The origin of osteoclasts: An immunohistochemical study on macrophages and osteoclasts in embryonic rat bone

Summary The origin of osteoclasts was studied in embryonic rat bone primordia using a set of monoclonal antibodies (ED1, ED2, and ED3) that exclusively recognize monocytes and macrophages. ED1 recognizes monocytes and macrophages. Mononuclear phagocytes which were ED1 positive were found in the perichondrium/periosteum of developing bone. These cells started to infiltrate the primordia when the cartilage became hypertrophic. During bone formation, multinucleated ED1-positive cells with the morphological characteristics of osteoclasts were found in the developing bone marrow cavity and against the bone collar. The present findings support the notion that osteoclasts arise by fusion of mononuclear phagocytes derived from blood monocytes.     

Species differences in the immunophenotype of osteoclasts and mononuclear phagocytes

Summary Human osteoclasts, in contrast to mononuclear phagocytes, are known to express a well-defined restricted range of myeloid antigens. To determine whether these antigenic differences are present in other species, we examined the immunophenotype of chicken and rabbit ostoeclasts, macrophages, macrophage polykaryons, and monocytes and compared them with similarly derived and cultured human cells. Human, rabbit, and avian osteoclasts reacted with monoclonal antibodies against human 1 integrins (CD29, CD49b, CD49d), 3 integrins (CD51, CD61), as well as human macrophage-associated antigen CD68. Avian osteoclasts also reacted for CD11a/18 and CD14 which are not present on human osteoclasts. Avian and mammalian monocytes, macrophages, and macrophage polykaryons expressed all the above antigens. Both avian and human macrophage polykaryons produced by culture of peritoneal macrophages reacted with anti-CD51 antibodies indicating that expression of the vitronectin receptor alone does not distinguish between these cells in vitro.     

Isolation of osteoclasts from fetal rat long bones

Summary Cell isolates containing multinucleate osteoclasts were obtained from longitudinally split fetal rat long bones by treatment with testicular hyaluronidase. The total yield of osteoclasts and the osteoclast enrichment of the isolate were increased if the intact bones were first cultured for 72 h. Even greater enhancement was obtained if the bones were treated with 1,25-dihydroxycholecalciferol [1,25(OH)₂D₃] during the culture period. This technique resulted in a cell population containing approximately 15% osteoclasts in yields greater than 50 osteoclasts per long bone. The yield of osteoclasts and the percentage of osteoclasts correlated well with the extent of bone resorption induced by 1,25(OH)₂D₃. The effectiveness of several isolation procedures was compared using the 1,25(OH)₂D₃-treated long bones. Conventional digestion with 1 mg/ml crude collagenase gave a much poorer yield of osteoclasts than simply agitating the split long bones. Hyaluronidase plus EDTA was not significantly different from EDTA alone. Even with milder procedures, however, the isolated osteoclasts were damaged as judged by their failure to exclude trypan blue. The osteoclasts are obviously very fragile cells. The isolation technique coupled with May-Grunwald-Giemsa staining permitted reliable determination of the median number of nuclei per osteoclast. This parameter was the same in uncultured bones or in bones cultured for 72 h in control media. Treatment with 1,25(OH)₂D₃ increased the nuclear number. At lower levels of bone resorption, nuclear number did not increase, but it was significantly greater in more highly resorbed bones.     

辛伐他汀抑制甲状旁腺素相关肽诱导的破骨细胞生成和功能

目的:探讨辛伐他汀对骨髓来源的破骨细胞形成和功能的影响。方法:取Balb/C雄性小鼠双侧股骨和胫骨的骨髓,以不含血清的α-MEM培养液洗涤并收集骨髓细胞,再将细胞重新悬浮于含100 m l/L胎牛血清的α-MEM培养液中,细胞计数后,配成1.5×109/L的细胞悬液,同时加入甲状旁腺素相关肽(PTHrP)和不同剂量的辛伐他汀(10-7、10-6、10-5mol/L)于24孔培养板进行培养,并设阳性对照(只加PTHrP)和阴性对照(PTHrP和辛伐他汀都不加)组,每组均有1孔放置骨磨片1片,培养6 d后;去除上清,抗酒石酸(TRAP)染色检测培养板底部破骨细胞形成;骨磨片用甲苯胺蓝染色,电镜检测骨磨片的吸收陷窝。结果:小鼠骨髓细胞在PTHrP的诱导下获得大量的TRAP染色阳性的破骨细胞,骨磨片有吸收陷窝形成;用辛伐他汀(10-7、10-6mol/L)和PTHrP共同培养下TRAP染色阳性的破骨细胞形成数量均明显减少(P<0.01),辛伐他汀在10-5mol/L时则无TRAP染色阳性的破骨细胞形成;辛伐他汀在10-7mol/L时骨磨片有吸收陷窝的形成但少于阳性对照组(P<0.01),在10-6、10-5mol/L时骨磨片则无吸收陷窝的形成。结论:辛伐他汀对小鼠骨髓来源的破骨细胞的形成有着明显的抑制作用,并且呈剂量依赖关系。     

两种方法培养大鼠破骨细胞的比较研究

目的比较分析直接分离培养的Wistar大鼠破骨细胞（osteoclast，OC）和诱导培养的Wistar大鼠破骨样细胞（osteoclast-like cell，OLC）的形态和功能的差异，为体外药物干预试验奠定基础。方法采用两种培养法，即从新生（24h内）的Wistar大鼠的四肢长骨骨髓腔内壁机械分离成熟OC直接培养和10^-8mol／L的1，25（OH）2D3诱导4周龄Wistar大鼠骨髓单核细胞形成OLC的方法，对获得的OC／OLC进行形态和破骨功能观察。结果两种方法都培养出了抗酒石酸酸性磷酸酶（tartrate resistant acid phosphatase，TRAP）染色阳性的多核细胞，诱导法获得的破骨细胞数量较多（P〈0．05）。成熟OC与诱导获得的OLC形态特征相似，但后者在骨片上形成的陷窝较小而浅。结论直接分离培养法可获得骨吸收功能较活跃的OC，但数目较少，适合骨吸收功能分析、破骨迁移黏附、凋亡研究及单细胞分子生物学研究。1，25（OH）2D3诱导鼠骨髓单核细胞形成的OLC数量较多，但骨吸收功能较差，适合用于破骨细胞分化发育过程的研究。     

钛颗粒诱导破骨细胞分化过程中活化T细胞核因子c1的表达

目的探讨钙调磷酸酶（CN）／活化T细胞核因子（NFAT）途经在磨损颗粒诱导破骨细胞分化调节中的作用。方法MTT法检测11R—VIVIT肽和钛（Ti）颗粒对小鼠骨髓单核／巨噬细胞系细胞（BMMs）活力的影响;RT—PCR法分析Ti颗粒诱导破骨细胞分化过程中NFATcl mRNA表达,并观察1R—VIVIT肽对Ti颗粒诱导NFATcl表达的影响;TRAP染色进行破骨细胞分化鉴定。结果11R—VIVIT肽和Ti颗粒均不影响体外培养中的BMMs活力;Ti颗粒显著刺激破骨细胞分化（P〈0.01）和NFATcl mRNA表达;应用11R—VIVIT肽阻断CN／NFAT途径可显著抑制Ti颗粒诱导的NFATcl表达。结论Ti颗粒刺激BMMs向破骨细胞分化;其作用机制可能与激活CN／NFAT途径有关。     

c-fos antisense DNA inhibits proliferation of osteoclast progenitors in osteoclast development but not macrophage differentiation in vitro

We previously reported that osteoclast formation in vitro, by coculture of mouse bone marrow and primary osteoblastic cells, occurs in two phases: proliferation of osteoclast progenitors followed by terminal differentiation into mature osteoclasts. Using this coculture system, we examined the effects of c-fos antisense and sense phosphorothioate oligonucleotides on osteoclast development and macrophage differentiation. Treatment with c-fos antisense for the first 4 days of coculture inhibited osteoclast formation in a dosedependent fashion. However, when c fos antisense was added during the second phase of coculture (4–6 days), osteoclast formation was unaffected. In contrast, c-fos antisense treatment had no effect on the appearance of F4/80 antigen-positive cells of the macrophage lineage in these cultures or on the induction by colony stimulating factor-1 of macrophage colony formation in cultures of mouse bone marrow cells in agar. Neither osteoclast differentiation nor macrophage appearance was inhibited by adding control c-fos sense in the cocultures. When c-fos antisense was added into an assay of bone resorption by mature osteoclasts, pit formation on dentine slices was unaffected. These results indicate that c-fos plays an important role in the proliferative phase of osteoclast progenitors in osteoclast development, but not in the terminal differentiation phase or in the bone resorbing activity of mature osteoclasts. c-fos antisense specifically inhibited osteoclast formation but had no effect on macrophage development.     

痩素对小鼠破骨细胞分化及功能的影响

目的 观察瘦素（leptin）对体外骨髓诱导培养的小鼠破骨细胞分化和功能的作用效应,探索leptin和骨吸收之间的关联.方法 建立由巨噬细胞集落刺激因子（M-CSF）和骨保护素配体（RANKL）为共同细胞因子的小鼠破骨细胞骨髓诱导培养体系,将不同浓度的leptin作用于破骨细胞.实验中根据培养液中是否加入M-CSF和RANKL并依据leptin浓度的不同分为：A组,M-CSF和RANKL;B组,M-CSF、RANKL和leptin（80 ng/ml）;C组,M-CSF、RANKL和leptin（160 ng/ml）;D组,M-CSF、RANKL和leptin（240 ng/ml）;E组,M-CSF、RANKL和leptin（320 ng/ml）;F组,M-CSF、RANKL和leptin（400 ng/ml）;同时设立空白对照组G组.于作用后第7天取细胞玻片进行抗酒石酸酸性磷酸酶（TRAP）染色,观察破骨细胞并计数;于第10天取出骨片进行甲苯胺蓝染液染色,在光镜和扫描电镜观察骨吸收陷窝形态.结果：诱导培养的小鼠破骨细胞形态特征明显;A组在破骨细胞数量与D、E、F组相比较有明显的统计学差异（P＜0.05）;A组骨吸收面积比与B、C、D、E、F组都有明显的统计学差异（P＜0.05）.结论：leptin抑制体外培养的破骨细胞的分化和骨吸收功能.     

不同浓度葡萄糖对大鼠骨髓破骨细胞分化的影响

目的观察不同浓度葡萄糖对大鼠骨髓破骨细胞（Osteoclasts OC）分化的影响，探讨糖尿病骨质疏松的发病机制。方法用M—CSF、RANKL诱导大鼠骨髓单个核细胞分化为OC，同时给予不同浓度的葡萄糖（0、5．5、15、25mmol／L）干预，通过观察抗酒石酸酸性磷酸酶（TRAP）染色阳性OC数、TRAP活性测定、OC膜表面RANK mRNA表达量来分析葡萄糖对破骨细胞分化的影响。结果①高浓度葡萄糖组（25mmol/L）培养7d时TRAP染色阳性OC数及TRAP活性测定均高于对照（0mmol／L）组、葡萄糖5．5mmol／L组（P〈0．01）；与对照组比较高浓度葡萄糖组培养3d时TRAP染色阳性OC数明显增多（P〈0．01）。②葡萄糖呈浓度依赖性上调OC膜表面RANK mRNA的表达，其中高浓度葡萄糖组培养的各时间点与其他3组比较差异有统计学意义（P〈0．01，P〈0．05）。结论①高浓度葡萄糖可促进破骨细胞的分化，其促进作用始于诱导分化的早期。②骨髓微环境中高浓度葡萄糖可引起OC分化增多，可能是糖尿病骨质疏松的发病机制之一。     

Downregulation of colony-stimulating factor-1 (CSF-1) binding by CSF-1 in isolated osteoclasts

Colony-stimulating factor-1 (CSF-1), also called macrophage colony-stimulating factor, is the growth factor for the cells of the mononuclear phagocytic system. Further-more, CSF-1 is essential in osteoclastogenesis and also affects mature osteoclasts. The receptor for CSF-1 was demonstrated on cells of the osteoclast lineage, with highest levels on the mature cells. This study investigated whether the binding of CSF-1 to isolated rat osteoclasts is modulated by the growth factor itself. Exposure of osteoclasts to CSF-1 for 1 hour virtually abolished binding of the growth factor. After removal of CSF-1, binding sites were restored within 4 hours. This recovery was blocked by cycloheximide, indicating the dependence on new protein synthesis for reex-pression of receptors on the cell surface. The observed downregulation of CSF-1 binding sites might be a mechanism to control the effects of the growth factor on mature osteoclasts.     

The origin of the osteoclast

The origin of the osteoclast remains controversial even though investigations using light microscopy, tissue culture, electron microscopy, microcinematography, autoradiography, parabiosis, quail chick nuclear markers, giant lysosomal markers in beige mice, Y chromosomes, bone marrow cell culture, and monoclonal antibodies have been performed since its discovery. Concepts of the origin of the osteoclast have been changing. The classic concept was that the osteoclast originated from connective tissue cells. Others hypothesized that it originated from mature hematopoietic cells, particularly from monocyte or macrophage cells. A recent concept proposed an origin from hematopoietic stem cells. The hematopoietic stem cell was believed to differentiate into two cell lineages: one of monocytes and the other of preosteoclasts. The authors' concept, based on experimental observations as well as alternate interpretations stemming from experimental reports of other researchers, proposes an origin from local, nonhematopoietic, possibly perivascular mesenchymal cells. However, the relationship of the hematopoietic stem cells to perivascular mesenchymal cells in the periosteum at an early stage of enchondral ossification is still not well known. Therefore, the origin of the osteoclast also remains uncertain.     

鸡破骨细胞分离培养方法的建立

为了寻找一种获得大量、纯化、有活力的破骨细胞的方法，我们对已经建立的兔破骨细胞体外分离培养方法的基础上加以改进。利用冲洗的方法，从２８天的鸡的长骨中得到细胞团１破骨细胞后，又对鸡的长骨相继进行胶原酶和胰蛋白酶的消化，得到细胞团２和细胞团３破骨细胞。将这些分离的细胞与盖玻片或骨磨片共同培养，通过相差显微镜观察到：这些分离的多核巨细胞能够运动，并能在骨磨片上形成吸收陷窝。另外，这些细胞对酸性磷酸酶染色呈阳性反应，而酸性磷酸酶是鉴别破骨细胞的标志。表明此分离培养鸡破骨细胞的实验技术是成功的。通过此方法获得的鸡破骨细胞比用以往方法获得的兔破骨细胞量多，且活力强。本方法的建立，为进一步研究骨吸收机理奠定了基础。     

福莫特罗和ICI118551对大鼠成熟破骨细胞骨吸收功能的影响

目的研究选择性β2肾上腺素能激动剂福莫特罗(Formoterol)和阻滞剂ICI118551对体外培养大鼠成熟破骨细胞(osteoclast,OC)功能的影响,探讨β2肾上腺素能受体信号对骨代谢的影响。方法取清洁级出生24h内的SD乳大鼠,长骨干骨髓腔内壁机械分离成熟OC后分别加入不同浓度(10-5mol/L～10-9mol/L)的Formoterol和ICI118551,以抗酒石酸酸性磷酸酶(TRAP)染色观察破骨细胞形态,甲苯胺蓝染色计数骨片上的骨吸收陷窝数目,Image-ProPlus6.0图像软件分析骨片上骨吸收陷窝面积。结果破骨细胞与骨片共培养6天,不同浓度的Formoterol与对照组相比均可增加骨片上OC的骨吸收陷窝数目和面积;随着ICI118551浓度的提高骨片上骨吸收陷窝的数目和面积逐渐减少。结论β2肾上腺素能受体激动剂可促进体外培养OC的骨吸收功能,阻滞剂对OC的骨吸收功能有抑制作用,且呈剂量依赖性。     

The effects of bisphosphonates on the resorption cycle of isolated osteoclasts

Binding sites for wheat germ agglutinin (WGA)-lectin have been shown to become revealed in the demineralized resorption lacunae that osteoclasts excavate on bone substrate. Peroxidase-conjugated WGA-lectin, which binds to bone matrix glycoconjugates and proteoglycans, was used in pit formation assays to assess the activity of isolated osteoclasts cultured on either 3-amino-1,1-hydroxy-propylidene-bisphosphonate (APD)-or dichloromethylene bisphosphonate (Cl₂MBP)-covered bone slices. Immunofluorescence and histochemical techniques were also used to study the effects of bone-bound bisphosphonates on isolated rat osteoclasts. Neither APD nor Cl₂MBP interfered with the special organization of actin or vinculin in osteoclasts when the cells were initializing their resorption cycle. After 24 hours of culture, the number of resorbing osteoclasts increased strongly on control slices, but remained low on either APD- or Cl₂MBP-treated slices. At this time, the actin and vinculin rings in osteoclasts also started to exhibit abnormal, more diffuse staining. Both bisphosphonates studied resulted in signs of cytotoxicity: the number of osteoclasts decreased on APD- or Cl₂MBP-covered bone during the course of the study and those remaining attached exhibited severe cytoplasmic retractions. The total areas of resorption remained at significantly lower levels in both experimental groups studied, and this was due to decreases in both the number and sizes of individual resorption pits. The size of the most extensive lacunae detected on the Cl₂MBP slices did not exceed 5x10³ m², whereas on the control slices, resorption pits bigger than 15x10^{3 2} were frequently discovered.     

雌激素通过调节骨髓来源的成骨细胞所产生的细胞因子抑制破骨细胞功能

目的：通过观察雌激素对骨髓来源的成骨细胞所产生的细胞因子的调节作用，探讨雌激素抑制破骨细胞功能的机制。方法：在诱导小鼠骨髓细胞分化为成骨细胞后，于成骨细胞培养中加入雌激素，应用抗体荧光免疫标记法检测成骨细胞培养液中IL－1、IL－6和TNF－α水平。结果：与对照组比较，经0．01－1nM雌激素处理后， 成骨细胞所产生的IL－1和IL－6水平呈浓度依赖性下降，TNF－α水平则无明显变化。结论：雌激素具有调节成骨细胞分泌细胞因子功能，提示雌激素抑制破骨细胞作用机制源自调控成骨细胞旁分泌。     

The alteration of osteoclast morphology by diphosphonates in bone organ culture

Two diphosphonates alter the morphology of the osteoclast, as they inhibit the calcium⁴⁵ release from bones stimulated to resorb by lipopolysaccharide. Disodium dichloromethylene diphosphonate was more potent than disodium ethane-1-hydroxy-1,1-diphosphonate in both inhibiting⁴⁵calcium release and altering osteoclast morphology. Alteration in the morphology of osteoclasts is associated with little or no change in the morphology of the surrounding non-osteoclast cells. These results indicate a specific morphological effect of diphosphonates on osteoclasts.     

Characterization of Circulating Human Osteoclast Progenitors: Development of In Vitro Resorption Assay

Several cell surface markers were used to isolate monocytes as osteoclast progenitors with an immunomagnetic cell separation system. Use of this system with specific monocyte antibodies produced 99% pure monocytes. When purified monocytes were cultured on bovine bone slices in the presence of receptor activator of nuclear factor-B (RANKL), macrophage-colony stimulating factor (M-CSF), tumor necrocis factor alpha (TNF-), and dexamethasone for 14 days, CD14⁺ CD11b⁺, and CD61⁺ monocytes had approximately 90-, 30- and 20-fold higher osteoclast formation capacities/plated cells compared to the control culture. CD15⁺ monocytes generated few tartrate-resistant acid phosphatase-positive multinucleated cells (TRACP+ MNC), and CD169⁺ monocytes generated no TRACP+ MNC. This suggests, that there are various subsets of monocytes in the blood circulation and that they have different capacities in osteoclast formation. These results show that circulating human osteoclast progenitors can be efficiently purified by immunomagnetic cell separation system using anti-CD14, -CD11b, and -CD61 antibodies. These purified monocyte fractions had different ability to give rise to osteoclasts. CD169 was not found to be suitable for osteoclast progenitor isolation. Optimal concentration of dexamethasone for osteoclast formation and bone resorption was 10 nM. To develop a human resorption assay, osteoclasts were first induced for 7 days, whole media were replaced, cultures were continued for additional 3 days and C-terminal telopeptide of type I collagen was determined from culture media. This assay was shown to be functional, since two well-known resorption inhibitors, bafilomycin A₁ and calcitonin, dose-dependently inhibited the resorption activity of osteoclasts.     

The lifespan of osteoclasts: Experimental studies using the giant granule cytoplasmic marker characteristic of beige mice

Osteoclasts are large multlnucleated skeletal cells that form by fusion of bloodborne mononuclear precursors. Fusion with mononuclear precursors occurs throughout life, and survival of Osteoclasts is believed to be dependent upon continued replenishment by fusion. This study examined osteoclast lifespan, defined as maximal survival without fusion, in normal mice irradiated to eliminate host stem cells and rescued with stem cells from beige (bg) mice whose osteoclasts have a distinctive phenotype. Osteoclasts of donor phenotype appeared during the second week and progressively increased so that by the sixth week no osteoclasts of host phenotype were present. Radiation alone did not produce any change in osteoclast phenotype. These data are interpreted to indicate that the maximal survival of osteoclasts without fusion of precursors is less than 6 weeks.     

Recruitment of osteoclast precursors by stromal cell derived factor-1 (SDF-1) in giant cell tumor of bone.

Giant cell tumor (GCT) of bone is a unique bone lesion that is characterized by an excessive number of multinucleated osteoclasts. GCT consists of neoplastic stromal cells, multinucleated osteoclasts and their precursors, thus serving as a naturally occurring human disease model for the study of osteoclastogenesis. It still remains unclear how stromal cells of GCT recruit osteoclast precursors. In the present study, we characterized the cellular components of GCT and confirmed the presence of CD14(+)-monocytes/CD68(+)-macrophages and CD34(+)-hematopoetic stem cells that express CXCR4, a specific receptor for SDF-1; SDF-1 gene expression and presence of SDF-1 protein were confirmed by real time RT-PCR, in situ hybridization, and immunohistochemistry in the GCT tissue and cultured cells. SDF-1 was present at 25-50 ng/ml in the conditioned media from the GCT cultures, which is in the range of physiological chemotactic concentration. Migration of osteoclast precursors was 2.5-fold higher in response to GCT conditioned media compared to the control media; and migration was inhibited by an average of 36% with anti-SDF-1 neutralizing antibody or competing recombinant SDF-1. These results suggest that SDF-1 is one of the significant chemoattractant factors involved in the recruitment of hematopoietic osteoclast precursor cells during tumor-induced osteoclastogenesis.     

The generation of osteoclasts from RAW 264.7 precursors in defined,serum-free conditions

Osteoclasts are the unique cell type capable of resorbing bone. The discovery of the TNF-ligand family member, RANKL, has allowed more reliable study of these important cells. The mouse monocytic cell line, RAW 264.7, has been shown to readily differentiate into osteoclasts upon exposure to recombinant RANKL. Unlike primary osteoclast precursors, there is no requirement for the addition of macrophage colony stimulating factor (M-CSF). However, to date, their differentiation has always been studied in the context of added foetal calf serum (FCS). FCS is a complex and largely undefined mixture of growth factors and matrix proteins, and varies between batches. For this reason, osteoclastogenesis would ideally be studied in the context of a defined, serum-free medium. RAW 264.7 cells were cultured in serum-replete α-MEM or serum-deprived medium (SDM) shown previously to support the growth of human osteoclasts in a co-culture with normal osteoblasts. In SDM, in the presence of recombinant RANKL, RAW 264.7 cells readily differentiated into tartrate resistant acid phosphatase (TRAP) positive multinucleated osteoclast-like cells, a process that was enhanced with the addition of 1α,25-dihydroxyvitamin D₃ (1,25D). While the osteoclasts grown in SDM were smaller in size compared with those derived in serum-replete media, their resorptive capacity was significantly increased as indicated by a twofold increase in average resorption pit size. In conclusion, we describe a defined model for studying osteoclast differentiation and activity in the absence of serum, which will be ideal for studying the role of agonistic and antagonistic molecules in this process.