Histochemical evidence of osteoclastic degradation of extracellular matrix in osteolytic metastasis originating from human lung small carcinoma (SBC-5) cells

The aim of this study was to assess the dynamics of osteoclast migration and the degradation of unmineralized extracellular matrix in an osteolytic metastasis by examining a well-standardized lung cancer metastasis model of nude mice. SBC-5 human lung small carcinoma cells were injected into the left cardiac ventricle of 6-week-old BALB/c nu/nu mice under anesthesia. At 25-30 days after injection, the animals were sacrificed and their femora and/or tibiae were removed for histochemical analyses. Metastatic lesions were shown to occupy a considerable area extending from the metaphyses to the bone marrow region. Tartrate resistant acid phosphatase (TRAPase)-positive osteoclasts were found in association with an alkaline phosphatase (ALPase)-positive osteoblastic layer lining the bone surface, but could also be localized in the ALPase-negative stromal tissues that border the tumor nodules. These stromal tissues were markedly positive for osteopontin, and contained a significant number of TRAPase-positive osteoclasts expressing immunoreactivity for CD44. We thus speculated that, mediating its affinity for CD44, osteopontin may serve to facilitate osteoclastic migration after their formation associated with ALPase-positive osteoblasts. We next examined the localization of cathepsin K and matrix metallo-proteinase-9 (MMP-9) in osteoclasts. Osteoclasts adjacent to the bone surfaces were positive for both proteins, whereas those in the stromal tissues in the tumor nests showed only MMP-9 immunoreactivity. Immunoelectron microscopy disclosed the presence of MMP-9 in the Golgi apparatus and in vesicular structures at the baso-lateral cytoplasmic region of the osteoclasts found in the stromal tissue. MMP-9-positive vesicular structures also contained fragmented extracellular materials. Thus, osteoclasts appear to either select an optimized function, namely secreting proteolytic enzymes from ruffled borders during bone resorption, or recognize the surrounding extracellular matrix by mediating osteopontin/CD44 interaction, and internalize the extracellular matrices. Microsc.     

Measurement by vertical scanning profilometry of resorption volume and lacunae depth caused by osteoclasts on dentine slices

The resorption pit assay is classically used to evaluate osteoclast activity on bone or dentine slices that can be eroded by these cells. Two different types of cells were generated from peripheral blood mononuclear cells cultured in the presence of M-CSF + sRANKL or with M-CSF + LPS. At the end of the culture period (21 days), cells were discarded and the dentine slices stained with toluidine blue and examined with an NT9100 Wyco vertical scanning profilometer. The images of the dentine surface were corrected for tilt and the eroded volume was calculated on the whole images. The depth of the eroded pits was determined. The data files were used to reconstruct the surface of the slices by standardizing the ground level to compare both conditions. Osteoclasts generated with M-CSF + sRANKL were capable of resorbing a more important volume than those generated with M-CSF + LPS. In addition, the formers were able to resorb the dentine matrix more deeply. Data provided by the microscope were used to reconstruct three-dimensional images of the dentine slices with pseudo colours varying with the depth of erosion. Vertical scanning profilometry, a technique used to measure the roughness of polished or etched surfaces in metallurgic industry, can be used to accurately measure the eroded volume and the mean erosion depth done by osteoclasts in the resorption pit assay.     

Quantitative evaluation of bone resorption activity of osteoclast‐like cells by measuring calcium phosphate resorbing area using incubator‐facilitated and video‐enhanced microscopy

Quantitative evaluation of the ability of bone resorption activity in live osteoclast‐like cells (OCLs) has not yet been reported on. In this study, we observed the sequential morphological change of OCLs and measured the resorbing calcium phosphate (CP) area made by OCLs alone and with the addition of elcatonin utilizing incubator facilitated video‐enhanced microscopy. OCLs, which were obtained from a coculture of ddy‐mouse osteoblastic cells and bone marrow cells, were cultured on CP‐coated quartz cover slips. The CP‐free area increased constantly in the OCLs alone, whereas it did not increase after the addition of elcatonin. This study showed that analysis of the resorbed areas under the OCL body using this method enables the sequential quantitative evaluation of the bone resorption activity and the effect of several therapeutic agents on bone resorption in vitro. Microsc. Res. Tech, 2009. © 2008 Wiley‐Liss, Inc.     

Histological evaluation on bone regeneration of dental implant placement sites grafted with a self-setting alpha-tricalcium phosphate cement

This study aimed to evaluate the histological characteristics of the new bone formed at dental implant placement sites concomitantly grafted with a self-setting tricalcium phosphate cement (BIOPEX-R). Standardized defects were created adjacent to the implants in maxillae of 4-week-old male Wistar rats, and were concomitantly filled with BIOPEX-R. Osteogenesis was examined in two sites of extreme clinical relevance: (1) the BIOPEX-R-grafted surface corresponding to the previous alveolar ridge (alveolar ridge area), and (2) the interface between the grafting material and implants (interface area). At the alveolar ridge area, many tartrate-resistant acid phosphatase (TRAPase)-reactive osteoclasts had accumulated on the BIOPEX-R surface and were shown to migrate toward the implant. After that, alkaline phosphatase (ALPase)-positive osteoblasts deposited new bone matrix, demonstrating their coupling with osteoclasts. On the other hand, the interface area showed several osteoclasts initially invading the narrow gap between the implant and graft material. Again, ALPase-positive osteoblasts were shown to couple with osteoclasts, having deposited new bone matrix after bone resorption. Transmission electron microscopic observations revealed direct contact between the implant and the new bone at the interface area, although few thin cells could still be identified. At both the alveolar ridge and the interface areas, newly formed bone resembled compact bone histologically. Also, concentrations of Ca, P, and Mg were much alike with those of the preexistent cortical bone. In summary, when dental implant placement and grafting with BIOPEX-R are done concomitantly, the result is a new bone that resembles compact bone, an ideal achievement in reconstructive procedures for dental implantology.     

Matrix metalloproteinases (MMP) and cathepsin K contribute differently to osteoclastic activities

The best established proteolytic event of osteoclasts is bone matrix solubilization by the cysteine proteinase cathepsin K. Here, however, we draw the attention on osteoclastic activities depending on matrix metalloproteinases (MMPs). We discuss the observations supporting that MMPs contribute significantly to bone matrix solubilization in specific areas of the skeleton and in some developmental and pathological situations. Our discussion takes into account (1) the characteristics of the bone remodeling persisting in the absence of cathepsin K, (2) the ultrastructure of the resorption zone in response to inactivation of MMPs and of cathepsin K in different bone types, (3) bone resorption levels in MMP knockout mice compared to wild-type mice, (4) the identification of MMPs in osteoclasts and surrounding cells, and (5) the effect of different bone pathologies on the serum concentrations of specific collagen fragments believed to discriminate between cathepsin K and MMP cleavage. Next, we provide evidence that MMPs are very critical for osteoclast migration, thereby controlling also the cell-matrix interactions required for cell attachment/detachment. The evidence supporting this role is based on a model of osteoclast recruitment in primitive long bones, an assay of osteoclast invasion through collagen gel, and the effect of proteinase inhibitors/knockouts in these models. Furthermore, we mention observations indicating a role of MMPs in initiation of bone resorption. Finally, we emphasize the many distinct ways MMPs may alter focally the extracellular environment thereby regulating the osteoclast behavior. Although the understanding of MMPs in osteoclast biology is rapidly expanding, it is suspected that important roles remain to be discovered.     

Adrenergic regulation of bone metabolism: possible involvement of sympathetic innervation of osteoblastic and osteoclastic cells

It has been demonstrated that human osteoblastic as well as osteoclastic cells are equipped with adrenergic receptors and neuropeptide receptors and that they constitutively express diffusible axon guidance molecules that are known to function as a chemoattractant and/or chemorepellent for growing nerve fibers. These findings suggest that the extension of axons of sympathetic and peripheral sensory neurons to osteoblastic and osteoclastic cells is required for the dynamic neural regulation of local bone metabolism. Recently, bone resorption modulated by sympathetic stimulation was demonstrated to be associated with ODF (osteoclast differentiation factor) and OCIF (osteoclastogenesis inhibitory factor) produced by osteoblasts/stromal cells. This review summarizes the evidence implicating sympathetic neuron action in bone metabolism. The possible function of osteoclastogenesis, which could result in the initiation of sympathomimetic bone resorption, is also discussed.     

Intracellular membrane trafficking in bone resorbing osteoclasts

There is ample evidence now that the two major events in bone resorption, namely dissolution of hydroxyapatite and degradation of the organic matrix, are performed by osteoclasts. The resorption cycle involves several specific cellular activities, where intracellular vesicular trafficking plays a crucial role. Although details of these processes started to open up only recently, it is clear that vesicular trafficking is needed in several specific steps of osteoclast functioning. Several plasma membrane domains are formed during the polarization of the resorbing cells. Multinucleated osteoclasts create a tight sealing to the extracellular matrix as a first indicator of their resorption activity. Initial steps of the sealing zone formation are alpha(v)beta(3)-integrin mediated, but the final molecular interaction(s) between the plasma membrane and mineralized bone matrix is still unknown. A large number of acidic intracellular vesicles then fuse with the bone-facing plasma membrane to form a ruffled border membrane, which is the actual resorbing organelle. The formation of a ruffled border is regulated by a small GTP-binding protein, rab7, which indicates the late endosomal character of the ruffled border membrane. Details of specific membrane transport processes in the osteoclasts, e.g., the formation of the sealing zone and transcytosis of bone degradation products from the resorption lacuna to the functional secretory domain remain to be clarified. It is tempting to speculate that specific features of vesicular trafficking may offer several potential new targets for drug therapy of bone diseases.     

Effect of low-level laser therapy on intramembranous and endochondral autogenous bone grafts healing

The aim of this study was to evaluate the healing process of intramembranous (IM) and endochondral (EC) bone grafts under low‐level laser therapy (LLLT). Male rabbits underwent onlay autogenous bone grafts (1 cm in diameter) retrieved from the calvaria and iliac crest and fixed on parietal bones, divided into four groups: Calvaria (C), Iliac (I), Calvaria + LLLT (C+L), and Iliac + LLLT (I+L). Animals from C+L and I+L Groups had their grafts exposed to LLLT (AlGaAs–808 nm, CW, 30 mW, 0.028 cm² average laser beam area), 15 s irradiation time (16 J/cm² per point–total of 64 J/cm² per session). After 7, 14, 30, and 60 days, grafts were retrieved and resorption pattern analyzed by means of morphometry and TRAP‐positive osteoclasts detection. Differences in the resorption levels of iliac grafts were observed, presenting 40% in I group against 8% in I+L grafts at the 14th day of evaluation (P < 0.05). After 30 days, resorption was maintained at 41% in I group, whereas I+L presented 15% in the same period (P = 0.0591). No significant differences were noted in the rates of calvaria grafts resorption in all periods. A significant higher number of osteoclasts on the grafts' surface was observed in C+L Group at day 30, in comparison with C group. In I+L Group, prevalence of osteoclasts was marked at day 7 (P < 0.05) in comparison to I Group. In general, it was concluded that biomodulative effects of LLLT did not significantly affect healing and resorption processes of autogenous bone grafts from EC and IM origins. Microsc. Res. Tech. 75:1237–1244, 2012. © 2012 Wiley Periodicals, Inc.     

Osteoclastic bone resorption around intraosseous screws in rat and pig mandibles

This report describes osteoclastic bone resorption around intraosseous fixation screws in rat and pig mandibles. These screws supported distraction devices and provided for neutral fixation following the distraction period. Progressive clinical instability of bicortical screws and radiographic and histologic evidence of osseous resorption were frequent findings. In rats, clinical evaluation revealed screw loss and/or loosening in 50% of the rats between 11 and 30 days of neutral fixation. Radiographic signs of resorption were apparent around 60% of the screws that were in place at the end of the observation period. The total rate of resorption or loss was 64 of 80 screws (80%). Histologic examination showed partial or extensive osteolysis around the screw holes in 87% of screws that were clinically fixed in the bone at sacrifice. In histologic sections of porcine specimens, osseous resorption around identifiable screw holes was present in 75% of the cases, and showed progressive increase in resorption with time. Osteoclastic resorption was common around bicortical screws that were evaluated after they had served for osseous stability.     

弓网系统滑动电接触表面磨损特征研究

为探究弓网系统受电弓滑板在不同电流、接触压力和滑动速度条件下的磨耗特性,开展弓网载流磨损实验,探讨不同工况下摩擦副的载流效率、电弧能量、摩擦因数和温度;分别使用金相显微镜与表面粗糙度仪对实验后的滑板磨损区域进行分块多次测量,使用一种基于Otsu大津算法的图像处理方法,将凹坑部分从形貌图像中提取并进行标记计算,统计分析不同工况下凹坑面积、数量和粗糙度参数（包括轮廓算术平均偏差和最大轮廓谷深）的变化情况。结果表明：随着电流的增大,凹坑面积和数量增加,粗糙度参数减小;随着接触压力的增大,凹坑面积减小,凹坑数量先减小后略有增大,粗糙度参数减小;随着滑动速度的增大,凹坑面积、数量和粗糙度参数都增大。     

Exosomes derived from osteoclasts under compression stress inhibit osteoblast differentiation

Orthodontic tooth movement is triggered by orthodontic force loading on the periodontal ligament and is achieved by alveolar bone remodeling, which is regulated by intimate crosstalk between osteoclastogenesis and osteoblast differentiation. Whether the communication between osteoclasts and osteoblasts is influenced by orthodontic compression stress requires further clarification. In this study, osteoclasts were differentiated for 10 days. On day 4 of differentiation, the number of pre-osteoclasts peaked, as determined by the increased expression of RANK and the number of multinucleated cells. After 24 h of compression stress loading, on day 4, the number of osteoclasts increased, and the optimal magnitude of stress to promote osteoclastogenesis was determined as 1 g/cm2. Moreover, the results of RNA-sequencing analysis showed that the miRNA expression profile changed markedly after compression loading and that many of the altered miRNAs were associated with cell communication functions. A series of indirect co-culture experiments showed an inhibitory effect of osteoclasts on osteoblast differentiation, especially after compression. Next, we added osteoclast-derived exosomes to hPDLSCs during osteoblast differentiation. Exosomes derived from osteoclasts under compression (cEXO) showed a greater inhibitory effect on osteoblast differentiation, compared to exosomes from osteoclasts without compression (EXO). Therefore, we analyzed differentially expressed miRNAs associated with bone development functions in exosomes: miR-223-5p and miR-181a-5p were downregulated, whereas miR-133a-3p, miR-203a-3p, miR-106a-5p, and miR-331-3p were upregulated; these altered expressions may explain the enhanced inhibitory effect of compression stress.     

Ultrastructural and immunohistochemical study of the effect of sodium alendronate in the progression of experimental periodontitis in rats

The aim of the present research was to investigate the ultrastructural aspects and the immunoexpression of receptor activator of NFκB ligand (RANKL) and osteoprotegerin (OPG) on experimental periodontal disease of alendronate (ALN)‐treated rats. Male Wistar rats received daily injections of 2.5 mg/kg body weight of ALN during 7 days previously and 7, 14, and 21 days after the insertion of a 4.0 silk suture into the gingival sulcus around the right upper second molar. Specimens were fixed in 0.1% glutaraldehyde + 4% formaldehyde under microwave irradiation, decalcified in 4.13% EDTA and paraffin embedded for TRAP histochemistry and immunohistochemistry for RANKL and OPG, or embedded in Spurr epoxy resin for TEM analysis. ALN reduced the activity of osteoclasts and significantly decreased the resorption of the alveolar crest. In the control group the alveolar crest appeared resorbed by TRAP‐positive osteoclasts, which presented ultrastructural features of activated cells. The immunoexpression of RANKL was not inhibited by the drug; however, the expression of OPG was increased in the treated animals. The alveolar crest of ALN‐treated specimens at 21 days showed signs of osteonecrosis, like empty osteocyte lacunae, the exposed bone regions and bacterial infection. The results showed that ALN treatment in individuals with periodontal disease represents a risk of osteonecrosis because of the reduced activity of osteoclasts resultant of the increased immunoexpression of OPG. Microsc. Res. Tech. 77:902–909, 2014. © 2014 Wiley Periodicals, Inc.     

High dose glucocorticoid hampers bone formation and resorption after bone marrow ablation in rat

We analyzed the effect of glucocorticoid on bone regeneration after bone marrow ablation in tibiae of 8-week-old rats. Methylprednisolone sodium succinate (MPSS) was injected intramuscularly at a dose of 100 mg/kg/day for 3 days. Tibiae on days 1, 3, 5, 7, 10, 12, and 14 after ablation were subjected to tartrate-resistant acid phosphatase staining, immunohistochemistry, in situ hybridization, and transmission electron microscopy (TEM), and measurement of the volume of newly-formed bone and the osteoclast number. MPSS significantly decreased the newly-formed bone volume on day 7, and immature bone still remained on day 10 in the MPSS-treated group. The volume of this bone was significantly higher than that in the control group. However, there were no differences between the groups in the osteoclast number, the expression of mRNAs for osteoblast differentiation markers, and alkaline phosphatase and cathepsin K judged by immunohistochemistry. TEM findings showed no difference in the form of osteoblasts, whereas osteoclasts in the MPSS-treated group had less developed ruffled borders, compared to those in the control group. These results suggest that MPSS treatment affects neither the differentiation nor the shape of osteoblasts, and does not change the osteoclast number or the cathepsin K level. However, high dose MPSS inhibits both bone formation and resorption during bone regeneration after rat tibial bone marrow ablation, and inhibits ruffled border formation in osteoclasts. These data will be useful to develop bone regenerative therapies for bone diseases due to high dose steroid administration.     

Single-cell RNA sequencing reveals classical monocytes are the major precursors of rat osteoclasts

To dissect which subset of bone marrow monocyte is the major precursor of osteoclast, 3-month-old rat bone marrow was obtained for single-cell RNA sequencing. A total of 6091 cells were acquired for detailed analysis, with a median number of 1206 genes detected per cell and 17,959 genes detected in total. A total of 19 cell clusters were recognized, with the main lineages identified as B cells, Granulocytes, Monocytes, T cells, Erythrocytes and Macrophages. Monocytes were further divided into classical monocytes and non-classical monocytes. Compared with non-classical monocytes, classical monocytes highly expressed osteoclast differentiation related genes Mitf, Spi1, Fos and Csf1r. Additionally, biological processes of classical monocytes were related to osteoclast differentiation. qPCR revealed differentially expressed genes of classical monocytes played a role in osteoclast differentiation. In conclusion, classical monocytes were identified as the main precursors of osteoclasts in rats, and may contribute to osteoclast differentiation by regulating S100a4, S100a6, S100a10, Fn1, Vcan and Bcl2a1. The results of this study contribute to the understanding of the origin of osteoclasts and may provide potential biomarkers for early diagnosis of bone metabolic diseases, as well as molecular and cellular targets for clinical intervention in bone metabolic diseases.     

Comparative study of decalcification versus nondecalcification for histological evaluation of one‐wall periodontal intrabony defects in dogs

Biological reactions between biomaterials and surrounding tissues, analyzed by histology, may be important predictors of clinical healing pattern and selection of slide preparation techniques requires a careful consideration regarding sample properties. In this study, we compared histology of bone specimens prepared with or without decalcification and performed histological and histomorphometrical assessments. For the histological evaluation, one‐wall intrabony defects were created around the mandibular molars of six adult dogs, filled with biphasic calcium phosphate, synthetic bone graft material/recombinant human bone morphogenic protein‐2, and healing pattern was histologically evaluated at 4 and 12 weeks. New bone formation in 5 × 4 × 4 mm defects and the length of new cementum, connective tissue attachments around the teeth and number of osteoclasts were measured by histomorphometric analysis. After decalcification, new cementum was easily observed and was significantly increased at week 4. In nondecalcified samples, significantly increased connective tissue attachments were seen at week 12. After 12 weeks, the number of countable multinucleated osteoclasts was significantly increased by 62% in nondecalcified versus calcified tissue sections (P = 0.030). Histomorphometric results may be significantly affected by histological preparation method and therefore, selecting the most appropriate histological preparation method is essential for reliable diagnosis and evaluation of bony samples in studies analyzing tissue regeneration. Microsc. Res. Tech. 78:94–104, 2015. © 2014 Wiley Periodicals, Inc.     

Natural frequency analysis of tooth stability under various simulated types and degrees of alveolar vertical bone loss

The aim of this study was to test natural teeth stability under various simulated types and degrees of alveolar vertical bone loss, as well as to assess the role that the surrounding bone played for maintaining tooth stability. A three-dimensional finite element model of the human maxillary central incisor with surrounding tissue, including periodontal ligament, enamel, dentin, pulp, and alveolar bone, was established. One side and multiple vertical bone loss were simulated by means of decreasing the surrounding bone level apically from the cemento-enamel junction in 1 mm steps incrementally downward for 10 mm. Natural frequency values of the incisor model with various types and degrees of bone loss were then calculated. The results showed that, with one-sided bone resorption, the model with labial bone loss had the lowest natural frequency decreasing rates (8.2 per cent). On the other hand, in cases of multiple bone loss, vertical bone resorption at the mesial and distal sides had more negative effects on tooth stability compared to vertical bone losses on facial and lingual sides. These findings suggest that the natural frequency method may be a useful, auxiliary clinical tool for diagnosis of vertical periodontal diseases.     

Differentiation and functions of osteoclasts and odontoclasts in mineralized tissue resorption

The differentiation and functions of osteoclasts (OC) are regulated by osteoblast-derived factors such as receptor activator of NFKB ligand (RANKL) that stimulates OC formation, and a novel secreted member of the TNF receptor superfamily, osteoprotegerin (OPG), that negatively regulates osteoclastogenesis. In examination of the preosteoclast (pOC) culture, pOCs formed without any additives expressed tartrate-resistant acid phosphatase (TRAP), but showed little resorptive activity. pOC treated with RANKL became TRAP-positive OC, which expressed intense vacuolar-type H(+)-ATPase and exhibited prominent resorptive activity. Such effects of RANKL on pOC were completely inhibited by addition of OPG. OPG inhibited ruffled border formation in mature OC and reduced their resorptive activity, and also induced apoptosis of some OC. Although OPG administration significantly reduced trabecular bone loss in the femurs of ovariectomized (OVX) mice, the number of TRAP-positive OC in OPG-administered OVX mice was not significantly decreased. Rather, OPG administration caused the disappearance of ruffled borders and decreased H(+)-ATPase expression in most OC. OPG deficiency causes severe osteoporosis. We also examined RANKL localization and OC induction in periodontal ligament (PDL) during experimental movement of incisors in OPG-deficient mice. Compared to wild-type OPG (+/+) littermates, after force application, TRAP-positive OC were markedly increased in the PDL and alveolar bone was severely destroyed in OPG-deficient mice. In both wild-type and OPG-deficient mice, RANKL expression in osteoblasts and fibroblasts became stronger by force application. These in vitro and in vivo studies suggest that RANKL and OPG are important regulators of not only the terminal differentiation of OC but also their resorptive function. To determine resorptive functions of OC, we further examined the effects of specific inhibitors of H(+)-ATPase, bafilomycin A1, and lysosomal cysteine proteinases (cathepsins), E-64, on the ultrastructure, expression of these enzymes and resorptive functions of cultured OC. In bafilomycin A1-treated cultures, OC lacked ruffled borders, and H(+)-ATPase expression and resorptive activity were significantly diminished. E-64 treatment did not affect the ultrastructure and the expression of enzyme molecules in OC, but significantly reduced resorption lacuna formation, by inhibition of cathepsin activity. Lastly, we examined the expression of H(+)-ATPase, cathepsin K, and matrix metalloproteinase-9 in odontoclasts (OdC) during physiological root resorption in human deciduous teeth, and found that there were no differences in the expression of these molecules between OC and OdC. RANKL was also detected in stromal cells located on resorbing dentine surfaces. This suggests that there is a common mechanism in cellular resorption of mineralized tissues such as bone and teeth.