Ultrastructural Cytochemical and Ultrastructural Morphological Differences Between Human Multinucleated Giant Cells Elicited by Wear Particles from Hip Prostheses and Artificial Ligaments at the Knee

The authors investigated the ultrastructural cytochemical features of multinucleated and mononuclear cells in periprosthetic tissues associated with bone resorption (osteolysis) and those in tissues adjoining failed artificial ligaments having no relation to bone resorption. Clinical specimens of granulation tissue of each type, respectively numbering 4 and 3, were stained for tartrate-resistant acid phosphatase (TRAP) reactions and examined by light and electron microscopy. Both periprosthetic granulation tissues and those adjoining artificial ligaments contained TRAP-positive multinucleated and mononuclear cells. Near joint prostheses, multinucleated cells, including some giant cells, showed TRAP activity and cytoplasmic features resembling osteoclasts, while others had features consistent with foreign-body giant cells, and still others showed degenerative changes. Near artificial ligaments, TRAP-positive multinucleated cells lacked osteoclastic features. At both sites, TRAP-positive multinucleated cells had phagocytised wear particles. TRAP-positive mononuclear cells at both sites also showed phagocytic cytoplasmic features, but not osteoclastic cytoplasmic features. Human mononuclear phagocytes and multinucleated giant cells induced by wear particles possess TRAP activity. Those multinucleated giant cells at sites of osteolysis developed osteoclastic cytoplasmic features and have a phagocytic function.     

Ultrastructural cytochemical and ultrastructural morphological differences between human multinucleated giant cells elicited by wear particles from hip prostheses and artificial ligaments at the knee

The authors investigated the ultrastructural cytochemical features of multinucleated and mononuclear cells in periprosthetic tissues associated with bone resorption (osteolysis) and those in tissues adjoining failed artificial ligaments having no relation to bone resorption. Clinical specimens of granulation tissue of each type, respectively numbering 4 and 3, were stained for tartrate-resistant acid phosphatase (TRAP) reactions and examined by light and electron microscopy. Both periprosthetic granulation tissues and those adjoining artificial ligaments contained TRAP-positive multinucleated and mononuclear cells. Near joint prostheses, multinucleated cells, including some giant cells, showed TRAP activity and cytoplasmic features resembling osteoclasts, while others had features consistent with foreign-body giant cells, and still others showed degenerative changes. Near artificial ligaments, TRAP-positive multinucleated cells lacked osteoclastic features. At both sites, TRAP-positive multinucleated cells had phagocytised wear particles. TRAP-positive mononuclear cells at both sites also showed phagocytic cytoplasmic features, but not osteoclastic cytoplasmic features. Human mononuclear phagocytes and multinucleated giant cells induced by wear particles possess TRAP activity. Those multinucleated giant cells at sites of osteolysis developed osteoclastic cytoplasmic features and have a phagocytic function.     

Ultrastructural Cytochemical and Ultrastructural Morphological Differences Between Human Multinucleated Giant Cells Elicited by Wear Particles from Hip Prostheses and Artificial Ligaments at the Knee

The authors investigated the ultrastructural cytochemical features of multinucleated and mononuclear cells in periprosthetic tissues associated with bone resorption (osteolysis) and those in tissues adjoining failed artificial ligaments having no relation to bone resorption. Clinical specimens of granulation tissue of each type, respectively numbering 4 and 3, were stained for tartrate-resistant acid phosphatase (TRAP) reactions and examined by light and electron microscopy. Both periprosthetic granulation tissues and those adjoining artificial ligaments contained TRAP-positive multinucleated and mononuclear cells. Near joint prostheses, multinucleated cells, including some giant cells, showed TRAP activity and cytoplasmic features resembling osteoclasts, while others had features consistent with foreign-body giant cells, and still others showed degenerative changes. Near artificial ligaments, TRAP-positive multinucleated cells lacked osteoclastic features. At both sites, TRAP-positive multinucleated cells had phagocytised wear particles. TRAP-positive mononuclear cells at both sites also showed phagocytic cytoplasmic features, but not osteoclastic cytoplasmic features. Human mononuclear phagocytes and multinucleated giant cells induced by wear particles possess TRAP activity. Those multinucleated giant cells at sites of osteolysis developed osteoclastic cytoplasmic features and have a phagocytic function.     

Co-culture with periodontal ligament stem cells enhanced osteoblastic differentiation of MC3T3-E1 cells and osteoclastic differentiation of RAW264.7 cells

Objectives: Periodontal ligament stem cells (PDLSCs) are characterized by having multipotential differentiation and immunoregulatory properties, which are the main mechanisms of PDLSCs-mediated periodontal regeneration. Periodontal or bone regeneration requires coordination of osteoblast and osteoclast, however, very little is known about the interactions between PDLSCs and osteoblast-like cells or osteoclast precursors. In this study, the indirect co-culture approach was introduced to preliminarily elucidate the effects of PDLSCs on differentiation of osteoblast-like cells and osteoclast precursors in vitro. Materials and methods: Human PDLSCs were obtained from premolars extracted and their stemness was identified in terms of their colony-forming ability, proliferative capacity, cell surface epitopes and multi-lineage differentiation potentials. A noncontact co-culture system of PDLSCs and preosteoblastic cell line MC3T3-E1 or osteoclast precursor cell line RAW264.7 was established, and osteoblastic differentiation of MC3T3-E1 and osteoclastic differentiation of RAW264.7 were evaluated. Results: PDLSCs exhibited features of mesenchymal stem cells. Further investigation through indirect co-culture system showed that PDLSCs enhanced ALP activity, expressions of ALP, Runx2, BSP, OPN mRNA and BSP, OPN proteins and mineralization matrix deposition in MC3T3-E1. Meanwhile, they improved maturation of osteoclasts and expressions of TRAP, CSTK, TRAF6 mRNA and TRAP, TRAF6 proteins in RAW264.7. Conclusions: PDLSCs stimulates osteoblastic differentiation of osteoblast precursors and osteoclastic differentiation of osteoclast precursors, at least partially, in a paracrine fasion.     

Morphological and histochemical comparison of the cells elicited by ectopic bone implants and tibial osteoclasts

Pellets of mineralized and demineralized bone and a composite mixture of mineralized and demineralized, devitalized bone particles were implanted subcutaneously on the dorsal body wall of young adult rats. Two weeks post-implantation, the pellets were removed and processed for histochemical and morphological analyses. Rat proximal tibia was also processed for evaluation. The levels of tartrate-resistant acid phosphatase (TRAP) activity in the multinucleated giant cells (MNGCs) from each of the three implants and from osteoclasts were assessed using an image analyzer. The osteoclasts from the proximal tibia and the majority of MNGCs from the demineralized implants demonstrated high levels of TRAP activity. MNGCs from the mineralized implants showed either a low level or absence of TRAP activity. Most MNGCs from the composite implants exhibited a low level of TRAP activity; however, there was a population of cells that demonstrated a high level of reaction product, similar to that seen in the tibia and demineralized implant. Morphologically, osteoclasts from the proximal tibia and from the osteogenic demineralized implant exhibited ruffled borders. A small population of MNGCs from the composite implant also revealed osteoclastic features. In summary, MNGCs from the mineralized implant did not exhibit a level of TRAP reaction product or morphology similar to osteoclasts, while the majority of cells from the demineralized implant and a subpopulation of the MNGCs elicited by the composite implant did demonstrate TRAP expression and morphology similar to osteoclasts. The expression of osteoclastic characteristics in cells at an ectopic site may be dependent on accessory signals from the skeletal microenvironment; such signals appear to be absent from or incomplete in the mineralized implants but appear to be present when demineralized bone particles are implanted.     

Effect of intermittent shear stress on mechanotransductive signaling and osteoblastic differentiation of bone marrow stromal cells

Perfusion culture of osteoprogenitor cells seeded within porous scaffolds suitable for bone tissue engineering is known to enhance deposition of a bone-like extracellular matrix, and the underlying mechanism is thought to involve flow-induced activation of mechanotransductive signaling pathways. Basic studies have shown that mechanotransduction is enhanced by impulse flow and may be mediated through autocrine signaling pathways. To test this, an intermittent flow regimen (5 min on/5 min off ) that exerts impulses on adherent cells and permits accumulation of secreted factors in the cell microenvironment was compared to continuous flow for its ability to stimulate phosphorylation of ERK and p38, synthesis of prostaglandin E2 (PGE2), and expression of mRNA for collagen 1alpha1 (Col-1alpha1), osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin (OCN). Studies were performed using bone marrow stromal cells cultured in osteogenic media, and parallel-plate flow chambers were used to exert a shear stress of 2.3 dyn/cm2 on cell layers. Results show that continuous flow significantly enhanced phosphorylation of ERK and p38 after 30 min relative to intermittent flow, while intermittent flow significantly increased accumulation of PGE2 in the circulating medium by 24 h relative to continuous flow. Neither continuous nor intermittent flow affected mRNA expression of Col-1alpha1 and OPN after 4 h, but when monolayers were stimulated for 24 h and then allowed to differentiate under static conditions for an additional 13 days, expression of Col-1alpha1, OPN, BSP, and OCN under continuous and intermittent flow was similar and significantly elevated relative to static controls. This study demonstrates that the variation of perfusion regimen modulates mechanotransductive signaling.     

Sol-gel bioactive glasses support both osteoblast and osteoclast formation from human bone marrow cells

We have examined the ability of bioactive sol-gel glass ceramics to support both osteoblast and osteoclast differentiation from human bone marrow cells (HBMC). Nucleated cells from human bone marrow were cultured on tissue culture plastic and on two sol-gel coatings: A2 glass-ceramic containing 54 mol % CaO/40 mol % SiO(2) and S2 glass-ceramic containing 16 mol % CaO/80 mol % SiO(2). Osteoblast differentiation was followed by measuring alkaline phosphatase (ALP) activity, mRNA levels for ALP, osteopontin, RANK ligand (RANKL), and immunofluorescent co-localization of ALP and RANKL. Osteoclasts were identified by morphology and positive staining for tartrate-resistant acid phosphatase (TRAP). ALP activity and mRNA levels were similar for cells on A2 coatings and on tissue culture plastic, but mRNA levels of osteopontin and RANKL were tenfold higher on A2 than on plastic. Cultures on A2 coatings also contained multinucleated osteoclasts staining positively for TRAP. In contrast, cells cultured on S2 coatings had the characteristics of more differentiated osteoblasts as measured by higher ALP expression. However, the levels of osteopontin and RANKL mRNA on S2 glass were lower than on A2 glass and there were fewer, weakly staining TRAP-positive multinucleate cells. Thus, sol-gel glass-ceramic materials differing in CaO/SiO(2) ratios can produce markedly different effects on the osteoblast and osteoclast differentiation from HBMC.     

The expression of bone matrix proteins induced by different bioimplants in a rabbit sinus lift model

This study aimed to analyze the expression of bone matrix proteins and CD31 by immunohistochemistry after maxillary sinus grafting with different bioimplants in a rabbit model. Rabbit demineralized bone matrix (DBM), partially purified bovine bone morphogenetic proteins (BMP), a mixture of BMP with DBM (BMP/DBM), or particulated autogenous bone was grafted into the maxillary sinuses of 42 rabbits. Animals were sacrificed at 2 and 8 weeks. Immunohistochemistry was used to investigate the expression of type 1 collagen (COL1), osteonectin (ON), osteocalcin (OC), bone sialoprotein (BSP), osteopontin (OPN), and CD31. Sinuses grafted with BMP were filled with trabeculae of woven bone that was strongly immunoreactive for COL1, OC, ON, and BSP. BMP/DBM showed strongly positive immunoreactivity for these proteins within the newly formed bone, but weak immunoreactivity in the DBM particles. Immunoreactivity for COL1, OC, ON, and BSP in DBM sinuses was only seen in the osteoblasts rimming the grafted bone particles. The staining of autogenous bone graft sinuses was similar to those grafted with DBM. OPN staining was detected in autogenous bone graft, BMP/DBM, and BMP bioimplants. CD31 staining was strongest in BMP and BMP/noncollagenous matrix proteins sinuses. These results suggest that exogenous BMP enhances not only osteogenesis but also angiogenesis, an important part of bone repair.     

Effect of beta-tricalcium phosphate particles with varying porosity on osteogenesis after sinus floor augmentation in humans

This study examines the effect of two beta-tricalcium phosphate (TCP) particulate bone grafting materials with varying porosity on bone formation and on osteogenic marker expression 6 months after sinus floor augmentation. Unilateral sinus grafting was performed in 20 patients using a combination (4:1 ratio) of beta-TCP particles with 35% porosity (TCP-C) or 65% porosity (TCP-CM) and autogenous bone chips. At implant placement cylindrical biopsies were sampled and processed for immunohistochemical analysis of resin embedded sections. Sections were stained for collagen type I (Col I), alkaline phosphatase (ALP), osteocalcin (OC) and bone sialoprotein (BSP). Furthermore, the area fraction of newly formed bone as well as the particle area fraction were determined histomorphometrically first, apically close to the Schneiderian membrane and second, in the center of the cylindrical biopsies. In the TCP-CM patient group a larger amount of bone formation and particle degradation was observed in the apical area and thus at the largest distance from the crestal bone compared to the TCP-C group. Good bone bonding behaviour was observed with both materials. This was accompanied by expression of ALP, Col I, BSP and OC in the newly formed bone and osteogenic mesenchym in contact with the degrading particles. Both TCP materials supported bone formation in the augmented sinus floor. Six months after implantation of both types of beta-TCP particles, bone formation and matrix mineralization was still actively progressing in the tissue surrounding the particles. Consequently, a greater porosity appears to be advantageous for enhancing bone formation and particle degradation.     

Expression of bone sialoprotein and osteopontin in breast cancer bone metastases

Bone sialoprotein (BSP) and osteopontin (OPN) are prominent, mineral-associated proteins in the extracellular matrix of bone that have been implicated in the metastatic activity of cancer cells. The expression of BSP, which is normally restricted to mineralizing tissues, has been observed in cancers with a high propensity for forming bone metastases. To investigate the relationship between BSP expression and the formation of bone metastases we have conducted an initial study of the expression of BSP in 10 intraductal breast carcinoma bone metastases using immunostaining and in situ hybridization, and compared the expression with OPN. The metastases were characterized by the infiltration of tumour cells into bone with extensive bone resorption evident. Moderate to strong staining for BSP was observed in all (100%) carcinomas, which also expressed BSP mRNA as determined by in situ hybridization. Variable staining for BSP was also observed in the mineralized bone and expression of BSP mRNA could be observed in osteoblastic cells on the bone surface and in some osteocytes at sites of bone remodelling. Contrary to a previous report, BSP expression could be demonstrated by PCR in three breast cancer cell lines, MCF-7, T47-D and MDA-MB-231. Moreover, in sub-cutaneous tumours formed by MDA-MB-231 breast cancer cells injected into athymic mice, higher immunostaining for BSP was seen in large ulcerating tumours in which mineral deposits were formed. In contrast to BSP, staining for OPN in bone metastases was generally restricted to the interface between tumor cells and bone surface of the carcinomas. While OPN staining was also observed in the cytoplasm of osteoclasts, which showed strong hybridization to a digoxygenin-labelled OPN cRNA probe, expression of OPN was not clearly detectable in the tumour cells. These studies provide the first demonstration of BSP expression by tumour cells in bone metastases and support the concept that BSP may have a role in targeting metastatic cells to bone. Expression of OPN in bone metastases appears to be related to increased bone resorptive activity by osteoclasts. This revised version was published online in July 2006 with corrections to the Cover Date.     

Bone morphogenetic proteins 5 and 6 stimulate osteoclast generation

Bone regeneration is required for fracture-healing, and different procedures have been used to promote osteogenesis. Recently, BMP-2 has been shown to induce bone formation in vivo and has been tested in clinical trials. A recent in vitro study evaluated the osteogenic activity of 14 BMPs on osteoblastic progenitor cells with an osteogenic hierarchical model in which BMP-2 and BMP-6 may play an important role in inducing osteoblast differentiation. Although the relative osteoinductive activity of each BMP is important, bone regeneration is a process consisting of bone formation and bone resorption. Therefore, it remains unclear which effects BMP-5 and -6 have on the generation of osteoclasts and by which mechanism osteoclastogenesis is stimulated. To compare osteoclastic potency of each BMP, primary murine bone marrow cells were treated with human recombinant BMP-2, BMP-5, or BMP-6 and 1,25-(OH)2 vitamin D3 and stained for the TRAP enzyme. Osteogenic activity of BMP-5 was determined by measuring induction of ALP-activity and proliferation after incubation with primary murine osteoblasts. For elucidating the molecular mechanism, primary bone marrow cells with various concentrations of OPG were added to the TRAP assay and mRNA levels of RANKL and OPG were measured after stimulation with BMP-5. The presented data show that BMP-5 and BMP-6, unlike BMP-2, enhanced the formation of murine TRAP+/MNCs in a biphasic curve. BMP-5 and -6 were less potent in stimulating osteoclastogenesis compared to BMP-2. Concerning the effects of BMP-5 on osteoblasts, there was a dose-dependent increase of ALP activity and proliferation up to a maximum dose of 300 ng/mL. At the mRNA level, BMP-5 increased the RANKL/OPG ratio. In conclusion, this study demonstrates that in contrast to BMP-2, BMP-5 and -6 influences the generation of osteoclasts in a biphasic mode. Both proteins might be very important regulators of bone homeostasis, and therefore, potent candidates for future treatment strategies of bone regeneration.     

Identification of cell types responsible for bone resorption in rheumatoid arthritis and juvenile rheumatoid arthritis.

Focal resorption of bone at the bone-pannus interface is common in rheumatoid arthritis (RA) and juvenile rheumatoid arthritis (JRA) and can result in significant morbidity. However, the specific cellular and hormonal mechanisms involved in this process are not well established. We examined tissue sections from areas of bone erosion in patients with RA and JRA. Multinucleated cells (MNCs) were present in resorption lacunae in areas of calcified cartilage and in subchondral bone immediately adjacent to calcified cartilage, as previously described. mRNA for the calcitonin receptor (CTR) was localized to these MNCs in bone resorption lacunae, a finding that definitively identifies these cells as osteoclasts. These MNCs were also positive for tartrate-resistant acid phosphatase (TRAP) mRNA and TRAP enzymatic activity. Occasional mononuclear cells on the bone surface were also CTR positive. Mononuclear cells and MNCs not on bone surfaces were CTR negative. The restriction of CTR-positive cells to the surface of mineralized tissues suggests that bone and/or calcified cartilage provide signals that are critical for the differentiation of hematopoietic osteoclast precursors to fully differentiated osteoclasts. Some MNCs and mononuclear cells off bone and within invading tissues were TRAP positive. These cells likely represent the precursors of the CTR-TRAP-positive cells on bone. Parathyroid hormone receptor mRNA was present in cells with the phenotypic appearance of osteoblasts, in close proximity to MNCs, and in occasional cells within pannus tissue, but not in the MNCs in bone resorption lacunae. These findings demonstrate that osteoclasts within the rheumatoid lesion do not express parathyroid hormone receptor. In conclusion, the resorbing cells in RA exhibit a definitive osteoclastic phenotype, suggesting that pharmacological agents that inhibit osteoclast recruitment or activity are rational targets for blocking focal bone erosion in patients with RA and JRA.     

Bone formation and resorption of highly purified beta-tricalcium phosphate in the rat femoral condyle

The aim of this study was to examine the chronological histology associated with highly purified beta-tricalcium phosphate (beta-TCP) implanted in the rat femoral condyle. Specimens were harvested on days 4, 7, 14, 28 and 56 after implantation, and were analyzed by tartrate-resistant acid phosphatase (TRAP) staining, immunohistochemistry of the ED1 protein as a marker of the phagocyte system, and in situ hybridization with digoxigenin-labeled alpha1 chain of type I procollagen (COL1A1), osteopontin and osteocalcin. beta-TCP was resorbed in a chronological manner. Although new bone was not observed on day 4, fibroblast-like cells around beta-TCP were positive for COL1A1 and osteopontin mRNA. New bone formation presented after day 7. In the double-staining for OPN and ED1 on day 7, most cells around beta-TCP were positive for either osteopontin mRNA or ED1 protein. However, there were some doubly positive multinucleated cells, suggesting that they belonged to the mononuclear phagocyte system. After day 28, the implanted region was replaced with bone marrow. Multinucleated TRAP-positive and ED1-positive cells which adhered to beta-TCP at all stages seemed to be osteoclasts and they continuously resorbed beta-TCP. beta-TCP has a good biocompatibility since both bioresorption and bone formation started at an early stage after implantation.     

Similarities between giant cell tumor of bone, giant cell tumor of tendon sheath, and pigmented villonodular synovitis concerning ultrastructural cytochemical features of multinucleated giant cells and mononuclear stromal cells

The authors investigated ultrastructural cytochemical features of multinucleated and mononuclear stromal cells in giant cell tumor of bone (GCTB), giant cell tumor of tendon sheath (GCTTS), and pigmented villonodular synovitis (PVNS). Specimens of each tumor, respectively numbering 4, 4, and 3, were stained for tartrate-resistant acid phosphatase (TRAP) reactions and examined with an electron microscope. In GCTB and GCTTS, multinucleated cells, including some relatively small giant cells, showed TRAP activity and cytoplasmic features characteristic of osteoclasts, and also sometimes abundant rough endoplasmic reticulum and siderosomes. A few giant cells with macrophage-like features and slight TRAP activity were demonstrated in GCCTS and PVNS. In each tumor type, mononuclear cells showing TRAP activity shared cytoplasmic features with osteoclast-like multinucleated giant cells, while some others had macrophage-like features, and still others were poorly differentiated; a few mononuclear cells showed cell-to-cell contact. Ultrastructural similarities of TRAP-positive mononuclear cells in the three tumor types, and those between TRAP-positive multinucleated cells in GCTB and GCTTS, suggest a common cell lineage capable of multinucleated giant cell formation in the 3 tumors, despite differing histogenesis.     

Genotype-phenotype relationship in human ATP6i-dependent autosomal recessive osteopetrosis

Autosomal-recessive osteopetrosis is a severe genetic disease caused by osteoclast failure. Approximately 50% of the patients harbor mutations of the ATP6i gene, encoding for the osteoclast-specific a3 subunit of V-ATPase. We found inactivating ATP6i mutations in four patients, and three of these were novel. Patients shared macrocephaly, growth retardation and optic nerve alteration, osteosclerotic and endobone patterns, and high alkaline phosphatase and parathyroid hormone levels. Bone biopsies revealed primary spongiosa lined with active osteoblasts and high numbers of tartrate-resistant acid phosphatase (TRAP)-positive, a3 subunit-negative, morphologically unremarkable osteoclasts, some of which located in shallow Howship lacunae. Scarce hematopoietic cells and abundant fibrous tissue containing TRAP-positive putative osteoclast precursors were noted. In vitro osteoclasts were a3-negative, morphologically normal, with prominent clear zones and actin rings, and TRAP activity more elevated than in control patients. Podosomes, alphaVbeta3 receptor, c-Src, and PYK2 were unremarkable. Consistent with the finding in the bone biopsies, these cells excavated pits faintly stained with toluidine blue, indicating inefficient bone resorption. Bone marrow transplantation was successful in all patients, and posttransplant osteoclasts showed rescue of a3 subunit immunoreactivity.     

外周血单个核细胞诱导培养破骨细胞

目的：从人外周血单个核细胞诱导培养获得高产量高纯度破骨细胞,为破骨细胞的体外研究提供丰富的细胞来源。 方法：从外周血分离单个核细胞贴壁培养,采用0.25％胰蛋白酶／0.02％EDTA联合消化,纯化后以RANKL和M-CSF加以诱导(A组),并与未经消化之传统方法（B组）比较。结果：与B组相比,A组可获得(1 426±204)个破骨细胞（P<0.05）,0.25％胰蛋白酶／0.02％EDTA联合消化可使破骨细胞纯化率达90％。诱导生成的破骨细胞TRAP染色阳性,功能试验显示具有噬骨能力。结论：联合消化结合RANKL/M-CSF诱导培养法可产生大量的破骨样细胞,方法简便而且经济实用。     

Nanotexturing of titanium-based surfaces upregulates expression of bone sialoprotein and osteopontin by cultured osteogenic cells

Bone formation around implants is influenced by surface geometry. Since cell/matrix/substrate interactions associated with cell signaling occur in the nanoscale dimension, we have evaluated the influence of nanotexturing of titanium-based surfaces on the expression of matrix proteins by cultured osteogenic cells at initial time points. Cells were obtained by enzymatic digestion of newborn rat calvaria and grown on titanium and titanium alloy discs with nanotextured or machined surfaces, and on glass coverslips for periods of 6 h, 1 day, and 3 days, under standard culture conditions. Cultures were processed for single or dual immunolabeling with monoclonal and/or polyclonal antibodies against bone sialoprotein (BSP), fibronectin (FN), osteopontin (OPN), type-I pro-collagen, or tubulin, followed by corresponding fluorophore-conjugated secondary antibodies. Some samples were processed for scanning electron microscope analysis of morphology and immunogold labeling. After 6 h, nanotextured surfaces exhibited up to a nine-fold increase in the proportion of cells with peripheral OPN labeling. At day 3, the proportion of OPN and BSP labeled cells was higher, and the intensity of immunoreactivity dramatically increased. No significant differences were observed in the expression pattern and the proportion of cells immunoreactive for FN or type-I pro-collagen. Our results demonstrate that nanotexturing of titanium-based surfaces upregulates the early expression of BSP and OPN in osteogenic cell cultures.     

Effects of neridronic acid on osteoclasts derived by physiological dual-cell cultures

Increased osteoclastic activity is observed in many osteopathic disorders - including postmenopausal osteoporosis, Paget's disease, primary bone tumours, lytic bone metastases, multiple myeloma and rheumatoid arthritis - that involve increased bone resorption and a loss of bone mass. Bisphosphonates are highly effective inhibitors of bone resorption that selectively affect the osteoclasts. The aim of this study was to obtain more information about the mechanism of action of bisphosphonates such as neridronic acid using a dual-cell culture model. As a model of osteoclastogenesis we used a murine monocyte/macrophage cell line RAW 264.7 type CRL 2278 co-cultured with murine osteoblasts. The monocyte-osteoblast system allows physiological experimentation of bone anti-resorption drugs, simulating bone turnover in pathologies such as osteoporosis. The direct actions of neridronic acid on cell proliferation and functionality in the co-culture model were examined using tartrate-resistant acid phosphatase (TRAP) assay, immunohistochemical localization of actin, and transmission and scanning electron microscopy (SEM). Results showed that the percentage of TRAP-positive cells, an early marker of osteoclastic differentiation, was significantly higher in control cultures than in co-cultures treated with variable concentrations of neridronic acid. Neridronic acid induced dramatic morphological changes, characterized by the loss of the ruffled border. The actin ring associated with the plasma membrane of the cells treated with neridronic acid was shown to break down. The tissue-specific targeting of neridronic acid to bone mineral suggests that it may inhibit bone resorption by direct effects on osteoclasts or other bone cells in the immediate microenvironment of the osteoclasts. From our study, we conclude that structural alterations induced by neridronic acid in our co-culture system lead to decreased osteoclast function. This may encourage the use of neridronic acid to reduce bone resorption in the therapy of demineralizing metabolic bone disorders.