resorb dentine discs and carbonated calcium phosphate in vitro suggests a direct role of monocytes/macrophages in bone resorption and periprosthetic osteolysis. The finding supports our hypothesis and previous histomorphometric observations on the presence of such osteolytic macrophages in vivo around loosening prosthesis.

Topics: Acid Phosphatase; Animals; Bone Marrow Cells; Bone Resorption; Cell Adhesion; Cells, Cultured; Dentin; Femur; Immunohistochemistry; Isoenzymes; Macrophages; Monocytes; Osteolysis; Phagocytosis; Physical Stimulation; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Surface Properties; Tartrate-Resistant Acid Phosphatase

The purpose of this study was to determine whether macrophage depletion using clodronate liposomes diminishes wear-debris-induced inflammatory osteolysis in a murine osteolysis model. Ultra high molecular weight polyethylene (UHMWPE) particles were introduced into established air pouches on BALB/c mice, followed by implantation of calvaria bone from syngeneic littermates. Macrophages were depleted by the intraperitoneal injection of clodronate liposome (2 mg) 2 days before bone implantation and re-injection every 3 days (1 mg) until the sacrifice of the mice. Mice without clodronate liposome therapy or treated with empty liposome as well as mice injected with saline alone were included in this study as controls. Pouch tissues were collected 14 days after bone implantation for molecular and histology analysis. Our findings indicated that (1) macrophage depletion in clodronate-liposome-treated mice was achieved, as illustrated by F4/80 immunostaining in both pouch and spleen tissues; (2) clodronate-liposome treatment significantly reduced UHMWPE-induced tissue inflammation, with diminished pouch membrane thickness, reduced inflammatory cellular infiltration, and lowered interleukin 1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) expression; (3) clodronate-liposome treatment markedly reduced the number of TRAP(+) cells in pouch tissues and protected against bone collagen depletion. In conclusion, this study demonstrates that macrophage depletion using clodronate-liposome reduces UHMWPE particle-induced inflammatory osteolysis. This observation supports the hypothesis that macrophages contribute to the severity of UHMWPE particles induced inflammatory osteolysis, and suggest that macrophage depletion represents a viable therapeutic approach to the prevention and treatment of patients with aseptic loosening.

Topics: Acid Phosphatase; Animals; Bone and Bones; Bone Resorption; Clodronic Acid; Collagen; Female; Implants, Experimental; Inflammation; Interleukin-1beta; Isoenzymes; Liposomes; Macrophages; Membranes; Mice; Mice, Inbred BALB C; Models, Animal; Molecular Weight; Osteoclasts; Osteolysis; Polyethylene; Protective Agents; Spleen; Tartrate-Resistant Acid Phosphatase; Tumor Necrosis Factor-alpha

Orthodontic force induces osteoclastogenesis in vivo. It has recently been reported that administration of an antibody against the macrophage-colony-stimulating factor (M-CSF) receptor c-Fms blocks osteoclastogenesis and bone erosion induced by tumor necrosis factor-alpha (TNF-alpha) administration. This study aimed to examine the effect of an anti-c-Fms antibody on mechanical loading-induced osteoclastogenesis and osteolysis in an orthodontic tooth movement model in mice. Using TNF receptor 1- and 2-deficient mice, we showed that orthodontic tooth movement was mediated by TNF-alpha. We injected anti-c-Fms antibody daily into a local site, for 12 days, during mechanical loading. The anti-c-Fms antibody significantly inhibited orthodontic tooth movement, markedly reduced the number of osteoclasts in vivo, and inhibited TNF-alpha-induced osteoclastogenesis in vitro. These findings suggest that M-CSF plays an important role in mechanical loading-induced osteoclastogenesis and bone resorption during orthodontic tooth movement mediated by TNF-alpha.

Topics: Acid Phosphatase; Animals; Antibodies, Monoclonal; Biomarkers; Bone Resorption; Cell Differentiation; Immunoglobulin G; Isoenzymes; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Models, Animal; Osteoclasts; Osteolysis; Receptor, Macrophage Colony-Stimulating Factor; Receptors, Tumor Necrosis Factor, Type I; Receptors, Tumor Necrosis Factor, Type II; Stress, Mechanical; Tartrate-Resistant Acid Phosphatase; Tooth Movement Techniques; Tumor Necrosis Factor-alpha

To study the effect of doxycycline (DOX) on osteoclastogenesis, mature osteoclast fate and function, wear particles-induced osteoeolysis, and to provide some foundation for treating aseptic loosening and osteolysis after joint arthroplasty.. Osteoclasts were generated from mouse bone marrow monocytes with the receptor activator of NF-kappaB ligand and the macrophage colony stimulating factor. DOX at a concentration of 5, 10, 15, and 20 microg/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 3 d in 24-well plates or on bone slices. DOX at a concentration of 5, 10, 15, and 20 microg/mL was respectively added to the medium. After TRAP staining, the osteoclasts were counted, resorption on bone slices was quantified, and the area was calculated after toluidine blue and Mayer-hematoxylin staining. Polymethyl methacrylate (PMMA) or ultra-high molecular weight polyethylene (UHMWPE) particles were implanted on the calvariae of C57BL/J6 mice. DOX, at a dose of 2 and 10 mg x kg(-1) x d(-1), was respectively given intraperitoneally for 7 d. Seven days later, the calvariae were removed and processed for pathological analysis.. DOX treatment effectively inhibited in vitro osteoclastogenesis, affected the fate of mature osteoclasts, and inhibited mature osteoclasts, causing bone resorption. In vivo data indicated that DOX strongly inhibited PMMA or UHMWPE-induced osteolysis and osteoclastogenesis.. DOX can effectively inhibit osteoclastogenesis and affect mature osteoclast fate and suppress wear particles induced by osteolysis and osteoclastogenesis. DOX might be useful in the treatment or prevention of wear particles-induced osteolysis and aseptic loosening for its effect on osteoclast generation and mature osteoclast fate and function.

Topics: Acid Phosphatase; Animals; Anti-Bacterial Agents; Bone Marrow Cells; Bone Resorption; Cell Survival; Doxycycline; Isoenzymes; Male; Mice; Mice, Inbred C57BL; Osteoclasts; Osteogenesis; Osteolysis; Particle Size; Polyethylenes; Polymethyl Methacrylate; Rabbits; Tartrate-Resistant Acid Phosphatase

Various studies indicate that periodontal ligament fibroblasts (PLF) have some similarities to osteoblasts, for example they have the capacity to induce the formation of osteoclast-like cells. Here, we investigated whether a second population of tooth-associated fibroblasts, gingival fibroblasts (GF), has similar osteoclastogenesis properties. PLF and GF were co-cultured with peripheral blood mononuclear cells (PBMC) in the presence and absence of dexamethasone and 1alpha,25dihydroxycholecalciferol (dex + vit D(3)) on plastic and on cortical bone slices. Tartrate resistant acid phosphatase (TRACP) positive multinucleated cells (MNCs) were more abundant in co-cultures with PLF than in GF-PBMC co-cultures, more abundant on plastic compared to bone and more abundant in the presence of dex + vit D(3). In line with these findings was an inhibition of MNC formation and not inhibition of existing osteoclasts by medium conditioned by GF. We next investigated whether expression of molecules important for osteoclastogenesis differed between the two types of fibroblasts and whether these molecules were regulated by dex + vit D(3). OPG was detected at high levels in both fibroblast cultures, whereas RANKL could not be detected. Resorption of bone did not occur by the MNCs formed in the presence of either fibroblast subpopulation, suggesting that the fibroblasts secrete inhibitors of bone resorption or that the osteoclast-like cells were not functional. The incapacity of the MNCs to resorb was abolished by culturing the fibroblast-PBMC cultures with M-CSF and RANKL. Our results suggest that tooth-associated fibroblasts may trigger the formation of osteoclast-like cells, but more importantly, they play a role in preventing bone resorption, since additional stimuli are required for the formation of active osteoclasts.

Topics: Acid Phosphatase; Adult; Bone Resorption; Carrier Proteins; Cell Differentiation; Cells, Cultured; Coculture Techniques; Dexamethasone; Fibroblasts; Flow Cytometry; Gingiva; Glycoproteins; Humans; Isoenzymes; Macrophage Colony-Stimulating Factor; Male; Membrane Glycoproteins; Osteoblasts; Osteoclasts; Osteolysis; Periodontal Ligament; Periodontium; Polymerase Chain Reaction; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Stromal Cells; Tartrate-Resistant Acid Phosphatase; Tooth

The aim of this study was to assess the dynamics of osteoclast migration and the degradation of unmineralized extracellular matrix in an osteolytic metastasis by examining a well-standardized lung cancer metastasis model of nude mice. SBC-5 human lung small carcinoma cells were injected into the left cardiac ventricle of 6-week-old BALB/c nu/nu mice under anesthesia. At 25-30 days after injection, the animals were sacrificed and their femora and/or tibiae were removed for histochemical analyses. Metastatic lesions were shown to occupy a considerable area extending from the metaphyses to the bone marrow region. Tartrate resistant acid phosphatase (TRAPase)-positive osteoclasts were found in association with an alkaline phosphatase (ALPase)-positive osteoblastic layer lining the bone surface, but could also be localized in the ALPase-negative stromal tissues that border the tumor nodules. These stromal tissues were markedly positive for osteopontin, and contained a significant number of TRAPase-positive osteoclasts expressing immunoreactivity for CD44. We thus speculated that, mediating its affinity for CD44, osteopontin may serve to facilitate osteoclastic migration after their formation associated with ALPase-positive osteoblasts. We next examined the localization of cathepsin K and matrix metallo-proteinase-9 (MMP-9) in osteoclasts. Osteoclasts adjacent to the bone surfaces were positive for both proteins, whereas those in the stromal tissues in the tumor nests showed only MMP-9 immunoreactivity. Immunoelectron microscopy disclosed the presence of MMP-9 in the Golgi apparatus and in vesicular structures at the baso-lateral cytoplasmic region of the osteoclasts found in the stromal tissue. MMP-9-positive vesicular structures also contained fragmented extracellular materials. Thus, osteoclasts appear to either select an optimized function, namely secreting proteolytic enzymes from ruffled borders during bone resorption, or recognize the surrounding extracellular matrix by mediating osteopontin/CD44 interaction, and internalize the extracellular matrices. Microsc.

Topics: Acid Phosphatase; Animals; Bone Neoplasms; Carcinoma, Small Cell; Cathepsin K; Cathepsins; Cytoplasmic Vesicles; Disease Models, Animal; Extracellular Matrix; Femur; Golgi Apparatus; Humans; Hyaluronan Receptors; Immunohistochemistry; Male; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Immunoelectron; Osteoclasts; Osteolysis; Osteopontin; Sialoglycoproteins; Tibia

Prosthetic wear debris-induced peri-implant osteolysis is a major cause of aseptic loosening after total joint replacement. In this condition, wear particles released from the implant components induce a granulomatous inflammatory reaction at the interface between implant and adjacent bone, leading to progressive bone resorption and loss of fixation. The present study was undertaken to characterize definitively the phenotype of osteoclast-like cells associated with regions of peri-implant focal bone resorption and to compare the phenotypic features of these cells with those of mononucleated and multinucleated cells associated with polyethylene wear particles. Peri-implant tissues were obtained from patients undergoing hip revision surgery for aseptic loosening after total joint replacement. Cells were examined for the expression of several markers associated with the osteoclast phenotype using immunohistochemistry, histochemistry, and/or in situ hybridization. CD68 protein, a marker expressed by multiple macrophage lineage cell types, was detected in mononucleated and multinucleated cells associated with polyethylene particles and the bone surface. Cathepsin K and tartrate-resistant acid phosphatase were expressed highly in both mononucleated and multinucleated cells associated with the bone surface. Levels of expression were much lower in cells associated with polyethylene particles. High levels of beta3 integrin protein were detected in cells in contact with bone. Multinucleated cells associated with polyethylene particles exhibited faint positive staining. Calcitonin receptor mRNA expression was detected solely in multinucleated cells present in resorption lacunae on the bone surface and was absent in cells associated with polyethylene particles. Our findings provide further evidence that cells expressing the full repertoire of osteoclast phenotypic markers are involved in the pathogenesis of peri-implant osteolysis after total joint replacement. They also demonstrate that foreign body giant cells, although believed to be phenotypically and functionally distinct from osteoclasts, express many osteoclast-associated genes and gene products. However, the levels and patterns of expression of these genes in the two cell types differ. We speculate that, in addition to the role of cytokines and growth factors, the substrate with which these cells interact plays a critical role in their differential phenotypic and functional properties.

Topics: Acid Phosphatase; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Arthroplasty, Replacement; Bone Resorption; Cathepsin K; Cathepsins; Humans; Isoenzymes; Macrophages; Osteoclasts; Osteolysis; Prosthesis Implantation; Receptors, Calcitonin; RNA, Messenger; Tartrate-Resistant Acid Phosphatase

In bone metastatic lesions, osteoclasts play a key role in the development of osteolysis. Previous studies have shown that macrophage colony-stimulating factor (M-CSF) is important for the differentiation of osteoclasts. In this study, we investigated whether an inhibitor of M-CSF receptor (c-Fms) suppresses osteoclast-dependent osteolysis in bone metastatic lesions. We developed small molecule inhibitors against ligand-dependent phosphorylation of c-Fms and examined the effects of these compounds on osteolytic bone destruction in a bone metastasis model. We discovered a novel quinoline-urea derivative, Ki20227 (N-{4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl}-N'-[1-(1,3-thiazole-2-yl)ethyl]urea), which is a c-Fms tyrosine kinase inhibitor. The IC(50)s of Ki20227 to inhibit c-Fms, vascular endothelial growth factor receptor-2 (KDR), stem cell factor receptor (c-Kit), and platelet-derived growth factor receptor beta were found to be 2, 12, 451, and 217 nmol/L, respectively. Ki20227 did not inhibit other kinases tested, such as fms-like tyrosine kinase-3, epidermal growth factor receptor, or c-Src (c-src proto-oncogene product). Ki20227 was also found to inhibit the M-CSF-dependent growth of M-NFS-60 cells but not the M-CSF-independent growth of A375 human melanoma cells in vitro. Furthermore, in an osteoclast-like cell formation assay using mouse bone marrow cells, Ki20227 inhibited the development of tartrate-resistant acid phosphatase-positive osteoclast-like cells in a dose-dependent manner. In in vivo studies, oral administration of Ki20227 suppressed osteoclast-like cell accumulation and bone resorption induced by metastatic tumor cells in nude rats following intracardiac injection of A375 cells. Moreover, Ki20227 decreased the number of tartrate-resistant acid phosphatase-positive osteoclast-like cells on bone surfaces in ovariectomized (ovx) rats. These findings suggest that Ki20227 inhibits osteolytic bone destruction through the suppression of M-CSF-induced osteoclast accumulation in vivo. Therefore, Ki20227 may be a useful therapeutic agent for osteolytic disease associated with bone metastasis and other bone diseases.

Topics: Acid Phosphatase; Animals; Bone Neoplasms; Cell Differentiation; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Isoenzymes; Mice; Mice, Transgenic; Osteoclasts; Osteolysis; Phenylurea Compounds; Protein Kinase Inhibitors; Proto-Oncogene Mas; Rats; Rats, Inbred F344; Rats, Nude; Receptor, Macrophage Colony-Stimulating Factor; Tartrate-Resistant Acid Phosphatase; Thiazoles

We developed a rodent model that mimics the osteoblastic and osteolytic changes associated with human metastatic prostate cancer. Microarray analysis identified MMP-7, cathepsin-K, and apolipoprotein D as being upregulated at the tumor-bone interface. MMP-7, which was produced by osteoclasts at the tumor-bone interface, was capable of processing RANKL to a soluble form that promoted osteoclast activation. MMP-7-deficient mice demonstrated reduced prostate tumor-induced osteolysis and RANKL processing. This study suggests that inhibition of MMP-7 will have therapeutic benefit in the treatment of prostate cancer-induced osteolysis.

Topics: Acid Phosphatase; Actins; Animals; Carrier Proteins; Disease Models, Animal; Down-Regulation; Gene Expression; Gene Expression Profiling; Glycoproteins; Humans; Isoenzymes; Male; Matrix Metalloproteinase 7; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Biological; Monocytes; Osteoclasts; Osteolysis; Osteoprotegerin; Parathyroid Hormone-Related Protein; Prostatic Neoplasms; RANK Ligand; Rats; Rats, Inbred F344; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Skull; Tartrate-Resistant Acid Phosphatase; Up-Regulation

TNF-alpha is the dominant cytokine in inflammatory osteolysis. Using mice whose BM stromal cells and osteoclast precursors are chimeric for the presence of TNF receptors, we found that both cell types mediated the cytokine's osteoclastogenic properties. The greater contribution was made, however, by stromal cells that express the osteoclastogenic cytokine M-CSF. TNF-alpha stimulated M-CSF gene expression, in vivo, only in the presence of TNF-responsive stromal cells. M-CSF, in turn, induced the key osteoclastogenic cytokine receptor, receptor activator of NF-kappaB (RANK), in osteoclast precursors. In keeping with the proproliferative and survival properties of M-CSF, TNF-alpha enhanced osteoclast precursor number only in the presence of stromal cells bearing TNF receptors. To determine the clinical relevance of these observations, we induced inflammatory arthritis in wild-type mice and treated them with a mAb directed against the M-CSF receptor, c-Fms. Anti-c-Fms mAb selectively and completely arrested the profound pathological osteoclastogenesis attending this condition, the significance of which is reflected by similar blunting of the in vivo bone resorption marker tartrate-resistant acid phosphatase 5b (TRACP 5b). Confirming that inhibition of the M-CSF signaling pathway targets TNF-alpha, anti-c-Fms also completely arrested osteolysis in TNF-injected mice with nominal effect on macrophage number. M-CSF and its receptor, c-Fms, therefore present as candidate therapeutic targets in states of inflammatory bone erosion.

Topics: Acid Phosphatase; Animals; Antibodies, Monoclonal; Bone and Bones; Bone Marrow Cells; Bone Resorption; CD4 Antigens; CD8 Antigens; Cell Separation; Cytokines; Dose-Response Relationship, Drug; Female; Flow Cytometry; Gene Expression Regulation; Inflammation; Interleukin-1; Isoenzymes; Macrophage Colony-Stimulating Factor; Macrophages; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Mice, SCID; Mice, Transgenic; NF-kappa B; Osteoclasts; Osteolysis; Rats; Receptor, Macrophage Colony-Stimulating Factor; Recombinant Fusion Proteins; RNA, Messenger; Signal Transduction; Stromal Cells; T-Lymphocytes; Tartrate-Resistant Acid Phosphatase; Time Factors; Tumor Necrosis Factor-alpha

RANKL (receptor activator of nuclear factor kappaB ligand) promotes osteoclast differentiation, stimulates osteoclast activity, and prolongs osteoclast survival and adherence to bone. Abnormalities of the RANKL/RANK/osteoprotegerin system have been implicated in a range of diseases, including osteoporosis. To date, no work has been done in osteolytic lesions of the facial skeleton. In this study, specimens of ameloblastomas, dentigerous cysts, odontogenic keratocysts, and radicular cysts were subjected to immunohistochemical analysis for RANKL and tartrate-resistant acid phosphatase (TRAP). Immunofluorescence staining for TRAP was visualized under confocal microscopy. All specimens demonstrated distinct positive immunoreactivity to RANKL and TRAP. The TRAP-positive cells also stained with in situ hybridization for human calcitonin receptor, a definitive marker for osteoclasts. Mononuclear pre-osteoclasts were observed to migrate from blood to the connective tissue stroma and multinucleate toward the bone surface. It can be concluded that RANKL plays a role in bone resorption in osteolytic lesions of the facial skeleton.

Topics: Acid Phosphatase; Ameloblastoma; Dentigerous Cyst; Facial Bones; Glycoproteins; Humans; Immunohistochemistry; In Situ Hybridization; Isoenzymes; Jaw Neoplasms; Odontogenic Cysts; Osteolysis; Osteoprotegerin; Radicular Cyst; Receptors, Calcitonin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Signal Transduction; Tartrate-Resistant Acid Phosphatase

Osteolysis and hypercalcemia are observed in 5-15%, and 10%, respectively, of malignant lymphoma patients during their clinical course. However, both osteolysis and hypercalcemia are uncommon at onset of the disease. We encountered a 24-year-old male non-Hodgkin's lymphoma patient who had multiple osteolytic lesion from the onset of the disease and repeated episodes of hypercalcemia during the clinical course. The patient died with refractory disease. We studied the expression of chemokines which might affect bone resorption using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Increased expressions of MIP-1alpha, MIP-1beta and RANKL, which are osteoclast-activating factors, were observed in the RNA derived from the patient's lymphoma cells. The secretion of osteoclast-activating factors such as MIP-1alpha by the tumor cells (and/or bone marrow stromal cells) might be involved in the etiology of osteolysis and hypercalcemia in some malignant lymphoma cases.

Topics: Acid Phosphatase; Adult; Biopsy; Bone Marrow Cells; Bone Resorption; Carrier Proteins; Chemokine CCL3; Chemokine CCL4; Chemokines; Fatal Outcome; Humans; Hypercalcemia; Immunophenotyping; Isoenzymes; Low Back Pain; Lymphoma, B-Cell; Lymphoma, Large B-Cell, Diffuse; Lymphoma, Non-Hodgkin; Macrophage Inflammatory Proteins; Magnetic Resonance Imaging; Male; Membrane Glycoproteins; Osteoclasts; Osteolysis; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Reverse Transcriptase Polymerase Chain Reaction; Tartrate-Resistant Acid Phosphatase

Giant cell granuloma (GCG) is an osteolytic tumour of the jaw which is characterised by the presence of both mononuclear and multinucleated (osteoclast-like) giant cell components. The nature of these component cells and the pathogenesis of the extensive osteolysis associated with this lesion is uncertain.. Using cell culture techniques and immunohistochemistry, we defined the phenotypic characteristics of the mononuclear and multinucleated cells present in four cases of GCG of the jaw. We also analysed the cellular and humoral factors associated with osteoclast formation and osteolysis in these tumours and determined whether GCG stromal cells are capable of supporting osteoclast formation.. GCG-derived giant cells expressed the phenotypic characteristics of osteoclasts (TRAP+, VNR+, and calcitonin responsive) and were capable of lacunar resorption. In addition to macrophages, the mononuclear cell population contained numerous spindle-shaped stromal cells which proliferated in culture and expressed RANKL; these GCG-stromal cells were capable of supporting human osteoclast formation from circulating monocyte precursors.. Our findings indicate that the giant cells in GCG of the jaw are osteoclast-like and formed from monocyte/macrophage precursors which differentiate into osteoclasts under the influence of RANKL-expressing mononuclear stromal cells found in this lesion.

Topics: Acid Phosphatase; Adult; Biomarkers; Bone Resorption; Calcitonin; Carrier Proteins; Cell Culture Techniques; Cell Differentiation; Cell Division; Child; Female; Giant Cells; Granuloma, Giant Cell; Humans; Isoenzymes; Macrophages; Male; Mandibular Diseases; Membrane Glycoproteins; Middle Aged; NF-kappa B; Osteoclasts; Osteolysis; Phenotype; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Vitronectin; Tartrate-Resistant Acid Phosphatase

The current study was undertaken to determine if the presence of titanium wear particulate deleteriously influences early osseointegration of posterolateral bone graft or disrupts an established posterolateral fusion mass.. Using an in vivo animal model to evaluate the effect(s) of titanium wear particulate on a posterolateral spinal arthrodesis based on serologic, histologic, and immunocytochemical analyses.. The effect of unintended wear particulate resulting from micromotion between the interconnection mechanisms in spinal instrumentation remains a clinical concern.. Thirty-four New Zealand White rabbits were randomized into two groups based on postoperative time periods of 2 months (Group 1, n = 14) and 4 months (Group 2, n = 20). Group 1 underwent a posterolateral arthrodesis at L5-L6 using tricortical iliac autograft or tricortical iliac autograft + titanium particulate. Group 2 received iliac autograft at the initial surgery and were reoperated on after 8 weeks and treated with posterolateral arthrodesis exposure alone or titanium particulate. Postoperative analysis included serologic quantification of systemic cytokines. Postmortem microradiographic, immunocytochemical, and histopathologic assessment of the intertransverse fusion mass quantified the extent of osteolysis, local pro-inflammatory cytokines, osteoclasts, and inflammatory infiltrates.. Serologic analysis of systemic cytokines indicated no significant differences in cytokine levels (P > 0.05) between the titanium or autograft treatments. Immunocytochemistry indicated increased levels of local cytokines (tumor necrosis factor-alpha) at the titanium-treated posterolateral arthrodesis sites at both time periods (P < 0.05). Osteoclast cell counts and regions of osteolytic resorption lacunas were higher in the titanium-treated versus autograft-alone groups (P < 0.05), and the extent of cellular apoptosis was markedly higher in the titanium-treated sites at both time intervals. Electron microscopy indicated definitive evidence of phagocytized titanium particles and foci of local, chronic, inflammatory changes in the titanium-treated sites.. Titanium particulate debris introduced at the level of a spinal arthrodesis elicits a cytokine-mediated particulate-induced response favoring pro-inflammatory infiltrates, increased expression of intracellular tumor necrosis factor-alpha, increased osteoclastic activity, and cellular apoptosis. The presence of titanium particulate debris, secondary to motion between spinal implants, may serve as the impetus for late-onset inflammatory-infectious complications and long-term osteolysis of an established posterolateral fusion mass in the clinical setting.

Topics: Acid Phosphatase; Animals; Apoptosis; Bone Transplantation; Cell Count; Cytokines; Ilium; Immunohistochemistry; Implants, Experimental; Inflammation; Lumbar Vertebrae; Macrophages; Models, Animal; Osteoclasts; Osteolysis; Particle Size; Rabbits; Radiography; Spinal Fusion; Titanium; Treatment Outcome; Tumor Necrosis Factor-alpha

Prosthesis failure due to wear debris-induced osteolysis remains a major clinical problem and the greatest limitation for total joint arthroplasty. Based on our knowledge of osteoclast involvement in this process and the requirements of receptor activator of NF-kappaB (RANK) signaling in osteoclastogenesis and bone resorption, we investigated the efficacy of RANK blockade in preventing and ameliorating titanium (Ti)-induced osteolysis in a mouse calvaria model. Compared with placebo controls we found that all doses of RANK:Fc above 1 mg/kg intraperitoneally (ip) per 48 h significantly inhibited osteoclastogenesis and bone resorption in response to Ti implanted locally. Complete inhibition occurred at 10 mg/kg ip per 48 h, yielding results that were statistically equivalent to data obtained with Ti-treated RANK-/- mice. We also evaluated the effects of a single injection of RANK:Fc on day 5 on established osteolysis and found that Ti-treated were still depleted for multinucleated tartrate-resistant acid phosphatase-positive (TRAP+) cells 16 days later. More importantly, this osteoclast depletion did not affect bone formation because the bone lost from the osteolysis on day 5 was restored by day 21. An assessment of the quantity and quality of the newly formed bone in these calvariae by calcein labeling and infrared (IR) microscopy, respectively, showed no significant negative effect of RANK:Fc treatment. These studies indicate that osteoclast depletion via RANK blockade is an effective method to prevent and reverse wear debris-induced osteolysis without jeopardizing osteogenesis.

Topics: Acid Phosphatase; Animals; Bone Matrix; Bone Remodeling; Bone Resorption; Female; Fluoresceins; Glycoproteins; Mice; Mice, Inbred CBA; Osteoclasts; Osteogenesis; Osteolysis; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Signal Transduction; Skull; Titanium

To determine whether particle size affects macrophage-osteoclast differentiation in vitro, latex beads of 0.1, 1, and 10 microm in diameter were added to a murine macrophage-UMR106 osteoblast-like cell coculture system. The extent of osteoclast differentiation was determined by assessing the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells on glass coverslips and the extent of lacunar resorption on dentine slices. The addition of particles, 1 and 10microm in size, to the cocultures resulted in a significant increase in the number of TRAP-positive osteoclast-like cells and in the resorption pit surface area compared with findings in control cultures to which no particles had been added. Particles 0.1 microm in size also stimulated osteoclast formation relative to the control; however, the difference was not significant. These results indicate that particles, particularly these 1 and 10microm in size, sizes which were phagocytosable, significantly enhanced the process of macrophage-osteoclast differentiation and suggest that particle size plays an important role in periprosthetic osteolysis.

Topics: Acid Phosphatase; Animals; Cell Differentiation; Coculture Techniques; Isoenzymes; Latex; Macrophages; Mice; Osteoclasts; Osteolysis; Particle Size; Tartrate-Resistant Acid Phosphatase

Tartrate-resistant acid phosphatase (TRAP) isoform 5b is a potential serum marker for osteoclastic activity. Biochemical assays for serum TRAP activity with para-nitrophenylphosphate (pNPP) have low specificity for bone because of hydrolysis by unrelated nontype 5 TRAPs of blood cells and by related isoform 5a. Our purpose was to increase the specificity of TRAP assay for osteoclastic activity by using naphthol-ASBI phosphate (N-ASBI-P) as a substrate for serum type 5 TRAP activity and heparin as an inhibitor of isoform 5a. TRAP activity in individual and pooled sera of normal subjects and patients with endstage renal disease (ESRD) and rheumatologic diseases was quantitated using pNPP and N-ASBI-P as substrate at pH 5.5 and 6.1. For some experiments, heparin (23U/ml) was added as a specific inhibitor of isoform 5a activity. Isoforms 5a and 5b were separated from serum pools by cation exchange chromatography and identified by nondenaturing polyacrylamide gel electrophoresis (PAGE). N-ASBI-P was selectively hydrolyzed by TRAP isoform 5b. TRAP assays with pNPP and N-ASBI-P correlated only in ESRD sera, which contained primarily isoform 5b. The two assays did not correlate in normal or rheumatic sera with significant amounts of 5a. Heparin inhibited isoform 5a activity approximately 50% but had little effect on isoform 5b activity. Biochemical assay of serum TRAP activity can be made specific for isoform 5b by using N-ASBI-P and heparin. This method can be adapted to simple microplate biochemical or immunochemical assays. This simplified method for assessment of osteoclastic TRAP 5b activity warrants a detailed investigation in diseases of bone metabolism.

Topics: Acid Phosphatase; Aniline Compounds; Arthritis, Rheumatoid; Biomarkers; Bone Remodeling; Chromatography, Ion Exchange; Clinical Enzyme Tests; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Heparin; Humans; Hydrogen-Ion Concentration; Hydrolysis; Isoenzymes; Kidney Failure, Chronic; Organophosphorus Compounds; Osteoclasts; Osteolysis; Sensitivity and Specificity; Substrate Specificity; Tartrate-Resistant Acid Phosphatase

Polyethylene debris from joint replacements may be transported in synovial fluid and be phagocytosed by macrophages. The activation and migration of macrophages may play important roles in osteolysis and implant loosening. Tissues from the bone-implant interface do not always contain wear debris, which may mean that osteolysis may not require direct contact with wear debris. We hypothesized that the release of polyethylene debris from the implants induces macrophage activation in the joint space. Then the activated macrophages release humoral factors, such as inflammatory cytokines, into the joint fluid. These cytokines may be transported to the bone marrow tissues around the implants where they stimulate the differentiation of the bone marrow cells into osteoclasts. Finally, the activated osteoclasts resorb the surrounding bone. To test this hypothesis, macrophages were stimulated by polyethylene particles. The levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were determined by enzyme-linked immunosorbent assay and were increased significantly. To test humoral interaction between macrophages and bone-marrow cells, a co-culture system was used in an in vitro model. With this system, two kinds of cells can be cultured together with humoral contact without the two cell types having to contact each other. We stimulated the macrophages with 5 microm of polyethylene particles and observed whether the bone marrow cells differentiated into the osteoclasts without contact with the macrophages. The numbers of osteoclasts were assessed using tartrate-resistant acid phosphatase (TRAP) staining. The numbers of TRAP-positive cells in the polyethylene particle-stimulated group were higher than in the control group. The ability of the TRAP-positive cells to resorb bone was confirmed by dentine pit formation assay. The results of this study support our hypothesis and suggest that one mechanism of osteolysis in failed joint arthroplasty is the more distant effects of pro-inflammatory cytokine release on osteoclast differentiation and/or activity.

Topics: Acid Phosphatase; Animals; Bone Marrow Cells; Bone Resorption; Cell Differentiation; Cells, Cultured; Coculture Techniques; Enzyme-Linked Immunosorbent Assay; Histocytochemistry; Interleukin-6; Isoenzymes; Macrophages; Macrophages, Peritoneal; Male; Mice; Mice, Inbred Strains; Microscopy, Electron, Scanning; Osteoclasts; Osteolysis; Polyethylene; Tartrate-Resistant Acid Phosphatase; Tumor Necrosis Factor-alpha

Interleukin-1beta and tartrate resistant acid phosphatase concentrations in synovial fluid aspirates were examined to determine if they could be used as indicators of increased synovial inflammation and an osteolytic reaction in patients having total knee arthroplasty. Synovial aspirates were obtained from seven patients with severely osteoarthritic knees that were scheduled for primary total knee arthroplasty and from 20 patients with knees scheduled for total knee arthroplasty revision. Eleven of the revision cases involved titanium alloy prostheses and nine involved cobalt chrome alloy prostheses. The interleukin-1beta and tartrate resistant acid phosphatase concentrations were obtained and compared between the group having primary total knee arthroplasty and the group having revision total knee arthroplasty. The knees having revision surgery had higher concentrations of interleukin-1beta and tartrate resistant acid phosphatase than did the knees having primary total knee arthroplasty. These results indicate a greater inflammatory and osteolytic response in knees having revision surgery. Although the osteoarthritic knees and the knees needing revision surgery in this study are considered to have an inflammatory state, it was only after total knee arthroplasty when particulate wear debris would be present that appreciable concentrations of interleukin-1beta and tartrate resistant acid phosphatase were produced.

Topics: Acid Phosphatase; Arthroplasty, Replacement, Knee; Biomarkers; Humans; Interleukin-1; Isoenzymes; Knee Prosthesis; Osteoarthritis, Knee; Osteolysis; Reoperation; Synovial Fluid; Tartrate-Resistant Acid Phosphatase

Osteoclasts (OCs), which form by fusion of hematopoietic precursor cells, are typically present in large numbers in giant cell tumors of bone (GCTBs). These tumors may, therefore, contain cells which secrete factors that stimulate recruitment and differentiation of OC precursors. Multinucleated cells resembling OCs also form in cultures of human cord blood monocytes (CBMs) stimulated by 1.25 dihydroxyvitamin D3, but these cells lack the ability to form bone resorption pits, the defining functional characteristic of mature OCs. CBMs may thus require additional stimulation to form OCs; we therefore investigated whether GCTBs are a source of such a stimulus. CBMs were stimulated in long term (21 day) culture by medium conditioned by explants of GCTBs; media collected within 15 days of explant (early-CM) and after 15 days (late-CM) were employed. We also cocultured CBMs with primary GCTB-derived stromal cells as well as immortalized bone marrow stroma-derived cells. CBMs stimulated by early-CM formed resorption pits on cortical bone slices; however, stimulation by late-CM resulted in virtually no resorption. Both early-CM and late-CM increased CBM proliferation, but not the proportion of vitronectin receptor positive or multinucleated cells. Coculture of CBMs with stromal cells of GCTBs or bone marrow did not result in bone resorption, although these stromal cells (most expressing alkaline phosphatase but progressively losing parathyroid hormone receptor expression) expressed mRNA for cytokines involved in OC differentiation, including macrophage-CSF, granulocyte-macrophage-CSF, IL-11, IL-6, and stem cell factor. Our results indicate that CBMs are capable of terminal OC differentiation in vitro, a process requiring 1,25 dihydroxyvitamin D3 as well as diffusible factor(s) which can be derived from GCTB. Stromal cells of GCTB may produce such factors in vivo, but do not support OC differentiation in vitro, possibly through phenotypic instability in culture.

Topics: Acid Phosphatase; Alkaline Phosphatase; Base Sequence; Bone Neoplasms; Bone Resorption; Cell Differentiation; Cytokines; DNA Primers; Fetal Blood; Giant Cell Tumors; Humans; Molecular Sequence Data; Monocytes; Osteocalcin; Osteoclasts; Osteolysis; Receptors, Parathyroid Hormone; Tumor Cells, Cultured

In an attempt to verify the nature of bone resorption processes on the bone surrounding radicular cysts, fragments of cysts with the adjacent bone tissue were studied by morphological, cytochemical and ultrastructural methods. Cyst fluid was analysed for its content of cytokines with osteolytic activity. The cyst wall exhibited several connective tissue extensions which penetrated the adjacent bone. Numerous multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells were seen at the tip of the intraosseous extensions of the cyst capsule and in direct contact with the bone tissue. Typical resorption lacunae were identified on the bone surface by scanning electron microscopy. Moreover, mononuclear TRAP-positive cells were seen within the cyst capsule. High levels of prostaglandin E2 (PGE2) and interleukin-6 (IL-6) were detected in the cyst fluids. In conclusion, active bone resorption may contribute significantly to the growth of these lesions within the jaws.

Topics: Acid Phosphatase; Adolescent; Adult; Aged; Aged, 80 and over; Alveolar Bone Loss; Child; Dinoprostone; Female; Humans; Interleukin-6; Male; Middle Aged; Osteoclasts; Osteolysis; Radicular Cyst

This report is the first cytochemical investigation of vanishing bone disease "Gorham's Disease" (Gorham and Stout 1955). The ultrastructural localization of non-specific alkaline phosphatase and of specific and non-specific acid phosphatase activity was studied in slices of tissue removed from a patient with this rare disorder. Sodium beta-glycerophosphate and phosphorylcholine chloride were used as substrates. Alkaline phosphatase was present around the plasma membranes of osteoblasts and associated with extracellular matrix vesicles in new woven bone. This is consistent with the proposed role for this enzyme (Robison 1923) and for matrix vesicles (Bonucci 1967) in the mineralization of bone (Bernard and Marvaso 1981). Concentrations of specific secretory acid phosphatase reaction product in the cytoplasm of degenerating osteoblasts may contribute to the imbalance between bone formation and resorption. Osteoclasts, while few in number, showed non-specific and specific acid phosphatase activity. The Golgi apparatus and heterophagic lysosomes of mononuclear phagocytes were rich in non-specific acid phosphatase. This was also present in the Golgi lamellae and lysosomes of endothelial cells. Acid phosphatase cytochemistry suggests that mononuclear phagocytes, multinuclear osteoclasts and the vascular endothelium are involved in bone resorption in this disease.

Topics: Acid Phosphatase; Alkaline Phosphatase; Bone and Bones; Cell Membrane; Histocytochemistry; Humans; Osteolysis; Osteolysis, Essential

Amyloidosis primarily involving bone is described in a 59-year-old male pateint. Well circumscribed lytic lesions of the skeleton raised the possibility of myelomatosis. The prolonged insidious course of the disease was uncomplicated by hypercalcemia, pathological fracture, or hematologic abnormalities. The clinical course, together with histological findings and strongly positive bone scan, were the distinguishing features. The osseous manifestations without plasma cell tumor appears to be a rare occurrence in amyloidosis.

Topics: Acid Phosphatase; Amyloidosis; Bone and Bones; Bone Resorption; Humans; Male; Middle Aged; Osteolysis; Paraproteinemias; Serum Amyloid A Protein; Technetium

The well-known clinical and radiological findings of idiopathic osteolysis (Hajdu-Cheney) are described in a 17-year old man. He also had retarded puberty, recurrent dislocation of the patella, dental anomalies and markedly elevated alkaline and acid serum phosphatase. The differential diagnosis of this rare condition and its relationship to other bone dysplasias is discussed.

Topics: Acid Phosphatase; Adolescent; Alkaline Phosphatase; Bone Resorption; Fingers; Humans; Male; Osteolysis; Puberty; Radiography; Syndrome; Toes; Tooth Abnormalities

Topics: Acid Phosphatase; Adolescent; Adult; Alkaline Phosphatase; Bone Neoplasms; Cell Membrane; Cell Nucleus; Cytoplasm; Endoplasmic Reticulum; Female; Giant Cell Tumors; Histocytochemistry; Humans; Lipid Metabolism; Lysosomes; Male; Microscopy, Electron; Middle Aged; Mitochondria; Osteolysis; Phagocytosis; Vacuoles