Two types of bone resorption lacunae in the mouse parietal bones as revealed by scanning electron microscopy and histochemistry

To understand the bone resorption process on the basis of the morphology of bone resorption lacunae, the inner surface of parietal bones in juvenile mice was exposed with a treatment of ultrasonic waves or NaOCl treatment and examined by scanning electron microscopy (SEM). The bone resorption lacunae were divided into two types (I and II) according to differences in morphological features of their walls; the wall of type I lacunae was covered with loose collagen fibrils, while that of type II lacunae was smooth with almost no fibrillar structures. Collagen fibrils in type I lacunae treated with ultrasonic waves differed in appearance from those treated with NaOCl; the collagen fibrils were thin and displayed a smooth surface in type I lacunae treated with ultrasonic waves, while they were thick and showed a rough surface in those treated with NaOCl-probably because superficial uncalcified collagen fibrils were digested with the chemical. The results indicated that type I lacunae occupied 77% of all of the bone resorption lacunae treated with ultrasonic waves, but 51% of those treated with NaOCl. This finding led to the idea that type I lacunae can be subdivided into two: lacunae (Ia), covered with partially calcified fibrils as well as superficial uncalcified fibrils; and lacunae (Ib), covered only with uncalcified fibrils. The presence of uncalcified fibrils in the bone resorption lacunae was further confirmed by backscattered electron (BSE) imaging of SEM. Histochemistry for acid phosphatase or immuno-histochemistry for cathepsin B or carbonic anhydrase in combination with SEM revealed that type I lacunae were located under osteoclasts but type II lacunae were not. These findings indicate that type I lacunae are in the process of bone resorption by osteoclasts, while type II lacunae are in the final stage of bone resorption and free from osteoclasts. Bone resorption may thus proceed in the order of Ia, Ib, and II.     

A delivery system specifically approaching bone resorption surfaces to facilitate therapeutic modulation of microRNAs in osteoclasts

Dysregulated microRNAs in osteoclasts could cause many skeletal diseases. The therapeutic manipulation of these pathogenic microRNAs necessitates novel, efficient delivery systems to facilitate microRNAs modulators targeting osteoclasts with minimal off-target effects. Bone resorption surfaces characterized by highly crystallized hydroxyapatite are dominantly occupied by osteoclasts. Considering that the eight repeating sequences of aspartate (D-Asp₈) could preferably bind to highly crystallized hydroxyapatite, we developed a targeting system by conjugating D-Asp₈ peptide with liposome for delivering microRNA modulators specifically to bone resorption surfaces and subsequently encapsulated antagomir-148a (a microRNA modulator suppressing the osteoclastogenic miR-148a), i.e. (D-Asp₈)-liposome-antagomir-148a. Our results demonstrated that D-Asp₈ could facilitate the enrichment of antagomir-148a and the subsequent down-regulation of miR-148a in osteoclasts in vivo, resulting in reduced bone resorption and attenuated deterioration of trabecular architecture in osteoporotic mice. Mechanistically, the osteoclast-targeted delivery depended on the interaction between bone resorption surfaces and D-Asp₈. No detectable liver and kidney toxicity was found in mice after single/multiple dose(s) treatment of (D-Asp₈)-liposome-antagomir-148a. These results indicated that (D-Asp₈)-liposome as a promising osteoclast-targeting delivery system could facilitate clinical translation of microRNA modulators in treating those osteoclast-dysfunction-induced skeletal diseases.     

Morphological study on cell-cell interaction between osteoclasts and osteoblasts

We reviewed morphological characteristics in cell-cell interaction between osteoclasts and the cells of osteoblast lineage. Heparan sulfate proteoglycans (HSPG) are localized in the intercellular space between osteoblasts and osteoclasts. HSPG is involved in reservation of heparin binding growth factors (HBGF), protection from proteolysis of HBGF, and ligand-receptor interaction in the case of fibroblast growth factors. HSPG may play an important role in cell-cell interaction between osteoblasts and osteoclasts by reserving HBGF and heparin binding adhesion molecules such as fibronectin. On the other hand, CD44, a hyaluronate receptor, and moesin are colocalized in the basolateral plasma membrane of osteoclasts. Calcitonin changes their localization in osteoclasts, suggesting that the CD44-moesin-actin filament system is engaged in the regulation of cell polarity. Although hyaluronates colocalize with CD44 in the basolateral membrane of osteoclasts, the precise intracellular signaling mechanism needs to be clarified in further research. Bone metabolism may be regulated by cell-cell and cell-matrix interaction among bone cells via adhesion molecule, extracellular matrices and cytokines.     

Morphological evidence of reduced bone resorption in the osteosclerotic (oc) mouse

Osteopetrosis, a metabolic bone disease characterized by a generalized sclerosis of the skeleton, is inherited as an autosomal recessive in a number of mammalian species. The pathogenesis of congenital osteopetrosis is mediated by a reduction in bone resorption as a result of decreased osteoclast function. This hypothesis is based on both functional and structural evidence of reduced bone resorption in all mutations examined to date. The present study examined the histology of cartilage and bone, the ultrastructure of osteoclasts, and the morphology of mineralized bone surfaces in a lethal osteopetrotic mutation, the osteosclerotic (oc) mouse. Histologically, epiphyseal cartilage growth plates, especially the hypertrophic zone, are markedly thickened in oc mice and metaphyses contain excessive osteoid, features characteristic of rickets. Transmission electron microscopy revealed that less than one-quarter of osteoclasts in oc mice demonstrated evidence of ruffled border formation compared with three-quarters of the osteoclasts in normal littermates. In mutants, ruffled borders were less elaborate and cytoplasmic processes penetrated into bone surfaces, suggesting that bone may be removed by mechanical rather than by enzymatic means. There was little morphological evidence of cartilage degradation and broad laminae limitantes persisted in mutants. Mineralized surfaces that undergo resorption in normal mice showed no evidence of bone resorption by scanning EM in mutants. The presence of a rachitic condition, the observations of reduced bone resorption, and the possible contribution of undermineralized matrices to decreased bone resorption are charcteristics of the osteosclerotic mutation which suggest that it is a unqiue csteopetrotic mutant in which to study both the development and regulation of skeletal metabolism.     

Effect of tetracyclines which have metalloproteinase inhibitory capacity on basal and heparin-stimulated bone resorption by chick osteoclasts

Several tetracyclines (TETs) are potent inhibitors of collagenase (CGase) and can inhibit connective tissue degradation in a variety of inflammatory and degenerative disorders. The role of CGase in bone resorption by osteoclasts (OC) remains unclear. Disaggregated OCs from chick embryos were cultured for 24 h on devitalized bovine cortical bone±heparin in the presence of various TETs. Doxycycline (Dox) inhibited pit formation in a dose-dependent manner. CMT, a TET derivative which inhibits matrix metalloproteinases (MMPs) but is not antimicrobial, also inhibited chick OC bone resorption. Heparin markedly stimulated bone resorption at 5 g/ml, which was reversed by Dox, 5 g/ml. TETs can reversibly inhibit both basal and heparin-stimulated bone resorption by chick OCs. These findings suggest that MMPs may play a role in osteoclastic bone resorption, and that safe and effective inhibitors of MMPs, including certain TETs, might have a potential therapeutic role.The Long Island Campus for the Albert Einstein College of Medicine     

Mechanisms of promoting the differentiation and bone resorption function of osteoclasts by Staphylococcus aureus infection

Bone infection is a common and serious complication in the field of orthopedics, which frequently leads to excessive bone destruction and fracture nonunion. Staphylococcus aureus (S. aureus) infection affects bone cell function which, in turn, causes bone destruction. Bone is mainly regulated by osteoblasts and osteoclasts. Osteoclasts are the only cell type with bone resorptive function. Their over-activation is closely associated with excessive bone loss. Understanding how S. aureus changes the functional state of osteoclasts is the key to effective treatment. By reviewing the literature, this paper summarizes several mechanisms of bone destruction caused by S. aureus influencing osteoclasts, thereby stimulating new ideas for the treatment of bone infection.     

Involvement of receptor-interacting protein 140 in estrogen-mediated osteoclasts differentiation,apoptosis, and bone resorption

Estrogen withdrawal following menopause results in an increase of osteoclasts formation and bone resorption, which is one of the most important mechanisms of postmenopausal osteoporosis. Recently, growing evidence has suggested that receptor-interacting protein 140 was implicated in estrogen-regulated metabolic disease, including fat metabolism and lipid metabolism. However, little is known regarding the role of receptor-interacting protein 140 in the regulation of bone metabolic by estrogen. In the present study, Western blotting disclosed that estrogen brings a significant increasing expression of receptor-interacting protein 140 in osteoclasts, but not in osteoblasts and bone marrow mesenchymal stem cells. Furthermore, analysis of TRAP staining and bone resorption assay showed that depletion of receptor-interacting protein 140 could significantly alleviate the inhibitory effects of estrogen on osteoclasts formation and bone resorption activity. Moreover, estrogen could induce osteoclasts apoptosis by increasing receptor-interacting protein 140 expression through the Fas/FasL pathway. Taken together, receptor-interacting protein 140 might be a critical player in estrogen-mediated osteoclastogenesis and bone resorption.     

Disaggregated osteoclasts increase in resorption activity in response to roughness of bone surface.

The roughness of the bone matrix surface affects osteoblastic differentiation. However, the effect of the roughness of the matrix surface on osteoclastic bone resorption remains to be studied. We examined the latter effect using disaggregated osteoclasts from neonatal rats. The resorption pit number and the total pit area on the rough surface were not different from those on smooth surfaces after 1 day, but they were 2 or more times higher after 3 days. The number of osteoclasts was not different on bone slices with either smooth or rough surfaces at 3 days. The alkaline phosphatase (ALP)-positive osteoblasts were relatively rare in both types of slices at first, then the number and the diameter of the enzyme-positive cells and the clusters preferentially increased on the rough bone slices. When hydroxyurea was added to the culture in order to suppress the proliferation and the subsequent differentiation of osteoblastic cells on rough surfaces, the increase in resorption on the rough surfaces was effaced; however, this agent had little affect on resorption of the smooth surfaces. The addition of ALP-positive cells to disaggregated osteoclasts increased bone resorption on the smooth surface. The results suggest that osteoblast development and subsequently bone resorption by osteoclasts is enhanced by the roughness of matrix surfaces.     

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

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

Osmotic membrane stretch increases cytosolic Ca(2+) and inhibits bone resorption activity in rat osteoclasts

Although the importance of mechanical stress on bone metabolism is well known, the intracellular mechanisms involved are not well understood. To evaluate the role of mechanical stress on osteoclastic function, we investigated the effects of membrane stretch induced by osmotic cell swelling on cytosolic Ca(2+) and bone resorption activity in freshly isolated rat osteoclasts. The intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by fura-2 microspectrofluorimetry. Exposure to hypotonic solution (211-151 mOsm) caused cell swelling and reversibly increased [Ca(2+)](i) in the osteoclasts. This [Ca(2+)](i) increase was abolished by the omission of extracellular Ca(2+), but was not affected by the depletion of intracellular Ca(2+) stores. Gd(3+) and La(3+) inhibited the swelling-induced [Ca(2+)](i) increase, while nifedipine and Bay K 8644 did not. Neither protein kinase A inhibitors (Rp-cAMP, H-89) nor protein kinase C inhibitors (staurosporine, chelerythrine) affected the [Ca(2+)](i) increase. Membrane depolarization was not essential for the [Ca(2+)](i) increase either. To assess the effects of membrane stretch on the bone resorption activity of osteoclasts, we investigated actin ring formation, the intracellular structure responsible for bone resorption in osteoclasts. Hypotonic stimulation acutely disrupted actin ring formation in an extracellular Ca(2+)-dependent manner, and this disruption was prevented by Gd(3+). Moreover, Ca(2+) ionophore (ionomycin) also induced disruption of the actin rings. These results indicate that mechanical stress inhibits osteoclastic bone resorption activity, possibly via the elevation of [Ca(2+)](i) through stretch-activated, non-selective cation channels.     

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.     

柚皮甙干预鼠颅顶骨破骨细胞的形成及功能

背景：柚皮甙能诱导骨形态发生蛋白2的基因表达,促进成骨细胞系的增殖和分化。体外细胞实验提示柚皮甙有抑制破骨细胞的形成及抗骨质疏松的作用。 目的：建立含有柚皮甙的鼠颅顶骨培养模型,观察不同剂量下柚皮甙对破骨细胞增殖分化的影响。 方法：实验选取出生4 d的SD乳鼠颅顶骨,在分别含有0,1,10,100 mg/L 的柚皮甙的培养基中培养,于培养的第1,3,7,10天测定柚皮甙对鼠颅顶骨中破骨细胞标志酶抗酒石酸酸性磷酸酶阳性细胞数及培养基中钙离子浓度的影响。 结果与结论：第1天各组间抗酒石酸酸性磷酸酶阳性细胞数及培养基中钙离子浓度无明显变化,第3,7天各组间抗酒石酸酸性磷酸酶阳性细胞数随柚皮甙质量浓度增加而减少,而培养基中钙离子浓度变化明显增加,到第10天这种变化趋势最为明显。说明柚皮甙能影响鼠颅顶骨中抗酒石酸酸性磷酸酶阳性细胞数量及其功能,并且这种作用成明显的时间剂量相关性。提示柚皮甙不仅能促进成骨细胞的增殖,同时也能减少破骨细胞的分化或加速其凋亡。     

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。     

Relationships between tooth eruption, occlusion and alveolar bone resorption: cytological and cytochemical studies of bone resorption on rat incisor alveolar bone facing the enamel

The rat labial incisor alveolar bone facing the enamel and bearing the occlusal force was examined by electron microscopy after being compared with the lingual alveolar bone by histological and scanning electron microscopic (SEM) observations. On the labial side, shallow resorptive lacunae were recognized all over the bone surface; these were mainly covered by osteoclasts and some mononuclear cells. The cement line was absent from the bone matrix. On the lingual side, residues of Sharpey's fibers, the bone formation surface and deep resorptive lacunae were observed by SEM. Histologically, bone remodeling areas showing both osteoclasts and active bone-forming osteoblasts on the bone surface, as well as many cement lines in bone matrix, were recognized. Furthermore, electron microscopic and cytochemical studies demonstrated that mononuclear cells located close to osteoclasts displayed osteoblastic characteristics such as alkaline phosphatase activity, a developed Golgi apparatus, and a rough endoplasmic reticulum. These findings indicate that continuous bone resorption occurs on the labial bone surface, while active bone remodeling occurs on the lingual surface. Even on the labial surface, osteoblastic cells close to osteoclasts seem to play an important role in the differentiation and or activation of osteoclasts.     

Pathogenesis of osteopetrosis in the microphthalmic mouse: Reduced bone resorption

Bone resorption, stimulated by injection of parathyroid extract, was measured in vivo in microphthalmic mice as the rate of release of ³H from bone after incorporation of ³H-proline. Bone resorption in these mice, which inherit osteopetrosis, was less than 10% of that in normal littermates. Autoradiography confirmed the reduction in removal of radioactive bone matrix and in bone growth in microphthalmic mice. The ability of these mice to raise the serum calcium concentration in response to PTE was also reduce. These results, that bone resorption is reduced in microphthalmic mice, are discussed in relation to the pathogenesis and cure of the disease.     

Different inhibitory actions of immunomodulating agents and immunosuppressive agents on bone resorption of mouse calvaria

In this study, we have investigated the in vitro effects of the immunomodulators lobenzarit and traxanox and a newly synthesized immunosuppressant, mizoribine, as well as cyclosporin A, on bone resorption using neonatal mouse calvariae labelled with 45Ca. As stimulators of bone resorption, bovine parathyroid hormone (PTH), lipopolysaccharide (LPS), interleukin 1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) were used. Lobenzarit, traxanox, mizoribine and cyclosporin A inhibited or tended to inhibit bone resorption stimulated by PTH, LPS, IL-1 beta or TNF-alpha in a dose-dependent manner. Basal bone resorption was inhibited by immunosuppressant cyclosporin A or mizoribine, while immunomodulators lobenzarit and traxanox failed to inhibit basal bone resorption. Removal of lobenzarit from the culture medium resulted in the recovery of bone resorptive activity. These results suggest that the inhibitory effect of immunomodulators on bone resorption is reversible and nonselective. Also, it raises the possibility that immunomodulators and immunosuppressants may affect bone resorption by different mechanisms.     

Bone-cell changes in estrogen-induced bone-mass increase in mice: dissociation of osteoclasts from bone surfaces

Early studies in certain avian and mammalian species have described estrogen-associated bone-cell changes, which were based on bone cells that were neither quantified nor identified histochemically (osteoclasts). In the experiments described here, weanling female mice were given a pharmacological dose of 17 beta-estradiol benzoate (1 mg/week) for 1 and 4 weeks, and changes in osteoclasts and osteoblasts were assessed in tibial metaphyses and diaphyses. In the proximal metaphysis, the number of osteoclasts/mm surface length was significantly reduced by estrogen at 1 week (43%) and 4 weeks (64%), which was accompanied by significant increases in the number of osteoclasts in the marrow space not in contact with bone surfaces (no./mm2: 382% and 999%, respectively). These increases, along with observations of decreased osteoclast size (19%), of changes in osteoclast morphology, and of numerous acid-phosphatase-positive fragments in the marrow space, suggest that estrogen treatment causes the dissociation and disintegration, and thus decreased activity, of osteoclasts. The above changes were accompanied by more than 48% increases in the number of trabecular osteoblasts. In the diaphysis, the number of endosteal osteoclasts was significantly decreased by estrogen at 1 week (32%), but was not significantly changed at 4 weeks. These changes were attended by significant increases in the number of osteoclasts in the marrow space not in contact with bone surfaces (no./mm2: 393% at 1 week and 342% at 4 weeks). The number of endosteal osteoblasts was also increased by estrogen at 1 week (132%), and so was the size of endosteal osteoblasts (39% at 1 week and 81% at 4 weeks). Comparable results were obtained when a lower dose of 17 beta-estradiol benzoate (20 micrograms/week) was given to ovariectomized mice. The increase in bone mass and its associated cell changes following estrogen treatment were also found in athymic nude mice, suggesting that these bone/bone cell changes are independent of the thymus.     

Activation of osteoclasts and the repopulation of bone surfaces following hibernation in the bat, Myotis lucifugus

In previous studies of the adult bat, Myotis lucifugus, bone loss that occurred during the hibernating period was attributed to osteocytic osteolysis, rather than osteoclastic activity. We have used histochemistry and light and electron microscopy to determine the functional state of the skeleton during the hibernating period. We find that during hibernation the marrow cavity of the long bones is filled with lipid deposits interspersed with vascular sinusoids containing mononuclear cells and red blood cells. The lipid deposits are found within fat cells and at extracellular sites. Hematopoietic cells are absent, osteoclasts are absent, and the bone surfaces are covered with quiescent bone-lining cells. Osteocytes retain their structural integrity and maintain canalicular systems with the bone lining cells and with other osteocytes. During the arousal period, osteoclasts reappear on the bone surface, followed in time by increased numbers of osteoblast-like cells. Perivascular cells undergo structural hypertrophy. Many mononuclear cells are now found in extravascular sites. The lipid content in the marrow is gradually reduced and replaced by hematopoietic cells. Each of these events occurs in a repeatable sequence that is related to the state of hibernation of Myotis lucifugus.