Electron microscopy of developing calvaria reveals images that suggest that osteoclasts engulf and destroy osteocytes during bone resorption

Summary It is generally accepted that osteoclasts are responsible for the breakdown and removal of bone matrix constituents. However, very little is known about the fate of osteocytes during bone resorption. In the present study we have examined sites of bone destruction in calvaria of young rats aged 4–9 days in the hope of obtaining information on the fate of osteocytes. Decalcified glutaraldehyde-formaldehyde-fixed specimens were prepared for ultrathin section electron microscopy. When sequentially arranged, the images obtained suggest that osteoclasts engulf and destroy osteocytes during bone degradation. We propose that the following sequence of events takes place when a lacuna is opened up by an osteoclast: (1) When the osteoclast comes in contact with an osteocyte, the villi of the ruffled border become flat and broad. (2) Long osteoclastic extensions surround the osteocyte. (3) The osteocyte is subsequently internalized with apparent degradation.     

The effects of high dose of parathyroid hormone on fetal osteoclasts and their precursors in vivo: An ultrastructural-cytochemical study

Background: It is not well known how the immediate precursors of osteoclast develop into osteoclasts in the fetus. This ultrastructural-cytochemical study was designed to clarify the formation process of the osteoclasts and their increased activities in the fetal mouse limb buds after administration of high dose parathyroid hormone (PTH). Methods: Twenty-four or forty-eight hours after the high doses of PTH were injected into amniotic fluid of the pregnant C₃H mice, the femoral limb buds of embryos were dissected out. Tartrate-resistant acid phosphatase (TRAP) reactions were performed while preparing specimens for electron microscopy. Results: Both control and PTH-given preosteoclasts and osteoclasts exhibited TRAP-positivities in dense bodies and vesicles. As effects of PTH, a binucleated preosteoclast of tandem fashion was observed. More osteoclastic hyperactivities were observed in the diaphyseal bone marrow. An osteoclast with a large cytoplasm exhibited two sets of clear zones and ruffled borders. Some osteoclasts demonstrated prominent amoeboid figures, while other osteoclasts developed large cytoplasmic vacuoles, which contained pieces of calcified chondroid bars. Conclusions: Our results revealed the progression of maturation from young preosteoclasts to osteoclasts. An existence of a peculiar binucleated preosteoclasts suggested one of the processes for multinucleation of the osteoclast. Quite remarkable osteoclastic hyperactivities were obviously the effects of high dose PTH. Our results also indicated the endophagocytic ability of the osteoclast. How PTH affected the osteoclasts and their precursors in the diaphyseal bone marrow can be speculated. © 1995 Wiley-Liss, Inc.     

Osteoclasts contain macrophage and megakaryocyte antigens

The origin and mechanism of formation of the osteoclast remains controversial. Although it is known to be derived from a circulating mononuclear percursor, the identity of this cell is unknown. Using a panel of monoclonal antibodies raised against macrophage and other marrow-derived cells, we determined the immunocytochemical staining of human osteoclasts in both fetal bone metaphyseal imprints and frozen sections. Osteoclasts and marrow mononuclear cells were stained by three broad spectrum antimacrophage antibodies, EBM-11, Y182a and BM2. T310, an antibody which stains macrophages and T helper cells, and C17, an antimegakaryocyte antibody, also stained osteoclasts. EBM-11, Y182a and BM2 also stained megakaryocytes in bone imprints as well as normal bone marrow smears. The presence of macrophage-associated antigens in osteoclasts, megakaryocytes and bone marrow mononuclear cells indicates that they are phenotypically similar to macrophages.     

白细胞介素1刺激破骨细胞性骨吸收作用的实验研究

目的：了解白细胞介素1β（IL－1β）在破骨细胞性骨吸收中的刺激作用及其相应的作用机制。方法：分别将新生大鼠破骨细胞单独以及和成骨细胞联合接种于预置象牙片的培养板中。24h后培养液中加入不同浓度的IL－1β。继续培养48h,然后取出象牙片,超声处理后行甲苯胺蓝染色,光镜下观察骨吸收陷窝的数目并计算其部面积。结果：IL－1β能够明显增加坡骨细胞和成骨细胞联合培养组象牙片上吸收陷窝的数目和面前,且刺激作用呈剂量依赖性,但对单独培养的破骨细胞无明显刺激作用。结论：IL－1β的刺激骨吸收作用由成骨细胞所介导,而非直接作用于破骨细胞。     

Etidronate (EHDP) Inhibits Osteoclastic-Bone Resorption, Promotes Apoptosis and Disrupts Actin Rings in Isolate-Mature Osteoclasts

Bisphosphonates, therapeutic reagents against tumoral bone diseases (Paget's disease or osteoporosis), are potent inhibitors of bone resorption. The mechanisms by which they directly act on mature osteoclasts remain unclear. Using a recently developed technique for isolation of highly purified mammalian mature osteoclasts, we demonstrated that etidronate [ethane-1-hydroxy-1,1-diphosphonate (EHDP), 1-hydroxy-1,1-ethylidenebisphosphonate], inhibited directly osteoclastic bone-resorbing activity by pit assay. In addition, EHDP also directly induced apoptosis and disrupted actin rings in osteoclasts. The data support previous data on non-purified osteoclasts and results in vivo. Received: 26 June 1997 / Accepted: 27 July 1998     

Endosteal surfaces in hyperparathyroidism: An enzyme cytochemical study on low-temperature-processed,glycol-methacrylate-embedded bone biopsies

Summary Alkaline phosphatase (AIP) and tartrate-resistant acid phosphatase (TRAP) activities have been studied comparatively in needle biopsies of the iliac crest of four cases of secondary hyperparathyroidism (renal osteodystrophy). AIP activity was associated with the plasma membrane of osteoblasts and their processes, of reticular cells of bone marrow and of young osteocytes of osteoid borders and woven bone. Moreover, it was detected in the fibroblast-like cells of the endosteal fibrosis. These cells were orderly in arrangement and were parallel to the endosteal surfaces near zones of bone formation. They were disorderly near zones of bone resorption. A strong TRAP-positive reaction was present in osteoclasts and mononuclear cells of endosteal fibrosis and in osteocytes located near active osteoclasts and in woven bone. These results suggest that the socalled fibrosis of hyperparathyroidism, rather than representing reparative, inert tissue, consists of osteoblastlike cells, probably precursors of osteoblasts derived by parathormone-stimulated proliferation of AIP-positive stromal cells of bone marrow, and of TRAP-positive, mononuclear cells, probably preosteoclasts. Moreover, they show that TRAP activity can be present in osteocytes, probably under stimulation by the same factors which stimulate osteoclast activity. The histochemical demonstration of AIP and TRAP facilitates the morphological diagnosis of metabolic bone disease and may improve knowledge of bone physiopathology.     

中药含药血清对体外培养鼠破骨细胞的影响

目的 :研究骨康抑制破骨细胞性骨吸收的机理。方法 :新鲜牛皮质骨经锯式切片机横切成 5 0um厚0 .5cm× 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SD大鼠四肢长骨分离破骨细胞。原代破骨细胞培养 2 4小时后 ,加入含药血清培养 7天。结果 :发现培养的细胞具有典型破骨细胞的形态特点。结果可见破骨细胞能在骨片表面形成吸收陷窝 ,其形态多样 ,随时间延长 ,陷窝扩大 ,数量增多 ,而骨康含药血清可抑制骨陷窝数量。结论 :这说明含药血清可直接抑制破骨细胞的功能 ,骨康抑制骨吸收的功能可能与其直接抑制破骨细胞功能有关。     

A novel compound RS-0466 reverses beta-amyloid-induced cytotoxicity through the Akt signaling pathway in vitro

In order to determine whether 5-[bis(carboxymethyl) amino]-2-carboxy4-cyano-3-thiopheneacetic acid distrontium salt (S12911-2) inhibits bone resorption by acting on the differentiation and/or function of osteoclasts, its effects were assessed on the 1,25-dihydroxyvitamin D(3)-induced expression of carbonic anhydrase II and vitronectin receptor in chicken bone marrow cells, and on the resorbing activity of authentic rat osteoclasts cultured on bone slices. S12911-2 dose-dependently inhibited, after a 6-day exposure, the expression of carbonic anhydrase II and vitronectin receptor in stimulated osteoclasts (46% and 40%, respectively, at 10(-3) M Sr(2+), P<0.05). A pre-incubation of bone slices with S12911-2 induced a dose-dependent inhibition of bone resorbing activity from 32% at 10(-4) M Sr(2+) to 66% at 10(-3) M Sr(2+) (P<0.05 in each case). A continuous incubation (10(-3) M Sr(2+)) induced a greater inhibition of bone resorbing activity (73%, P<0.05). The inhibition of bone resorption obtained specifically with S12911-2 is related to an inhibition of the differentiation and resorbing activity of the osteoclasts.     

Denosumab Can Prevent Collapse in Patients with Early‐Stage Steroid‐Induced Osteonecrosis of the Femoral Head by Inhibiting Osteoclasts and Autophagy

ObjectiveThe osteoclastic bone resorption inhibitors might have positive effect in preventing femoral head collapse in patients with osteonecrosis of the femoral head (ONFH). However, as a novel osteoclastic inhibitor, whether denosumab can prevent collapse in steroid‐induced ONFH remains unknown. This study aims to evaluate the treatment effect of denosumab and the potential protective mechanism.MethodsThis was a retrospective study. A total of 161 patients with steroid‐induced ONFH who underwent denosumab treatment were reviewed, and 209 untreated patients were selected as controls. Their clinical characteristics and radiological exam results were obtained. Patients were treated with 60 mg denosumab every 6 months for 2 years. The primary outcome was the incidence of femoral head collapse at 2 years after the initial diagnosis of ONFH. Secondary outcomes included the Harris hip score, progression of osteosclerosis, increase in necrotic area, bone marrow oedema relief, and bone mineral density increase in the femoral head. The Mann–Whitney U test and chi‐square tests were performed to identify the differences between the continuous and categorical variables, respectively. A multivariate logistic regression model was built to identify the factors associated with the treatment effect of denosumab.ResultsThe incidence of femoral head collapse was 42.24% (68/161) in the denosumab group and 54.07% (113/209) in the control group (χ ² = 5.094, p = 0.024; relative risk = 0.787, 95% CI = 0.627–0.973). The excellent‐good rates of the Harris hip score were 63.98% (103/161) in the denosumab group and 44.98% (94/209) in the control group (χ ² = 13.186, p < 0.001). The incidence of osteosclerosis progression in the denosumab group was 55.28% (89/161), which was significantly higher than that in the control group (43.54%, 91/209, χ ² = 5.016, p = 0.025). Meanwhile, a significant increase in bone mineral density was identified in 29.19% (47/161) and 7.18% (15/209) of patients in the denosumab and control groups, respectively (χ ² = 31.600, p < 0.001). The osteoclastic cytoplasm expression of LC3‐II was more positive in the control group than in the denosumab group (immunohistochemistry scoring: 3.58 ± 2.27 vs 6.33 ± 2.64, Z = −2.684, p = 0.007). A total of three independent factors were considered to be associated with the positive treatment effect of denosumab, the time of first denosumab administration (OR = 2.010, 95% CI = 1.272–3.177), osteosclerosis (OR = 1.583, 95% CI = 1.024–2.445), and the necrotic area before denosumab administration (medium necrotic area: OR = 2.084, 95% CI = 1.245–3.487; large necrotic area: OR = 2.211, 95% CI = 1.255–3.893).ConclusionsThe current study demonstrated that denosumab had a positive effect on preventing femoral head collapse in patients with steroid ONFH. This effect might be closely associated with the inhibition of osteoclasts and their autophagy.     

重组人生长激素对去势大鼠正畸牙牙周组织改建的影响

目的:探讨重组人生长激素(recombinanthumangrowthhormone,rh-GH)对去势(ovariectomy,OVX)大鼠正畸牙移动后牙周组织细胞变化的影响。方法:30只7周龄SPF级雌性Wistar大鼠随机分为3组:对照组、去势盐水组(OVX-NS组)和去势生长激素组(OVX-GH组)。将去势摘除双侧卵巢和腹部皮下注射rh-GH作为不同处理因素,观察第15天和第30天时3组大鼠正畸牙移动引起牙槽骨受压侧破骨细胞数的变化,并进行组织学观察。用SPSS12.0软件包对数据进行完全随机设计资料的方差分析。结果:对3组大鼠不同时间正畸牙牙槽骨受压侧破骨细胞计数进行比较,发现同一时间3组数据的差异有统计学意义(P<0.05);OVX-NS组比OVX-GH组显著增多(P<0.05),对照组最少;同组相比,第15天处死的大鼠与第30天处死的大鼠其破骨细胞数无显著差异(P>0.05);牙周组织学观察见,OVX-GH组牙周膜的损伤和创伤性炎症较OVX-NS组明显减轻。结论:去势(成年)大鼠腹部皮下注射rh-GH,能够减少其正畸牙移动引起的受压侧破骨细胞数目,同时可促进牙周组织因正畸力所致的病理性变化的恢复,对成年正畸治疗有良好的协同作用。