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.     

Bone resorption by isolated human osteoclasts in vitro: effects of calcitonin

Human osteoclasts were isolated from 12- to 17-week-old fetal tissue and from transiliac crest bone biopsies for an in vitro study of their biology. A hypodermic needle was used to flush either the fetal long bones or the trabeculae of the iliac crest bone biopsy with tissue culture medium and the resulting cell suspension sedimented briefly either onto the surface of plastic tissue culture dishes, for time-lapse microcinematography, or onto slices of devitalized bovine cortical bone for quantitative assay of bone resorption. The osteoclasts were motile, tartrate-resistant acid phosphatase positive and capable of excavating pits in slices of devitalized bovine cortical bone. Human calcitonin, at doses of 1 ng/ml and 1 microgram/ml, caused a 70% inhibition of bone resorption by human fetal osteoclasts over a 24 h period but had no apparent effect on the morphology or motility of either fetal or adult osteoclasts.     

Tissue inhibitor of metalloproteinases 1 and 2 directly stimulate the bone-resorbing activity of isolated mature osteoclasts.

Tissue inhibitor metalloproteinases 1 (TIMP-1) and 2 have been reported to inhibit bone resorption. However, here, we report the direct action of both TIMP-1 and TIMP-2 on isolated rabbit mature osteoclasts to stimulate their bone-resorbing activity at significantly lower concentrations (approximately ng/ml) than those (approximately microg/ml) required for the inhibition of bone resorption. The cell population used in this study consisted of a mature osteoclast population with >95% purity. TIMP-1 (approximately 50 ng/ml) and TIMP-2 (approximately 8-10 ng/ml) increased the pit area excavated by the isolated mature osteoclasts. The stimulatory effects of TIMPs were abolished by simultaneous addition of anti-TIMP antibodies. At higher concentrations, the stimulation of bone resorption decreased reversely to the control level. The magnitude of the stimulatory effect of TIMP-2 was more than that of TIMP-1. Metalloproteinase inhibitors such as BE16627B and R94138 could not replace TIMPs with respect to the bone-resorbing activity, suggesting that the osteoclast-stimulating activity of TIMPs was independent of the inhibitory activity on matrix metalloproteinases (MMPs). TIMPs stimulated tyrosine phosphorylation of cellular proteins in the isolated mature osteoclasts. Both herbimycin A, an inhibitor of tyrosine kinases, and PD98059 and U0126, inhibitors of mitogen-activated protein kinase (MAPK), completely blocked the TIMP-induced stimulation of osteoclastic bone-resorbing activity. On the plasma membrane of osteoclasts, some TIMP-2-binding proteins were detected by a cross-linking experiment. These findings show that TIMPs directly stimulate the bone-resorbing activity of isolated mature osteoclasts at their physiological concentrations and that the stimulatory action of TIMPs is likely to be independent of their activities as inhibitors of MMPs.     

In vitro resorptive activity of isolated chick osteoclasts: effects of carbonic anhydrase inhibition

A potent inhibitor of carbonic anhydrase, 5-[3-hydroxybenzoyl]thiophene-2-sulfonamide (HTS), was shown to cause a 37% reduction in the area of resorption pits formed by isolated chick osteoclasts when used at a dose of 10(-7) M. HTS at doses of 10(-9) and 10(-7) M was also effective in reducing acid formation by the osteoclasts (14 and 36%, respectively). Additionally, the effect of HTS was found to be readily reversed by removing the agent, showing that it does not exert a toxic effect on the cells. This study indicates that the inhibitory effect of HTS on bone resorption is at the level of the acid-forming mechanism in osteoclasts and supports the view that carbonic anhydrase has a central role in the process.     

重组人骨保护素对体外培养兔破骨细胞的影响

目的观察重组人骨保护素(recombinanthumanosteoprotegerin,rhOPG)对体外培养兔破骨细胞(osteoclast,OC)生存和功能的影响。方法将rhOPG用于干预从新生兔分离出的OC,于干预后1、3、7d取细胞玻片、皮质骨片进行HE、Giemsa、甲苯胺蓝染色,并观察抗酒石酸酸性磷酸酶(TRAP)染色阳性细胞和骨片吸收陷窝,扫描电镜进一步观察吸收陷窝形态。结果分离培养的兔OC多核形态明显;rhOPG对TRAP阳性OC生存的影响,1、3d结果差别不明显,7d的结果差异有显著性(P<0.05);rhOPG能够明显地抑制OC在骨片上形成吸收陷窝,三个时点计数结果差异均有显著性(P<0.01)。结论rhOPG可明显地抑制体外培养兔OC的骨吸收功能。     

两种不同降钙素对体外培养破骨细胞影响的实验研究

目的 观察不同浓度鳗鱼降钙素与鲑鱼降钙素对体外分离培养的破骨细胞数量和活力的影响.方法 从新生幼兔四肢长骨中机械分离出破骨细胞,分别接种于盖玻片及骨薄片上,加入两种不同种类的降钙素,抗酒石酸酸性磷酸酶(TRAP)染色观察盖玻片上破骨细胞的数量和形态,JD801图像分析软件分析骨片上骨吸收陷窝的面积.结果 两种降钙素的各个浓度组(分别是10-12 mol/L、10-10 mol/L、10-8 mol/L)均对贴壁生长的破骨细胞数量有明显抑制作用,并呈剂量相关性.骨吸收陷窝面积分析结果显示,两类降钙素均对破骨细胞吸收功能有明显抑制作用,呈剂量相关性.无论是盖玻片培养还是骨薄片培养,益钙宁和密盖息各相同浓度组间两两对照比较差异均无显著性 (P>0.05).结论 两种不同种类降钙素对体外培养的破骨细胞数量和骨吸收活性的抑制能力相仿.     

Effect of retinoic acid on the resorptive activity of chick osteoclasts in vitro.

Mixed cell suspensions mechanically isolated from the long-bones of day 20 prehatch chicks were cultured for 24 h on bone and sperm whale dentine slices in the presence of 0, 10, 100, and 1000 nM retinoic acid (RA). Significant inhibitions in the numbers of discrete lacunae resorbed per dentine slice, and in the ratio of lacunae per tartrate-resistant acid phosphatase-positive multinuclear cell were observed with all concentrations of RA studied. Semi-automated, 3-dimensional analysis of 733 pits was performed on one series of experiments using a tandem scanning (confocal) microscope, interfaced to an image analyzing computer. The majority of lacunae were small and unilocular; the plan areas of 90% of control pits were below 500 microns 2. Small but statistically significant increases in lacunar areas, but not mean or maximum depths or volumes, were observed in the presence of 10 and 100 nM RA; however, these changes were much smaller than the magnitude of the decrease in pit numbers. Thus, the overall effect of RA in this system was inhibitory. Our findings contrast with the well known stimulatory action of retinoids on bone resorption both in vivo and in organ culture, but may parallel the inhibitory effects of prostaglandins observed in disaggregated osteoclast systems.     

Visualization of bisphosphonate-induced caspase-3 activity in apoptotic osteoclasts in vitro

Bisphosphonates inhibit osteoclast-mediated bone resorption by mechanisms that have only recently become clear. Whereas nitrogen-containing bisphosphonates affect osteoclast function by preventing protein prenylation (especially geranylgeranylation), non-nitrogen-containing bisphosphonates have a different molecular mechanism of action. In this study, we demonstrate that nitrogen-containing bisphosphonates (risedronate, alendronate, pamidronate, and zoledronic acid) and non-nitrogen-containing bisphosphonates (clodronate and etidronate) cause apoptosis of rabbit osteoclasts, human osteoclastoma-derived osteoclasts, and human osteoclast-like cells generated in cultures of bone marrow in vitro. Osteoclast apoptosis was shown to involve characteristic morphological changes, loss of mitochondrial membrane potential, and the activation of caspase-3-like proteases capable of cleaving peptide substrates with the sequence DEVD. Caspase-3-like activity could be visualized in unfixed, dying osteoclasts and osteoclast-like cells using a cell-permeable, fluorogenic substrate. Bisphosphonate-induced osteoclast apoptosis was dependent on caspase activation, because apoptosis resulting from alendronate, clodronate, or zoledronic acid treatment was suppressed by zVAD-fmk, a broad-range caspase inhibitor, or by SB-281277, a specific isatin sulfonamide inhibitor of caspase-3/-7. Furthermore, caspase-3 (but not caspase-6 or caspase-7) activity could be detected and quantitated in lysates from purified rabbit osteoclasts, whereas the p17 fragment of active caspase-3 could be detected in human osteoclast-like cells by immunofluorescence staining. Caspase-3, therefore, appears to be the major effector caspase activated in osteoclasts by bisphosphonate treatment. Caspase activation and apoptosis induced by nitrogen-containing bisphosphonates are likely to be the consequence of the loss of geranylgeranylated rather than farnesylated proteins, because the ability to cause apoptosis and caspase activation was mimicked by GGTI-298, a specific inhibitor of protein geranylgeranylation, whereas FTI-277, a specific inhibitor of protein farnesylation, had no effect on apoptosis or caspase activity.     

Platelets increase while serum reduces the differentiation and activity of osteoclasts in vitro

Platelets modulate formation of osteoclasts and osteoblasts, but research with different preparations of platelets remains inconclusive. Here, we assessed whether serum components modulate the effect of platelet preparations. In murine bone marrow cultures, osteoclastogenesis was investigated in the presence of platelet‐released supernatant (PRS), serum containing PRS (SC‐PRS), and serum. Osteoclastogenesis was quantified by the numbers of tartrate‐resistant acid phosphatase (TRAP)‐positive multinucleated cells, TRAP activity and resorption assays. Also human osteoclastogenesis assays were performed. Viability and proliferation were tested by MTT and ³[H]thymidine incorporation assays, respectively. Osteoblastogenesis was assessed by histochemical staining for alkaline phosphatase‐of murine bone marrow cultures and human MG63 cells. We found PRS to increase the number of TRAP⁺ multinucleated cells in the early phase and TRAP activity in the later phase of osteoclastogenesis. SC‐PRS and serum decreased the number and activity of TRAP⁺ multinucleated cells. Both serum containing preparations reduced viability and proliferation of hematopoietic progenitors. PRS decreased the numbers of alkaline phosphatase‐positive colonies while SC‐PRS and serum increased osteoblastmarkers in MG63. Proliferation of MG63 was stimulated by all preparations. These results show that activated platelets support osteoclastogenesis, while platelet preparations that contain serum components decrease osteoclastogenesis and increase osteoblastogenesis in vitro, suggesting that serum components modulate the effects of platelets on osteoclastogenesis and osteoblastogenesis. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1561–1569, 2013     

The effects of extracellular pH on bone resorption by avian osteoclasts in vitro

It has previously been reported that low extracellular pH stimulates the excavation of resorption lacunae by rodent osteoclasts in vitro. Using avian bone cells in a similar in vitro assay we have demonstrated that osteoclast activity is optimal at pH 7.20-7.40 and is inhibited at extremes of pH (less than 7.10 and greater than 7.60). Over the first 24 h of incubation at low pH there may be an increase in osteoclastic resorption but to a lesser extent than that reported for rodent cells. However, after 24-30 h in culture there is little or no further increase in bone resorption, presumably due to a cytotoxic effect of low pH acting either on the osteoclast directly or via nonosteoclastic bone cells. In contrast to a previous report, in which preincubation of wafers for 24 h had no effect on bone resorption, we found that preincubation of bone substrates at pH 6.50 for longer periods enhances subsequent resorption at pH 7.20.     

依降钙素对破骨细胞的作用观察

目的 观察了国产降钙素-依降钙素对破骨细胞体外骨吸收功能的影响。并与进口降钙素-益钙宁的作用比较，方法 由10日龄幼兔四肢长骨机械分离破骨细胞于199培养液（含10%NCS 5?S），分别接种于象牙薄切片和培养板，置37℃，5%CO2培养箱中培养，细胞骨架观察采用荧光标记法（罗达明标记的鬼笔环肽），骨片吸收陷窝计数采用甲苯胺蓝染色法，益钙宁为阳性对照，等量培养液为阴性对照，结果 依降钙素组破骨细胞F-actin聚集，变短，骨片培养3d，吸收陷窝计数的结果显示，10^-10mol/L以上浓度依降钙素对破骨细胞吸收功能有明显抑制作用，并呈剂量相关性，10^-1mol/L时的抑制作用高达90%；依降钙素和益钙宁的抑制作用呈高度相关（r=0.99）。结论 依降钙素具有明显的抑制体外培养破骨细胞骨吸收活性的作用，与益钙宁的作用相仿。     

Biological activity of chicken calcitonin: effects on neonatal rat and embryonic chick osteoclasts

Chicken calcitonin (cCT) has recently been synthesized according to nucleotide sequence data. We have investigated the in vitro effects of this hormone on the activity of disaggregated, neonatal rat and embryonic chick osteoclasts. While synthetic cCT inhibited bone resorption by neonatal rat osteoclasts at concentrations as low as 0.1 pg/ml, it failed to produce a significant reduction in bone resorption by embryonic chick osteoclasts, even at concentrations as high as 1 microgram/ml. Similarly, cCT at 1 pg/ml reproducibly produced the rapid and dramatic inhibition of rat osteoclast motility characteristic of other calcitonins, as judged by time-lapse video recording, but did not impair the motile behavior of chick osteoclasts at concentrations up to a million-fold higher. Previous studies showing that embryonic chick osteoclasts were unresponsive to synthetic salmon calcitonin left open the possibility that the native hormone was required to produce an inhibitory effect. We conclude that the osteoclast is not a target cell for calcitonin in the embryonic chick; further studies will be required to define a role, if any, for this potent but enigmatic hormone in birds.     

淫羊藿对体外培养破骨细胞的影响

目的 观察淫羊藿提取物对体外培养破骨细胞的影响。方法 以出生２ｄ的Ｗｉｓｔａｒ大鼠长骨为来源,培养已成熟生长的破骨细胞;以出生７￣９周的雄性Ｗｉｓｔａｒ大鼠骨髓为来源,在１．２５－（ＯＨ）２ＶｉｔＤ３作用下,培养出骨髓诱导而产生的破骨细胞。两种细胞培养过程中均分别加入２００μｇ／Ｌ的淫羊藿提取物。结果 皮质骨内层可培养出多核,体积大,抗酒石酸酸性磷酸酶（ＴＲＡＰ）染色阳性的破骨细胞,淫羊藿提取物对     

Generation of bone-resorbing osteoclasts from B220+ cells: its role in accelerated osteoclastogenesis due to estrogen deficiency.

Estrogen deficiency stimulates both osteoclastic bone resorption and pre-B lymphopoiesis, the interrelationships between which remain unknown. To investigate the involvement of an increase in the number of B220+ cells in accelerated osteoclastogenesis after estrogen deficiency, we first examined whether ovariectomy (OVX) increased the frequency of clonogenic osteoclast precursors in bone marrow. The results were that after OVX, the frequency of clonogenic osteoclast precursors is increased in bone marrow, suggesting that accumulated osteoclast precursors contribute to accelerated osteoclastogenesis. Further, we found that cocultures of B220+ cells purified from bone marrow cells and stromal ST2 cells in the presence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] gave rise to osteoclasts that can resorb bone and express calcitonin receptors. When the frequencies of clonogenic osteoclast precursors in the purified B220+ and B220- cell fractions were compared, it was found that the fractions gave rise to osteoclasts at similar frequencies, which rules out the possibility of cross-contamination and suggests that the two fractions contain comparable numbers of osteoclast precursors. Furthermore, we identified cells that are positive for both tartrate-resistant acid phosphatase (TRAP) and B220, not only in cocultures of B220+ and ST2 cells, but also in freshly isolated unfractionated bone cells. Therefore, it is concluded that at least a subfraction of B220+ cells are capable of generating osteoclasts and that the increase in the number of B220+ cells caused by estrogen deficiency may contribute to accelerated bone resorption by this novel osteoclastogenesis pathway.     

二甲双胍对破骨细胞体外分化的影响

目的 探讨二甲双胍对破骨细胞体外分化的影响及其可能机制.方法 采用RANKL诱导鼠巨噬细胞系Raw264.7细胞破骨分化模型,给予不同浓度的二甲双胍(400 μmol/L、800 μmol/L和1000μmol/L)和雷帕霉素(100 hmol/L)处理后,通过抗酒石酸酸性磷酸酶(tartrate-resistant Acid Phosphatase,TRAP)染色和破骨细胞骨架结构荧光染色观察破骨细胞数量,骨吸收培养板观察骨陷窝面积,RT-PCR技术检测破骨细胞特异性基因TRAP、组织蛋白酶K、降钙素受体和金属基质蛋白酶-9的表达,ELISA法检测肿瘤坏死因子-α(tumor necrosis factor,TNF-α)表达水平,Western-b1ot检测c-Fos蛋白以及哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin complex l,mTORC1)信号通路下游底物S6K1 Thr389、S6 Ser235/236、4EBP1 Thr37/46的表达及磷酸化水平.结果 二甲双胍和雷帕霉素均可使RANKL诱导的破骨细胞数量减少,抑制破骨细胞特异性基因的表达、抑制TNF-α、c-Fos蛋白以及mTORC1信号通路下游底物S6K1 Thr389、S6 Ser235/236、4E-BP1 Thr37/46的磷酸化,且二甲双胍的抑制作用具有浓度依赖性.结论 二甲双胍可抑制RANKL诱导的破骨前体细胞分化,其机制可能与抑制TNF-α和c-Fos蛋白的生成,以及抑制mTORC1信号通路激活有关.

Abstract：

Objective To investigate the effects of mefformin on the differentiation of osteoclastas well as relative mechanism.Methods Raw264.7 cells from the murine macrophage cell line was used.Receptor activator of NF-κB ligand (RANKL) was used to stimulate osteoclast differentiation from Raw264.7 cells.Osteoclast differentiation was assessed by tartrate-resistant acid phosphatase (TRAP) and actin fluorescence staining and counting the TRAP-positive cells after exposure to different concentrations of mefformin (0 μmol/L,400 μmol/L,800 μmol/L and 1000 μmol/L) or rapamicin (100 nmol/L) in the presence of 50 ng/ml RANKL for 5 days.Bone-resorbing activity was evaluated by BD BioCoatTM OsteologicTM Bone Cell Culture System.The expression of osteoclast-specific genes like TRAP,capthesin K,calcitonin receptor (CTR) and matrix metalloproteinase (MMP-9) was evaluated by RT-PCR.The expression of tumor necrosis factor-α(TNF-ct) S6K1Thr389,S6 Ser235/236,4E-BP1Thr37/46 and c-Fos protein was evaluated by ELISA kit and Western blot analysis,respectively.Results Mefformin dose-dependently inhibited RANKL-stimulated osteoclasts differentiation in Raw264.7 cell culture,as manifested by decrease of TRAP-positive multinucleated cells and pit erosion area,down-regulation of TRAP,cathepsin K,CTR and MMP-9 mRNA and reduction of TNF-α and c-Fos protein expression.Further study revealed that RANKL activated mTOR complex 1(mTORC1) signaling,while mefformin impaired RANKL-stimulated mTORC1 signaling.Rapamycin,an mTORCl-specific inhibitor and immunosuppressive macrolides could also prevent RANKL-induced osteoclast differentiation and bone resorption in vitro.Conclusion Mefformin inhibits osteoclastogenesis in vitro,which may due to reduction of TNF-α and c-Fos protein expression,and mTORC1 signaling is involved in this process.     

The active metabolite of leflunomide,A771726, inhibits both the generation of and the bone-resorbing activity of osteoclasts by acting directly on cells of the osteoclast lineage

Leflunomide is a disease-modifying antirheumatic drug that inhibits paw swelling and joint destruction in type II collagen-induced arthritis in mice and it also delays disease progression in patients with rheumatoid arthritis (RA), through inhibiting proliferation and cytokine production of T cells, via the blocking of de-novo pyrimidine biosynthesis by its active metabolite, A771726. However, the direct action of leflunomide on cells of osteoclast lineage responsible for bone destruction in RA remains to be clarified. In this study, we examined the effect of A771726 on osteoclast formation and bone-resorbing activity in vitro, using cultures of bone marrow-derived osteoclast progenitors and purified functionally mature osteoclasts, and then we elucidated the molecular mechanism of action of the effect of A771726 on osteoclasts. A771726 inhibited osteoclast formation from macrophage colony-stimulating factor (M-CSF)-dependent osteoclast progenitors in the presence of receptor activator of nuclear factor kappa B (NF-B) ligand (RANKL), without any other types of cells present, in a dose-related manner, similar to the inhibition in cultures of unfractionated bone marrow cells. In addition, A771726 suppressed bone resorption by isolated mature osteoclasts. These results indicate that A771726 directly and intrinsically inhibited the differentiation and function of osteoclast lineage cells without any mediation by other cells. The inhibition by A771726 was not restored by the simultaneous addition of uridine, and may be independent of the blockade of NF-B activation and the tyrosine phosphorylation of proteins. Thus, leflunomide, through its active metabolite, has the potential to prevent bone loss by directly inhibiting osteoclastogenesis and osteoclast function. This inhibition suggests a novel mechanism for leflunomide in the retardation of the joint destruction observed in RA patients.     

体外培养破骨细胞的功能观察

观察破骨细胞在离体状态下的骨吸收功能。新鲜牛皮质骨经锯式切片机横切成５０μｍ厚０．５×０．５ｃｍ大小骨片。参照已建立的破骨细胞分离培养方法，从新生Ｗｉｓｔａｒ大鼠四肢长骨分离破骨细胞，培养于２．５ｃｍ细胞培养皿内或上述骨片上，相差倒置显微镜或扫描电镜观察，可见培养的细胞具有典型破骨细胞的形态特点，接种子骨片上的破骨细胞分别培养１、３、５、７天，扫描电镜观察，可见破骨细胞能在骨片表面形成吸收陷窝，其形态多样，深浅不一，边界清晰，底面粗糙，而且随培养时间延长，陷窝扩大，数量增多。结论，破骨细胞在体外培养条件下具有良好的骨吸收功能，可进行药物干预的研究。     

The effects of inhibitors of cysteine-proteinases and collagenase on the resorptive activity of isolated osteoclasts

The effects of specific inhibitors of cysteine-proteinases ((Z-Phe-Ala-CHN2: benzyloxycarbonyl-phenyl-alanyl alanyl diazomethane and E-64: trans-epoxy-succinyl-L-leucylamido(4-guanidino)-butane) and collagenase and collagenase ((Cl-1: N-(3-N-benzyloxycarbonyl amino-1-R-carboxypropyl)-L-leucyl-O-methyl-L-tyrosine N-methylamide) have been tested on the osteoclastic resorption of dentine. Chick osteoclasts were cultured in the presence or absence of 12.5 microM Z-Phe-Ala-CHN2, 40 or 60 microM E-64, or 40 or 100 microM Cl-1 for 1 or 2 days. In addition, osteoclasts were cultured on oyster shell calcitostracum with or without 12.5 microM Z-Phe-Ala-CHN2. Specimens were studied by light microscopy to count cells and resorption features and by scanning electron microscopy (SEM) stereophotogrammetry for the measurement of the depths, plan-areas and volumes of resorption pits. The numbers, depths and volumes (but not the plan-areas) of the resorption pits in dentine were significantly reduced by Z-Phe-Ala-CHN2 and E-64. Thus, for a given plan-area, the volumes and the depths of resorption pits were smaller in these experimental groups compared with control dentine specimens. The overall inhibition of resorption was at least 75%. Cl-1 did not have this inhibitory effect on the numbers or sizes of resorption pits in dentine. When the oyster calcitostracum was used as a substrate for the osteoclasts, Z-Phe-Ala-CHN2 did not reduce the numbers or volumes of pits, but increased the plan-areas and prevented the formation of deeper pits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antagonistic effects of different classes of bisphosphonates in osteoclasts and macrophages in vitro.

Nitrogen-containing bisphosphonates, such as alendronate and ibandronate, inhibit bone resorption by preventing protein prenylation in osteoclasts, whereas non-nitrogen-containing bisphosphonates, such as clodronate, are metabolized to nonhydrolyzable analogs of ATP, resulting in osteoclast apoptosis. Because these two classes of bisphosphonates have different molecular mechanisms of action, we examined in vitro whether combined treatment with clodronate and alendronate would alter antiresorptive effectiveness. Although, in cultures of rabbit osteoclasts, the antiresorptive effect of 10 microM alendronate was increased by the addition of clodronate, the effect of higher concentrations of alendronate was not altered by addition of clodronate. Furthermore, the inhibition of protein prenylation in osteoclasts caused by higher alendronate concentrations was partially prevented by cotreatment with clodronate. As in osteoclasts, the inhibition of protein prenylation in J774 cells caused by alendronate or ibandronate treatment was dose-dependently prevented by cotreatment with clodronate. Furthermore, alendronate-induced J774 apoptosis was significantly inhibited in the presence of clodronate. The presence of clodronate also decreased the short-term cellular uptake of [14C]ibandronate. These observations suggest that combined treatment with clodronate could enhance the antiresorptive effect of a low concentration of nitrogen-containing bisphosphonate, but clodronate can also antagonize some of the molecular actions and effects of higher concentrations of nitrogen-containing bisphosphonates. The exact molecular basis for the antagonistic effects between bisphosphonates remain to be determined, but could involve competition for cellular uptake by a membrane-bound transport protein.