Rat Osteoclast Precursors In Vivo Express a Vitronectin Receptor and a Chloride-Bicarbonate Exchanger

In vivo osteoclast precursors, which are mononuclear, were previously found to express TRAP (tartrate-resistant acid phosphatase) and CTR (calcitonin receptor), like multi-nucleated osteoclasts. In vitro, they were found to express, in addition, VNR (vitronectin receptor) and CBE (chloride-bicarbonate exchanger). In order to ascertain that osteoclast precursors in vivo express VNR and CBE like their in vitro counterparts, we used immunohistochemistry to localize these molecules in developing long bones of neonatal rats. Frozen sections of metatarsals and phalanges of 1-2 day-old rats were stained for TRAP and mineralization using histochemistry or were reacted with polyclonal antibodies specific for either the β₃ chain of the VNR or synthetic sequences of the CBE. Both mature, multinucleated osteoclasts within the forming marrow cavity of metatarsals (as shown previously) and mononuclear osteoclast precursors located outside the bony collar of the phalangeal calcified rudiment (as shown here for the first time) expressed both TRAP, VNR and CBE. These findings suggest that mononuclear osteoclast precursors express many of the phenotypical markers of multinucleated osteoclasts prior to their fusion and multinucleation which may allow them to resorb bone, as suggested by in vitro observations of pit formation by preosteoclasts cultured on resorbable substances.     

周期性张应变对破骨前体细胞和破骨细胞增殖和抗酒石酸酸性磷酸酶的影响

目的研究不同强度的力学载荷对破骨细胞及其前体细胞增殖、分化和功能的影响。方法以破骨诱导液培养RAW264.7破骨前体细胞,同时施加3 d的周期性张应变,然后培养4 d;另外一组RAW264.7细胞以破骨诱导液培养4 d,将其诱导为破骨细胞,再施加3 d的周期性张应变。结果在不同张应变下,两组细胞增殖活性的变化大致相同,但细胞抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatage,TRAP)活性和破骨细胞(TRAP阳性多核细胞)数量的变化明显不同。在2 500με的中等强度张应变下,第1组的TRAP活性降幅和破骨细胞数量减幅均最高,而后者TRAP活性降幅和破骨细胞数量减幅均最低。结论不同张应变对分化初期破骨前体细胞和已分化出破骨细胞的破骨前体细胞的破骨分化和功能状态的影响有明显差异。     

Osteoclastic Invasion and Mineral Resorption of Fetal Mouse Long Bone Rudiments are Inhibited by Culture Under Intermittent Compressive Force

To study the effect of low-magnitude mechanical stimuli on mineralized matrix metabolism, fetal mouse long bone rudiments were cultured for 5d in the absence or presence of intermittent (0.3 Hz) compressive force (ICF) of 132 g/cm². ICF treatment stimulated mineralization of the diaphyseal bone collar as well as hypertrophic cartilage, but inhibited the release of ⁴⁵Ca from prelabeled rudiments. ICF also inhibited the migration of osteoclasts and their precursors from the periosteum into the diaphysis and the subsequent excavation of a primitive marrow cavity.

These data suggest that osteoclasts are sensitive to mechanical stimuli. Mechanical stimulation seems to protect the bone rudiment against osteoclastic attack and has a strong anabolic effect on mineral metabolism.     

Morphology of osteoclasts in resorbing fetal rat bone explants: Effects of PTH and AIF in vitro

Osteoclastic bone resorption was studied using ⁴⁵Ca-labeled fetal rat bones cultured in the presence of parathyroid hormone (PTH) and an anti-invasion factor (AIF) derived from bovine hyaline cartilage which is enriched in a collagenase inhibitor. The specific morphological expressions of osteoclasts cultured in PTH and AIF were observed in both light and electron microscopy and analyzed cytometrically. Stimulation of bone resorption with PTH revealed significant increases in the numbers and activity of osteoclasts, whereas bones cultured in the presence of AIF showed significant decreases in numbers of osteoclasts and altered cell features including the loss of osteoclast contact with bone surfaces. These structural modifications were evaluated with ⁴⁵Ca release data derived from matched-pair explants of fetal rat bones, revealing the existence of a relationship between resorptive states of the cultured bones and morphological expressions of osteoclastic activity.     

Morphology and biochemistry of bone remodeling: possible control by vitamin D, parathyroid hormone, and other substances

The effects of PTH and vitamin D on bone are the result of their direct and indirect effects on the functional cells of bone remodeling units and their precursors. These effects are probably modified or controlled by growth factors, cytokines, and PGs generated locally by the process of bone remodeling. Bone remodeling includes resorptive and bone forming phases, each with a longitudinal and a radial component of progression in time and space. Longitudinal resorption is rapid, prolonged and is probably carried out by osteoclasts utilizing hydrogen ions and lysosomal enzymes to remove mineral and organic components of bone in a highly localized and directed fashion. Individual osteoclasts are probably long-lived cells with a nuclear and perhaps a cytoplasmic turnover rate of 8%/day, with replenishment coming from preosteoclasts in the reversal zone. Radial resorption is slower and shorter than longitudinal resorption. It is carried out by reversal phase monocytes whose exact relationship to osteoclasts is not clear. Activated collagenase diffusing from osteogenic cells in the reversal zone could also play a role. The longitudinal rate of bone formation is probably a measure of the rate of proliferation and differentiation of osteogenic cells at the site at which they were activated. The radial rate of bone formation is a measure of how rapidly osteoblasts synthesize and mineralize bone matrix once they reach the resorption surface. PTH and vitamin D have no direct effects on mature osteoclasts. They may have direct stimulatory effects on proliferation and differentiation of osteoclast precursors and their fusion with osteoclasts but this is not clear because the ontogeny of osteoclasts vis a vis monocytes and other phagocytic cells is still not clear. It is likely that their effects to increase osteoclast precursors involve interactions among lymphocytes, monocytes, and hematopoietic stem cells at a distance from bone remodeling units and are mediated by 1,25(OH)2 vitamin D3 induced synthesis of cytokines and colony-stimulating factors. Stimulatory effects of PTH, vitamin D, PGs, and cytokines on osteoclasts are mediated by as yet undefined factors produced by osteoblasts. Osteoblasts stimulated by PTH could also inhibit osteoclasts by synthesizing and releasing PGs. PTH and vitamin D have diverse and often contradictory effects on the functional activity of osteoblast-like cells in vitro that are difficult to interpret because the relationship of these cells to osteoblasts in vivo is not clear.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of ionizing irradiation on formation and resorbing activity of osteoclasts in vitro

The effects of ionizing irradiation on the differentiation and activity of the osteoclast were investigated. Embryonic mouse metatarsal bones of different ages (14, 15, 16, 17 days) in which no osteoclasts had as yet been formed were irradiated with various x-ray doses and cultured until a marrow cavity became visible in the nonirradiated paired control bones. Bone growth and calcification were followed microscopically during culture. Irradiation caused a dose-dependent stunting of the longitudinal growth. Calcification was inhibited by high radiation doses (10 to 20 Gray (Gy), whereas a dose of 2.5 Gy stimulated the process in the early stages of long bone development. Histologic examination revealed complete inhibition of osteoclast formation in the 14- and 15-day-old bones after irradiation with 2.5 Gy or more. The number of osteoclasts in cultured older bones (16 days) was significantly reduced by irradiation, but osteoclast formation could not be completely prevented even by high dosages. Irradiation of explanted bone rudiments which were in a stage 1 day prior to the appearance of osteoclasts in vivo (17 days) did not significantly influence the formation of osteoclasts. Autoradiographic experiments using young bones showed that differentiation of osteoclast precursors into multinucleated osteoclasts is preceded by one or more divisions of the precursors in the periosteum. Furthermore, it was established from continuous 3H-thymidine-labeling experiments that in older bones (16 days) a part of the osteoclast nuclei originated from postmitotic osteoclast precursors. Irradiation mainly inhibited the appearance of labeled osteoclast nuclei in these bones. The results indicate that the osteoclast precursor, already present in the periosteum at an early stage of embryonic development, first proliferates and then differentiates into a mononuclear postmitotic preosteoclast. The proliferation is probably highly radiosensitive. Subsequently, the preosteoclasts fuse into multinucleated osteoclasts and invade the calcified hypertrophic cartilage zone. The resorbing activity of the osteoclast is less radiosensitive but can be inhibited by 5.0 Gy or more, as was established by morphometric and biochemical methods.     

Circulating Bone Marrow Osteoclast Precursors and Osteoclastogenesis in Patients with Type IIA and IIB Hyperlipidemias

Osteoclasts, the major source of calcium released during bone resorption, and their precursors preosteoclasts derive from bone marrow hemopoietic stem cells, granulocyte-macrophage colony-forming units. The factors responsible for commitment of colony-forming units into osteoclast precursors remain unknown. Studies of osteoclastogenesis in cultured blood mononuclear cells from patients with hyperlipidemia accompanied by atherosclerosis and calcification of vessels revealed high content of preosteoclasts in the blood.     

Differentiation kinetics of osteoclasts in the periosteum of embryonic bones in vivo and in vitro

Osteoclast progenitors are seeded via the blood stream in the mesenchyme surrounding embryonic long bone models long before the appearance of multinucleated osteoclasts. The proliferation and differentiation of these progenitors in embryonic mouse metatarsal bones was studied with acid phosphatase (AcP) histochemistry and 3H-thymidine autoradiography. In vivo, tartrate-resistant, acid phosphatase-positive, mononuclear cells appear in the periosteum (AcPP-P cells) at the age of 17 days (after conception). On day 18, AcP-positive, multinucleated osteoclasts invade the bone rudiment and start resorbing the calcified cartilage matrix, resulting in the formation of the marrow cavity. The kinetics of osteoclast formation in vitro was studied in metatarsal bones of embryonic mice of different ages cultured in the continuous presence of 3H-thymidine. In young bones (15 days), mainly proliferating, 3H-thymidine-incorporating progenitors gave rise to AcPP-P cell and osteoclast formation. In older bones (16 and 17 days) osteoclasts were progressively more derived from postmitotic, unlabeled precursors. Irradiation of the metatarsal bones with a radiation dose of 5.0 Gy prior to culture resulted in a selective elimination of the proliferating progenitors, whereas the contribution of postmitotic precursors in AcPP-P cell and osteoclast formation remained unchanged. The results demonstrate that in the periosteum of embryonic metatarsal bones a shift occurs from a population composed of proliferating osteoclast progenitors (15 days) to a population composed of postmitotic precursors (17 days) before multinucleated osteoclasts are formed (18 days). Obviously, postmitotic AcP-negative precursors, already present in 16-day-old bones, differentiate into precursors characterized by tartrate-resistant AcP activity, the preosteoclasts (17 days), which in their turn fuse into osteoclasts.     

The effect of parathyroid hormone, 1,25-dihydroxycholecalciferol and prostaglandins on the cytoplasmic activity of isolated osteoclastsxs

Osteoclasts were isolated on glass or plastic substrates and the effect of bone-active hormones and prostaglandins on their behaviour was observed. Untreated osteoclasts migrate across the substratum, actively extending pseudopodia which show intense “rippling” activity. Addition of parathyroid hormone (PTH), 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃), prostaglandins (PG) F_2α, E₂ and thromboxane B₂ caused no change in the behaviour or morphology of these osteoclasts. Prostacyclin, however, caused rapid cessation of cytoplasmic rippling activity followed by gradual pseudopodial retraction in a manner indistinguishable from that previously observed in the presence of calcitonin. The cytoplasmic quiescence induced by prostacyclin was seen at concentrations of 10^?9M and above. Quiescence was reversible and its duration depended upon the dose of the compound administered (prostacyclin decays rapidly). No such effect was observed on the behaviour of the other cell types contaminating the osteoclast cultures, nor on purified populations of osteoblasts or peritoneal macrophages. We also tested the ability of PTH, 1,25-(OH)₂D₃ and PGs to reverse calcitonin-induced osteoclast quiescence. We found that even high doses showed no detectable stimulation of quiescent isolated osteoclasts. This suggests that the undoubted ability of these hormones to stimulate osteoclastic activity in vivo is mediated indirectly, following a primary hormonal interaction with another cell type. We propose a model for the control of osteoclasis based on these observations. Prostacyclin may play the role of a local-acting hormone, produced by osteoblasts, which terminates osteoclasis when the requirements of local bone morphogenesis are fulfilled. Calcitonin exerts a direct inhibitory action on isolated osteoclasts and we suggest that this is opposed in vivo by an osteoclastic stimulator, released by osteoblasts. The hormones PTH, 1.25-(OH)₂,D₃, and PGE₂, stimulate osteoclastic activity indirectly following a primary interaction with osteoblasts, and may do so either by reducing the release of prostacyclin by osteoblasts or increasing formation of the unknown stimulator, or both.     

Osteoclastic invasion and mineral resorption of fetal mouse long bone rudiments are inhibited by culture under intermittent compressive force

To study the effect of low-magnitude mechanical stimuli on mineralized matrix metabolism, fetal mouse long bone rudiments were cultured for 5d in the absence or presence of intermittent (0.3 Hz) compressive force (ICF) of 132 g/cm2. ICF treatment stimulated mineralization of the diaphyseal bone collar as well as hypertrophic cartilage, but inhibited the release of 45Ca from prelabeled rudiments. ICF also inhibited the migration of osteoclasts and their precursors from the periosteum into the diaphysis and the subsequent excavation of a primitive marrow cavity. These data suggest that osteoclasts are sensitive to mechanical stimuli. Mechanical stimulation seems to protect the bone rudiment against osteoclastic attack and has a strong anabolic effect on mineral metabolism.     

Osteoclastogenic potential of bone marrow cells increases with age in elderly women with fracture

The most reliable explanation for decreasing bone mass in elderly women is an imbalance of osteoclastic resorption and osteoblastic formation resulting from a relative increase in osteoclastic resorption. However, it is not clear whether an increase in osteoclastic bone resorption with age is due to increased osteoclast formation or to osteoclastic bone resorption activity. In this study, using a human bone marrow culture system, we attempt to clarify the increase in osteoclast formation with age. The mononuclear cell-rich fraction from bone marrow, obtained from the proximal region of the femur from female elderly patients with fracture, were cultured for 14 days in the presence of 1,25 dihydroxyvitamin D(3). Tartrate-resistant acid phosphatase-positive multinucleated cells were counted as osteoclasts. In our investigation, human osteoclast formation in the bone marrow culture increased with age in elderly women (age 64-96 years). The osteoclast formation was positively correlated with macrophage-colony stimulation factor and prostaglandin E(2) production in bone marrow culture. Also, osteoclast formation ex vivo was negatively correlated with bone mineral density of the lumbar spine (L2-L4). The above results indicate that the osteoclastogenic potential of bone marrow cells increases with aging in elderly women with fracture, and suggest that a decrease in bone mass of elderly women may be due to an increase in osteoclast population associated with aging.     

PTH对破骨细胞骨吸收功能的影响及成骨细胞介导作用

采用分离、培养兔破骨细胞和成骨细胞的方法，体外研究甲状旁腺激素（ＰＴＨ）对破骨细胞骨吸收功能的影响，以及成骨细胞和破骨细胞之间的相互作用。结果表明，ＰＴＨ对破骨细胞的骨吸收功能无直接影响，但在成骨细胞参与下，ＰＴＨ对破骨细胞性骨吸收有明显的促进作用。说明成骨细胞在ＰＴＨ调节破骨细胞功能活动中有着重要的介导作用。     

Possible involvement of MIP-1alpha in the recruitment of osteoclast progenitors to the distal tibia in rats with adjuvant-induced arthritis

In the rat model of rheumatoid arthritis, a marked formation of osteoclasts is found in the distal tibia and the metatarsal bone. It was therefore postulated that osteoclast progenitors would be increased in the bone marrow cavities of rats with adjuvant-induced arthritis (AA rats). Bone marrow cells obtained from tibia of AA rats were cultured to form cells in the osteoclast lineage to access the number of osteoclast progenitors. Unexpectedly, only a suppressed level of osteoclast progenitors was detected in the diaphyseal bone marrow of tibia in AA rats. Distribution of osteoclast progenitors in the bone marrow cavity was examined, and it was shown that osteoclast progenitors accumulated in the distal tibia. Macrophage inflammatory protein (MIP)-1alpha, an osteoclastogenic CC chemokine, was expressed in ED-1-positive macrophages localizing in the distal tibia with marked bone destruction. Chemotaxis studies showed that MIP-1alpha expressed significant activity towards bone marrow cells. The suppressed level of osteoclastogenesis in bone marrow cells of AA rats was restored to a normal level by the addition of MIP-1alpha. It was suggested that MIP-1alpha is involved in the migration of osteoclast progenitors to the distal tibia as well as in osteoclastogenesis in AA rats. In these rats, in situ hybridization of the distal tibia with a high level of bone destruction showed significant expression of Receptor activator nuclear factor kappaB ligand (RANKL) messenger RNA in aggregates of multinucleated osteoclast-like cells present in the bone marrow cavity, a unique pathological feature for these rats. Migrated osteoclast progenitors are thought to be efficiently differentiated into osteoclasts in response to RANKL expressed by the aggregates of osteoclast-like cells under the influence of the MIP-1alpha. Such positive-feedback regulation of osteoclastogenesis could result in the highest recruitment of active osteoclasts in the area of marked bone destruction.     

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 evidence of reduced bone resorption in osteopetrotic (op) mice

Osteopetrosis, a metabolic bone disease in which a generalized accumulation of bone mass reduces or obliterates marrow cavities, is inherited as an autosomal recessive in several mammalian species. A recently discovered mutation in mice, the osteopetrosic (op) mutation, exhibits an elevation in bone matrix synthesis and a resistance to the hypercalcemic effects of exogenous parathyroid extract when young mutants are compared with normal littermates. This investigation examined the number, cytology, and ultrastructure of osteoclasts and the structure of bone surfaces in op mice in a morphologic assessment of bone resorption. Compared with normal littermates, op mice have a severe deficiency of osteoclasts, which also contain unusual toluidine blue-positive and electron-dense cytoplasmic inclusions and hypertrophy of clear zones and ruffled borders. Marrow spaces in op mice contained large numbers of megakaryocytes and large lipoid masses. Bone surfaces exhibiting evidence of resorption by scanning electron microscopy in normal littermates showed no evidence of resorption in op mice. Instead, these areas were characterized by morphologic features of bone formation. These data offer morphologic evidence of a reduction of bone resorption in this mutation. They are interpreted to mean (1) that op mice have a severe reduction in numbers or proliferative capacity of osteoclast precursors, which may be related to the cellular inclusions in the osteoclast population, and (2) that hypertrophy of the ruffled borders and clear zones of op osteoclasts is a compensatory attempt to increase bone resorption.     

Morphology of osteoclasts in resorbing fetal rat bone explants: effects of PTH and AIF in vitro.

Osteoclastic bone resorption was studied using 45Ca-labeled fetal rat bones cultured in the presence of parathyroid hormone (PTH) and an anti-invasion factor (AIF) derived from bovine hyaline cartilage which is enriched in a collagenase inhibitor. The specific morphological expressions of osteoclasts cultured in PTH and AIF were observed in both light and electron microscopy and analyzed cytometrically. Stimulation of bone resorption with PTH revealed significant increases in the numbers and activity of osteoclasts, whereas bones cultured in the presence of AIF showed significant decreases in numbers of osteoclasts and altered cell features including the loss of osteoclast contact with bone surfaces. These structural modifications were evaluated with 45Ca release data derived from matched-pair explants of fetal rat bones, revealing the existence of a relationship between resorptive states of the cultured bones and morphological expressions of osteoclastic activity.     

Inhibition of leukotriene function can modulate particulate-induced changes in bone cell differentiation and activity.

Aseptic loosening remains the major problem facing arthroplasty longevity with particulates from component materials touted as the cause of periprosthetic osteolysis. Proposed mechanisms in aseptic bone loss include: increased resorption, increased differentiation of osteoclasts (and/or macrophages locally), and decreased osteoblastic bone formation. Leukotrienes participate in osteoclastic bone resorption. We investigated inhibiting leukotrienes synthesis, using ICI 230487, to ameliorate the effects of particulates on osteoclast pit formation and also assessed the effects of alendronate, a bisphosphonate, on pit formation. Three particulates were used: ultra high molecular weight polyethylene (UHMWPE), polymethylmethacrylate (PMMA) and hydroxyapatite (HA). Osteoclast resorption was increased with UHMWPE, PMMA, and HA particles. Interventions with alendronate and ICI 230487 reduced particulate-induced osteoclast resorption. Both ICI 230487 and alendronate reduced osteoclast numbers at higher doses. To assess the effect of particulates on osteoclast and macrophage differentiation, mouse bone marrow was cultured and stained for tartrate resistant acid phosphatase colonies (TRAP+, osteoclasts) and nonspecific esterase positive colonies (NSE+, macrophage precursors). Particulates increased both TRAP+ and NSE+ colony formation. These increases were inhibited by ICI 230487. Particulates also inhibited osteoblast function assessed by the development of mineralized nodules and alkaline phosphatase positive (AP+) colony area. ICI 230487 partly protected osteoblast function from this particulate effect. Blockade of leukotriene production may prove a useful therapeutic intervention for particulate-induced aseptic loosening by inhibiting resorptive activity, reducing the pro-inflammatory cell populations induced and recruited by these particulates, as well as ameliorating the negative effects of inflammatory mediators on osteoblast function.Copyright 2001 John Wiley & Sons, Inc.     

Parathyroid hormone induces bone resorption in human peripheral blood mononuclear cells

Osteoclasts are known to derive from a macrophage colony-stimulating factor (M-CSF)-dependent precursor shared with macrophages. Cells capable of forming osteoclasts are present in peripheral blood. We characterized this population by incubating human peripheral blood mononuclear cells (PBMCs) with osteoclast-inductive UMR 106 cells, human macrophage colony stimulating factor (hM-CSF) and parathyroid hormone (PTH) or 1,25(OH)₂vitamin D3 on slices of devitalised cortical bone. We found that PBMCs were capable of substantial bone resorption, to levels comparable to those of haemopoietic tissue. Cells plated at very low densities and screened for the presence or absence of excavations revealed a linear relationship (r = 0.994) between the number of cells plated and the number of excavations formed. The limiting dilution analysis suggested that 1 in every 300–600 plated cells (0.15–0.3% of the PBMC population) had the capacity to resorb bone. The precursor was found in the rapidly adherent fraction, and typically generated very small numbers of excavations, suggesting that it was a relatively mature cell type. Co-cultures of PBMCs with UMR 106 cells would not generate osteoclasts without PTH/1,25(OH)₂vitamin D3, even with M-CSF, indicating that osteoclast-induction by stromal cells is not attributable to hormonal induction of M-CSF in UMR 106 cells, but that PTH induces some other activity, necessary for osteoclast but not macrophage formation, in UMR 106 cells. Osteoclasts did not form if PTH was omitted in the first few days of the culture period. Thus, osteoclasts appear to form not from cells committed to macrophage differentiation, but from a discrete subpopulation of relatively mature bipotential or osteoclast-committed precursors which, in the absence of an osteoclast-inductive stimulus, become irreversibly lost to the osteoclast lineage.     

Differentiation and activity of human preosteoclasts on chitosan enriched calcium phosphate cement

Chitosan associated to various scaffolds has been shown to promote growth and mineral rich matrix deposition by osteoblasts in vitro, whereas its influence on osteoclast differentiation, which plays also a central role in bone remodeling, has never been described. The purpose of this study was to investigate the differentiation and activity of human preosteoclastic cells on calcium phosphate cement containing 2% chitosan (Cementek^®/chitosan) compared to the Cementek^® alone. Human primary osteoclast precursors were cultured directly on both biomaterials in the presence of rhM-CSF and rhRANK-L for 7 days. Using LIVE/DEAD fluorescent assay, tartrate-resistant acid phosphatase staining, scanning electron microscopy and quantitative RT-PCR, we demonstrated that incorporation of chitosan to Cementek^® does not affect the proliferation and adhesion of preosteoclasts but inhibits the formation of TRACP positive cells and prevents the osteoclastic resorption of the composite biomaterial compared to Cementek^® alone. This inhibitory effect of chitosan on osteoclast resorption activity should have important implications on bone formation and bone remodeling after in vivo implantation. Indeed, based on the positive results obtained in vivo by several investigators, one can suggest that this property of chitosan can be beneficial for bone regeneration.