Rabbit Bone Cells Research Articles

Assay of in vitro osteoclast activity on dentine, and synthetic calcium phosphate bone substitutes

Resorption of synthetic bone substitute materials is essential for the integration of these materials into the natural bone remodeling process. Osteoclast behavior in the presence of calcium phosphate bioceramics (CaPB) is partially understood, and a better understanding of the underlying mechanisms is expected to facilitate the development of new synthetic bone substitutes to improve bone regeneration. In the present study, our aim was to investigate osteoclastic resorption of various synthetic CaPB. We used neonatal total rabbit bone cells to generate osteoclasts. Osteoclast-generated resorption on dentine and multiple CaPB was investigated by quantifying the surface resorbed and measuring tartrate resistant acid phosphatase (TRAP) enzyme activity. In this study, we observed that osteoclastic cells responded in a different way to each substrate. Both dentine and CaPB were resorbed but the quantitative results for the surface resorbed and TRAP activity showed a specific response to each substrate and that increased mineral density seemed to inhibit osteoclast activity.

Flutamide does not mediate testosterone effects on BMP4 response in wild‐type rabbit bone cells

We previously reported that a treatment of testosterone (T) and BMP4 led to decreased proliferation and differentiation in rabbit calvarial bone cells. A primary sex difference was noted; males exhibited greater differentiation for suture bone and females for non‐suture bone. Those results suggested that T decreased response for BMP4 treated cells. We hypothesized that this effect is mediated through the androgen receptor (AR). Cells were treated with the AR blocker, flutamide (F), along with T and BMP4 to determine cellular response. Calvarial cells (suture or non‐suture) from New Zealand White rabbits (N=14) were stimulated with 50ng/ml BMP4 and T (10–16 dose), with or without F treatments (10‐8, ‐10 or ‐12 dose). Proliferation and differentiation were assessed after 7 days. Differences between F treatments by dose, sex, and bone‐type were analyzed. F did not effect BMP4 alone, p>.05. The dose response was biphasic, p<.05. Non‐suture bone was more susceptible to this F‐dependent decrease in proliferation p<.05, and male derived cells exhibited a greater decrease, p<.05. F did not effect differentiation p>.05, by dose, cell type, or sex, p>.05. These results suggest the effect T has on reducing BMP4‐induced differentiation may not be mediated through the AR pathway. An alternative pathway (e.g., MAPK) should be explored for the direct effect of T on normal calvarial bone cell response.Grant Funding Source: Internal

Controlled release of bisphosphonate from a calcium phosphate biomaterial inhibits osteoclastic resorption in vitro

Calcium phosphate biomaterials such as calcium deficient apatite (CDA) have been contemplated as carrier for delivery of bisphosphonate in bone tissues. In the present work, we have investigated the in vitro biological properties of Zoledronate-loaded CDA. CDA was loaded with zoledronate according to a previously described coating process. 31P MAS NMR spectra demonstrated the effective loading of zoledronate onto CDA. Using 14C labeled zoledronate, we then demonstrated the in vitro release of zoledronate from CDA. In a first set of experiments, we confirmed that Zoledronate reduced the number of TRAP-, vitronectin receptor-, and F-actin ring-positive cells as well as the resorption activity of osteoclasts obtained from a total rabbit bone cell culture. Interestingly, Zoledronate-loaded CDA and its extractive solutions decreased the osteoclastic resorption. Finally, zoledronate-loaded CDA did not affect the viability and alkaline phosphatase activity of primary osteoblastic cells. These data demonstrate that CDA is effective for loading and release of zoledronate. The released zoledronate inhibited osteoclastic resorption without affecting osteoblasts. Our findings therefore suggest that such a drug delivery system would allow an increase in the efficiency of bisphosphonates by being locally available. Further experiments are now required to evaluate the in vivo antiresorptive activity of this concept.

Identification of angiogenin as the osteoclastic bone resorption-inhibitory factor in bovine milk

We identified, for the first time, the factor responsible for inhibiting osteoclast-mediated bone resorption in the basic protein fraction of bovine milk (milk basic protein, MBP). The protein was purified by a combination of ion and gel column chromatography from MBP, based on its activity to prevent unfractionated rabbit bone cells from forming pits on dentine slices. It was found to have a molecular weight of 15 kDa on SDS-PAGE, and the sequence of the N-terminal 25 amino acid residues was identical to that of bovine angiogenin. The purified bovine angiogenin inhibited the pit-forming activity of both unfractionated bone cells and purified osteoclasts in a dose-dependent manner, and the inhibitory activity was markedly suppressed by treatment with anti-bovine angiogenin antibody. The inhibitory activity was confirmed in mice both in vitro and in vivo. Treatment of osteoclasts with bovine angiogenin resulted in an impairment of the formation F-actin ring and a reduction in the mRNA levels of TRAP and cathepsin K, both known to be essential for the bone resorption activity of osteoclasts. These results suggest that bovine angiogenin is the substance mainly responsible for the inhibitory effect of bovine milk on osteoclast-mediated bone resorption, and that it exerts its activity by acting directly on the osteoclasts.

Myeloma Cell-Osteoclast Interaction Enhances Angiogenesis Together with Bone Resorption: A Role for Vascular Endothelial Cell Growth Factor and Osteopontin

Similar to osteoclastogenesis, angiogenesis is enhanced in the bone marrow in myeloma in parallel with tumor progression. We showed previously that myeloma cells and osteoclasts are mutually stimulated to form a vicious cycle to lead to enhance both osteoclastogenesis and tumor growth. The present study was undertaken to clarify whether myeloma cell-osteoclast interaction enhances angiogenesis and whether there is any mutual stimulation between osteoclastogenesis and angiogenesis. Myeloma cells and monocyte-derived osteoclasts were cocultured, and angiogenic activity produced by the cocultures was assessed with in vitro vascular tubule formation assays and human umbilical vascular endothelial cell (HUVEC) migration and survival. Osteoclastogenic activity was determined with rabbit bone cell cultures on dentine slices. Myeloma cells and osteoclasts constitutively secrete proangiogenic factors, vascular endothelial growth factor (VEGF) and osteopontin, respectively. A cell-to-cell interaction between myeloma cells and osteoclasts potently enhanced vascular tubule formation. Blockade of both VEGF and osteopontin actions almost completely abrogated such vascular tubule formation as well as migration and survival of HUVECs enhanced by conditioned medium from cocultures of myeloma cells and osteoclasts. Furthermore, these factors in combination triggered the production of osteoclastogenic activity by HUVEC. Osteoclast-derived osteopontin and VEGF from myeloma cells cooperatively enhance angiogenesis and also induce osteoclastogenic activity by vascular endothelial cells. These observations suggest the presence of a close link between myeloma cells, osteoclasts, and vascular endothelial cells to form a vicious cycle between bone destruction, angiogenesis, and myeloma expansion.

Quantitative and reliable in vitro method combining scanning electron microscopy and image analysis for the screening of osteotropic modulators

The increased generation and up-regulated activity of bone resorbing cells (osteoclasts) play a part in the impairment of bone remodeling in many bone diseases. Numerous drugs (bisphosphonates, calcitonin, selective estrogen receptor modulators) have been proposed to inhibit this increased osteoclastic activity. In this report, we describe a pit resorption assay quantified by scanning electron microscopy coupled with image analysis. Total rabbit bone cells with large numbers of osteoclasts were cultured on dentin slices. The whole surface of the dentin slice was scanned and both the number of resorption pits and the total resorbed surface area were measured. Resorption pits appeared at 48 h and increased gradually up to 96 h. Despite the observation of a strong correlation between the total resorption area and the number of pits, we suggest that area measurement is the most relevant marker for osteoclastic activity. Osteotropic factors stimulating or inhibiting osteoclastic activity were used to test the variations in resorption activity as measured with our method. This reproducible and sensitive quantitative method is a valuable tool for screening for osteoclastic inhibitors and, more generally, for investigating bone modulators.

Angiogenesis and Osteoclastogenesis Are Mutually Stimulated in Myeloma: A Role for VEGF and Osteopontin.

Angiogenesis and osteoclastogenesis occur in the bone marrow as myeloma (MM) progresses. Suppression of osteoclastogenesis by bisphosphonates reduced tumor burden as well as vascular density in the BM in myeloma animal models(Croucher et al. 2003). These evidences suggest a link between angiogenesis, osteoclastogenesis and MM tumor expansion which orchestrates a network of co-operative cellular interactions. We have previously demonstrated that a cell-cell interaction between osteoclasts (OCs) and MM cells potently enhanced MM cell growth and survival as well as angiogenesis. Such angiogenic effects were almost completely abrogated by antibodies against VEGF and osteopontin (OPN) in combination, suggesting VEGF and OPN as predominant angiogenic factors produced in MM bone marrow microenvironment. Angiogenesis is a multi-step process including migration, proliferation and sprouting in response to angiogenic stimuli. The impact of these factors on these angiogenic processes as well as on osteoclastogenesis by vascular cells is largely unknown. We therefore investigated roles for OPN and VEGF in combination in different steps in angiogenesis and in induction of osteoclastogenic activity by vascular endothelial cells. Conditioned media (CM) from co-cultures of peripheral blood mononuclear cell-derived OCs and RPMI8226 cells at 10 % enhanced survival of HUVEC under factor-deprived conditions 3-fold compared to control culture media, and transmigration of HUVEC 1.5-fold. Antibodies against VEGF and OPN each alone partially and both in combination mostly abrogated these effects of the CM. Consistently, VEGF and OPN co-operatively promoted survival and transmigration of HUVEC as well as angiogenesis in vitro. In order to further clarify a role for VEGF and OPN in a link between angiogenesis and osteoclastogenesis, we next examined osteoclastogenic activity produced by HUVEC upon VEGF and OPN stimulation using rabbit bone cell cultures on dentine slices. CM were collected from HUVEC cultured on either OPN- or type 1 collagen-coated wells in the presence or absence of VEGF. CM from OPN-coated wells with VEGF enhanced OC formation most potently(2.8-fold) compared to control culture media, while CM from OPN-coated wells without VEGF, type 1 collagen-coated wells with VEGF and type 1 collagen-coated wells without VEGF enhanced 2-, 1.8- and 1.2-fold, respectively, indicating co-operative actions of VEGF and OPN. Furthermore, such VEGF and OPN treatment was found to induce osteoclastogenic IL-8 by HUVEC most apparently among 125 different cytokines determined by cytokine antibody arrays. These results suggest that VEGF and OPN derived from MM cells and OCs cause angiogenesis as well as osteoclastogenesis at least in part through the enhancement of migration and survival and the induction of osteoclastogenic activity of vascular endothelial cells, thereby mutually stimulating angiogenesis and osteoclastogenesis. Therefore, VEGF and OPN may be a therapeutic target against MM bone marrow environment to ameliorate bone destruction along with MM expansion.

Bone remodelling and tumour grade modifications induced by interactions between bone and swarm rat chondrosarcoma.

Chondrosarcoma is currently defined as a malignant cartilage tumour arising de novo or within a pre-existing benign cartilage tumour. Chondrosarcoma can be surgically resected, but all grades have significant rates of local recurrence. The purpose of the present study was to develop an animal intraosseous chondrosarcoma model simulating the progression of human chondrosarcoma and elucidating its behaviour and biology. An intraosseous Swarm rat model was designed to assess interactions between bone and chondrosarcoma. A comparison of tumour grading was carried out according to transplantation site. The effects of chondrosarcoma cells (SRC cells) on the mineralisation capacities of osteoblasts and on osteoclast differentiation were studied in relation to modifications observed in vivo at the cellular level. Transplantation of Swarm rat chondrosarcoma within bone marrow or contiguous to induced periosteal lesions led to extensive bone remodelling with trabecular bone rarefaction and periosteal apposition. Transplantation in close contact to bone but without any periosteal lesion had no effect on bone, suggesting that bone healing factors interact with tumour development. With the intramedullary model, the development of tumours of different grade confirms that bone environment is an important factor in malignancy. A decrease of bone nodule formation was noted after cocultures of SRC cells with rat bone marrow, but there was no modification of osteoclast differentiation after cultures of total rabbit bone cells with SRC cells. These data reveal the importance of interactions between bone environment and tumour in inducing bone remodelling and variations in tumour malignancy.

Role for macrophage inflammatory protein (MIP)-1α and MIP-1β in the development of osteolytic lesions in multiple myeloma

Multiple myeloma (MM) cells cause devastating bone destruction by activating osteoclasts in the bone marrow milieu. However, the mechanism of enhanced bone resorption in patients with myeloma is poorly understood. In the present study, we investigated a role of C-C chemokines, macrophage inflammatory protein (MIP)–1α and MIP-1β, in MM cell-induced osteolysis. These chemokines were produced and secreted by a majority of MM cell lines as well as primary MM cells from patients. Secretion of MIP-1α and MIP-1β correlated well with the ability of myeloma cells to enhance osteoclastic bone resorption both in vitro and in vivo as well as in MM patients. In osteoclastogenic cultures of rabbit bone cells, cocultures with myeloma cells as well as addition of myeloma cell-conditioned media enhanced both formation of osteoclastlike cells and resorption pits to an extent comparable to the effect of recombinant MIP-1α and MIP-1β. Importantly, these effects were mostly reversed by neutralizing antibodies against MIP-1α and MIP-1β, or their cognate receptor, CCR5, suggesting critical roles of these chemokines. We also demonstrated that stromal cells express CCR5 and that recombinant MIP-1α and MIP-1β induce expression of receptor activator of nuclear factor-κB (RANK) ligand by stromal cells, thereby stimulating osteoclast differentiation of preosteoclastic cells. These results suggest that MIP-1α and MIP-1β may be major osteoclast-activating factors produced by MM cells.

Role for macrophage inflammatory protein (MIP)-1α and MIP-1β in the development of osteolytic lesions in multiple myeloma

AbstractMultiple myeloma (MM) cells cause devastating bone destruction by activating osteoclasts in the bone marrow milieu. However, the mechanism of enhanced bone resorption in patients with myeloma is poorly understood. In the present study, we investigated a role of C-C chemokines, macrophage inflammatory protein (MIP)–1α and MIP-1β, in MM cell-induced osteolysis. These chemokines were produced and secreted by a majority of MM cell lines as well as primary MM cells from patients. Secretion of MIP-1α and MIP-1β correlated well with the ability of myeloma cells to enhance osteoclastic bone resorption both in vitro and in vivo as well as in MM patients. In osteoclastogenic cultures of rabbit bone cells, cocultures with myeloma cells as well as addition of myeloma cell-conditioned media enhanced both formation of osteoclastlike cells and resorption pits to an extent comparable to the effect of recombinant MIP-1α and MIP-1β. Importantly, these effects were mostly reversed by neutralizing antibodies against MIP-1α and MIP-1β, or their cognate receptor, CCR5, suggesting critical roles of these chemokines. We also demonstrated that stromal cells express CCR5 and that recombinant MIP-1α and MIP-1β induce expression of receptor activator of nuclear factor-κB (RANK) ligand by stromal cells, thereby stimulating osteoclast differentiation of preosteoclastic cells. These results suggest that MIP-1α and MIP-1β may be major osteoclast-activating factors produced by MM cells.

Potential synergies between matrix proteins and soluble factors on resorption and proteinase activities of rabbit bone cells.

Human growth hormone (GH) has recently been found to stimulate osteoclastic resorption, cysteine-proteinase and metalloproteinase activities (MMP-2 and MMP-9) in vitro via insulin-like growth factor-I (IGF-I) produced by stromal cells. The present study investigated the effects of two extracellular matrix components (vitronectin and type-I collagen) on hGH- and hIGF-1-stimulated osteoclastic resorption and proteinase activities in a rabbit bone cell model. After 4 days of rabbit bone cell culture on dentin slices with vitronectin coating, hGH and hIGF-1 stimulated bone resorption and hIGF-1 upmodulated cysteine-proteinase activities. MMP-2 expression (but not resorption, cathepsin or MMP-9 activities) was upmodulated by hGH and hIGF-1 on dentin slices coated with type I collagen as compared to those without coating. Then, vitronectin was synergistic with hIGF-1 in the regulation of cysteine-proteinase production whereas collagen showed synergy with hGH and hIGF-1 in the regulation of MMP-2 production. Anti-alphavbeta3 totally abolished the effects of hGH and hIGF-1 on metalloproteinase release, but had no influence on cathepsin release. The results suggest that cysteine-proteinase modulation is not mediated by alphavbeta3 integrin (strongly expressed on osteoclastic surface) whereas the resorption process and metalloproteinase modulation are clearly mediated by this integrin. Our finding about the collagen coating also suggests that hGH- and hIGF-1-stimulated MMP-2 activity are mediated, along with alphavbeta3 integrin, by another adhesion molecule.

Human Growth Hormone Stimulates Proteinase Activities of Rabbit Bone Cells via IGF-I

Human growth hormone (hGH) and human insulin-like growth factor-I (hIGF-I) are known to have a marked influence on osteoclastic formation and bone resorption in an unfractionated rabbit bone cell model. This study investigated the effects of both of these factors on the induction of cysteine-proteinases and matrix metalloproteinase-2 (MMP-2) and MMP-9. After 4 days of rabbit bone cell culture, hGH and hIGF-I significantly modulated cathepsin, MMP-9 (latent form) and MMP-2 (active form) activities. Similar studies were performed in the presence of parathyroid hormone (hPTH). hPTH increased MMP-2 and MMP-9 activities whereas it had no effect on the production of cathepsins by bone cells. When neutralizing anti-hIGF-1 antiserum was added to the culture, the stimulatory effects of hGH were totally abolished, indicating that hGH-modulated cathepsin and metalloproteinase activities were partly mediated by local hIGF-I secretion. Cysteine-proteinase activities released by purified osteoclasts were very low and were not modulated by hGH and h-IGF-I. However, hIGF-I but not hGH increased MMP-2 and MMP-9 activities released by purified osteoclasts. It may be concluded that hGH markedly stimulates the expression of proteinases in total rabbit bone cells via local hIGF-I production by stromal cells. Cysteine-proteinase activities are mainly produced by non-osteoclastic cells, while MMP-2 and MMP-9 modulated by hIGF-I are mainly expressed by osteoclastic cells.

Growth Hormone Stimulatory Effects on Osteoclastic Resorption Are Partly Mediated by Insulin-like Growth Factor I: An In Vitro Study

This study investigated the possible role in vitro of insulin-like growth factor I (IGF-I) as a mediator of the effects of growth hormone (GH) on osteoclastic resorption in an unfractioned rabbit bone cell model. After 4 days of rabbit bone cell culture, human GH (hGH) (50 ng/mL) and human IGF-I (hIGF-I) (50 ng/mL) significantly increased the formation of osteoclast-like cells with a lower level than parathyroid hormone (50 ng/mL) or VD3 (10 −8 mol/L). As well as parathyroid hormone and 1-α,25-dihydroxyvitamin D 3, addition of hGH (1, 10, and 50 ng/mL) and hIGF-I (1, 10, and 50 ng/mL) stimulated the resorption activity of osteoclasts in terms of the percentage of dentin slice surface resorbed, number of lacunae per surface unit, and mean area of lacunae as compared to the control. When neutralizing antiserum against hIGF-I (4 μg/mL) was added at the start of culture, the stimulatory effects of hIGF-I and hGH on osteoclastic resorption activity were totally abolished. These results indicate that the effects of GH stimulation on osteoclastic resorption in vitro are mediated via local IGF-I secretion by stromal cells such as osteoblasts. As IGF-I receptors have recently been reported on rabbit osteoclasts, a direct action of IGF-I on mature osteoclasts could be envisaged. Further experiments will be required to determine the real level of IGF-I implicated in the stimulation of bone osteoclastic resorption.

Osteoclastic resorption of biphasic calcium phosphate ceramic in vitro.

Neonatal rabbit bone cells were cultured for 1 and 4 days on biphasic calcium phosphate ceramic specimens to study the osteoclastic resorption of the ceramic. Scanning electron microscopic studies after removal of stromal cells with pronase E and EDTA revealed many osteoclast-associated lacunae on the ceramic surface. The degraded crystals inside the lacunae appeared to have been dissolved by acids. Cellular degradation of the ceramic was clearly due to the extracellular process characteristic of osteoclastic resorption.

Osteoclastic resorption of calcium phosphate ceramics with different hydroxyapatite/β-tricalcium phosphate ratios

To study the influence of calcium phosphate ceramic solubility on osteoclastic resorption, neonatal rabbit bone cells were cultured for 2 days on hydroxyapatite (HA), β-tricalcium phosphate (β-TCP) and two types of biphasic calcium phosphate (BCP) with HA/β-TCP ratios of 25 75 and 75 25 . Solubility was regulated by varying the ratio of less-soluble HA and more-soluble β-TCP. After removal of stromal cells by pronase E treatment, ceramic surfaces were observed by scanning electron microscopy. Osteoclasts resorbed BCP most extensively, with an HA/β-TCP ratio of 25 75 , producing typical lobulated, zig-zag track-like resorption lacunae. On pure β-TCP, which had the highest solubility in acid, osteoclasts formed smaller discontinuous island-like lacunae. The resorption pattern may have been modified by the large number of calcium ions released into the acidic microenvironment at the osteoclast-ceramic interface. No resorption lacunae were found on the other specimens. The extent of osteoclastic resorption of calcium phosphate ceramics might, to a certain degree, be proportional to solubility, although this was not the case when solubility was very high. It would appear that ceramic solubility influences osteoclast resorption activity.

Isolation of highly enriched rabbit osteoclasts from collagen gels: A new assay system for bone-resorbing activity of mature osteoclasts

When unfractionated rabbit bone cells were seeded and incubated on a collagen gel, tartrate-resistant acid phosphatase (TRAP)-positive multinucleate cells (MNCs) adhered to the surface of the collagen gel more readily than other types of cells. Based on the differences in adherence of the MNCs onto the collagen gel, we developed a new method for the isolation of functionally mature osteoclasts with high purity. Sequential treatments with pronase E/EDTA and collagenase at a low concentration (0.01%) released most of the nonosteoclasts from the gel surface, whereas only MNCs still remained on the surface. After washing off the released cells, we could harvest a cell population highly enriched for osteoclasts from the collagen gel by a second digestion with collagenase. The yield of TRAP-positive MNCs was 40000–50000 cells per long bones from one rabbit. The purity of the TRAP-positive MNCs was reproducibly more than 95%. These cells, cultured on dentine slices, showed typical ultrastructural features of osteoclasts, i.e., a highly developed ruffled border and clear zone, and they excavated the dentine to form pits. In addition, the isolated MNCs showed specific binding to calcitonin. The dentine-resorbing activity appeared as early as 2h after plating and reached a plateau at 24h. The pit area increased in direct proportion to the number of osteoclasts plated. Calcitonin inhibited the dentine-resorbing activity, but parathyroid hormone or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] did not have any effect on it. Though these cells exhibited dentine-resorbing activity even in the absence of any support from stromal cells, this activity was enhanced by rabbit stromal cells in response to 1,25(OH)2D3. In addition, these stromal cells elongated the life span of the isolated osteoclasts without 1,25(OH)2D3. To assess the functions of authentic osteoclasts, we can now culture highly purified and functional osteoclasts under the desired conditions and in controlled cell numbers. This isolation method is also applicable to numerous molecular studies.

Quantification of the collagenolytic activity of isolated osteoclasts by enzyme-linked immunosorbent assay.

Difficulties in the geometrical definition and measurement of resorption pits is a major problem for the quantitative analysis of bone resorption by isolated osteoclasts cultured on bone or dentin substrates. In this study we developed an enzyme-linked immunosorbent assay (ELISA) for quantification of bone resorption in vitro, which specifically quantifies type I collagen fragments released into the culture medium by the resorptive action of bone cells cultured on slices of bone or dentin. A consistently high correlation between the formation of resorption pits and the release of antigenic collagen fragments was observed for isolated rabbit osteoclasts seeded at various densities and cultured for various periods on bovine, elephant, and human substrates. In a further support of the osteoclastic nature of the collagenolytic effects, a high consistency between pit formation and collagenolysis was also observed when the rabbit bone cells were cultured in the presence of very differently acting but typical inhibitors of pit formation, i.e., the carbonic anhydrase inhibitor acetazolamide, the cysteine proteinase inhibitor epoxysuccinyl-L-leucylamido-(4-guanodino)butane (E-64), the phosphatidyl-inositol 3-kinase inhibitor wortmannin, and the bisphosphonate ibandronate (BM 21.0955). In conclusion, the ELISA represents a simple, precise, and objective way to dynamically monitor bone resorption in vitro through quantification of the collagenolytic activity of isolated osteoclasts.

Osteoclastic resorption of apatite formed on apatite‐ and wollastonite‐containing glass‐ceramic by a simulated body fluid

We immersed mirror-polished apatite- and wollastonite-containing glass-ceramic (A-W GC) disks in a simulated body fluid (SBF) for 5 days to form bonelike apatite on their surface. Neonatal rabbit bone cells were cultured on these or on plain A-W GC disks for 10, 24, and 48 h. We observed the substrates by scanning electron microscopy after treating them with pronase E plus EDTA to remove all cells except osteoclasts. Osteoclasts with a non-motile appearance formed no lacunae on the plain A-W GC, whereas on the bonelike apatite formed on A-W GC by the SBF, actively moving osteoclasts made many tracklike resorption lacunae. These were evident even after 10 h of culture and became more extensive after longer culture periods. The bonelike apatite was therefore a more suitable medium than plain A-W GC for maintaining osteoclast activity. This study demonstrated in vitro osteoclastic resorption of bonelike apatite formed on A-W GC by an SBF. It suggests that the apatite layer, through which a surface-active ceramic bonds to bone in vivo, can be resorbed by osteoclasts and subjected to bone remodeling.