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.     

Transmission electron microscopic demonstration of vimentin in rat osteoblast and osteocyte cell bodies and processes using the immunogold technique

Background: The immunogold labeling technique and transmission electron microscopy were used to demonstrate the expression and position of the intermediate filament vimentin in rat osteoblast and osteocyte cell bodies and cell processes. Conventional light and transmission electron microscopic studies of bone cells demonstrated adjacent cell linkage to be mediated by osteoblast and osteocyte processes present within the canalicular system traversing the bone matrix. The cell processes were filled with densely packed filaments, many of which have been shown previously to be actin microfilaments. The appearance, however, of 10 nm diameter filaments in some cell processes and the fact that the intermediate filament vimentin has been defined in many cells of mesenchymal origin raised the possibility that some of these filaments might be vimentin. The ultrastructural colloidal gold immunochemical technique allowed for demonstration in situ of the expression of vimentin filaments plus accurate definition of their position. Methods: The studies were performed in newborn rat femoral and tibial diaphyseal cortical bone and in 1-week-old repair bone from 2.4 mm diameter defects made through the lateral cortex in 6-week-old rat femurs and tibias. The bone tissues for the immunochemical study were fixed in 1% glutaraldehyde, 4% paraformaldehyde, and 0.1 M phosphate buffer (pH 7.4) for 2 days. Decalcification was performed in 6% EDTA for 2–3 days. Infiltration involved use of Lowicryl resin K4M, and the embedding and curing processes were performed in a cryostat with temperatures ?30°C. An antivimentin monoclonal antibody was used for labeling using the postembedding technique. Effective antibody dilutions ranged from 1:10 to 1:200, with the dilutions of 1:25 and 1:100 showing the best combination of filament labeling with the least matrix background. The grids were exposed to 10 nanometer gold colloid conjugated goat anti-mouse IgM for demonstration of binding. Results: Vimentin immunolabeling was defined clearly in relation to filaments within the osteoblast and osteocyte cell body cytoplasm, throughout the entire length of the osteoblast and osteocyte cell processes, and in close relationship to the intercellular gap junctions which were present within the cell processes both close to the cell bodies and within the canaliculi well away from them. Conclusions: Immunogold labeling demonstrates the presence of the intermediate filament vimentin in osteoblast and osteocyte cell bodies and processes of rat bone. Vimentin distribution is not concentrated to specific areas, is present throughout the extent of the bodies and processes, and is seen immediately adjacent to gap junctions. © 1995 Wiley-Liss, Inc.     

Modeling Deformation-Induced Fluid Flow in Cortical Bone’s Canalicular–Lacunar System

To explore the potential role that load-induced fluid flow plays as a mechano–transduction mechanism in bone adaptation, a lacunar–canalicular scale bone poroelasticity model is developed and implemented. The model uses micromechanics to homogenize the pericanalicular bone matrix, a system of straight circular cylinders in the bone matrix through which bone fluids can flow, as a locally anisotropic poroelastic medium. In this work, a simplified two-dimensional model of a periodic array of lacunae and their surrounding systems of canaliculi is used to quantify local fluid flow characteristics in the vicinity of a single lacuna. When the cortical bone model is loaded, microscale stress, and strain concentrations occur in the vicinity of individual lacunae and give rise to microscale spatial variations in the pore fluid pressure field. Furthermore, loading of the bone matrix containing canaliculi generates fluid pressures in the contained fluids. Consequently, loading of cortical bone induces fluid flow in the canaliculi and exchange of fluid between canaliculi and lacunae. For realistic bone morphology parameters, and a range of loading frequencies, fluid pressures and fluid–solid drag forces in the canalicular bone are computed and the associated energy dissipation in the models compared to that measured in physical in vitro experiments on human cortical bone. The proposed model indicates that deformation-induced fluid pressures in the lacunar–canalicular system have relaxation times on the order of milliseconds as opposed to the much shorter times (hundredths of milliseconds) associated with deformation-induced pressures in the Haversian system.     

A review of the differing roles of dead and live osteocytes

BackgroundOsteocytes form a network through gap junction-coupled cell processes and canaliculi throughout bone; this network extends to the osteoblasts in the bone surface. The osteocyte network is considered to function in mechanosensing and mechanotransduction. However, the lack of suitable animal models makes it difficult to clarify the function of osteocytes.HighlightAny kind of osteocyte death results in necrosis, whereby the intracellular contents, including immunostimulatory molecules, which activate osteoclastogenesis, are released through the canaliculi to the bone surface. This leads to enhanced bone resorption in the damaged region of the bone. Overexpression of Bcl2 in osteoblasts reduces the number of osteoblast processes, resulting in a reduction in the numbers of osteocyte processes and canaliculi. The osteocytes gradually die without enhancement of bone resorption because a severe reduction in the number of canaliculi interrupts the release of intracellular contents to the bone surface. Bcl2 transgenic mice at 4 months of age, in which the osteocyte network is disrupted, are an appropriate mouse model for the evaluation of osteocyte function. These mice show that the osteocyte network enhances bone resorption and inhibits bone formation under physiological conditions, and that these osteocyte functions are augmented under unloaded conditions. Under such conditions, Rankl upregulation in osteoblasts and Sost upregulation in osteocytes are, at least in part, responsible for enhanced bone resorption and suppressed bone formation, respectively.ConclusionDead osteocytes induce bone resorption, while live osteocytes enhance bone resorption and inhibit bone formation under physiological conditions, while their functions are augmented under unloaded conditions.     

Alpinumisoflavone rescues glucocorticoid-induced apoptosis of osteocytes via suppressing Nox2-dependent ROS generation

Background

Long term use of glucocorticoids is one of the most common causes of secondary osteoporosis. Osteocyte, the most abundant cell type in bone, coordinates the function of osteoblast and osteoclast. This study evaluates the protective effect of alpinumisoflavone (AIF), a naturally occurring flavonoid compound, on dexamethasone (Dex)-induced apoptosis of osteocytes.

Osteocyte death during orthodontic tooth movement in mice

Objective:To investigate the time course of osteocyte death in a mouse model of orthodontic tooth movement (OTM) and its association to the caspase-3 activation pathway and osteoclast formation.Materials and Methods:Twenty-five male wild type CD-1 mice (8–12 weeks old) were loaded with an orthodontic appliance. A spring delivering 10–12 g of force was placed between the right first molar and the incisor to displace the first molar mesially. The contralateral unloaded sides served as the control. The animals were equally divided into five different time points: 6, 12, 24, and 72 hours and 7 days of orthodontic loading. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, caspase-3 immunostaining, and tartrate-resistant acid phosphatase (TRAP) staining was performed on histologic sections of the first molars. The labeling was quantified in osteocytes on the compression side of the alveolar bone at each time point.Results:TUNEL labeling significantly increased at 12, 24, and 72 hours after orthodontic loading; the peak was observed at 24 hours. Elevated caspase-3 labeling was noted at 12, 24, and 72 hours and 7 days after loading, although the increase was not significant. Significant osteoclast formation was initially evident after 72 hours and progressively increased up to 7 days.Conclusions:Osteocyte death during OTM peaks at 24 hours, earlier than initial osteoclast activation. However, only a slight trend for increased caspase-3 activity suggests that other mechanisms might be involved in osteocyte death during OTM.     

股骨头缺血坏死对股骨近端骨组织的影响

目的 :探讨股骨头缺血坏死患者的股骨近端骨改变。方法 :18例股骨头缺血坏死行全髋关节置换术的患者 ,对其股骨头和股骨近端四处松质骨标本进行组织学检查 ,并用 18例骨关节炎患者作为对照组 ,所有标本采用双盲方式进行检查。结果 :股骨头缺血坏死患者小粗隆下方 4cm处存在广泛的骨坏死 ,两组之间股骨近端的骨坏死程度有极显著性差异 (P <0 .0 0 1)。结论 :股骨近端的骨改变可能是股骨头缺血坏死行全髋关节置换早期失败的原因之一。     

不同直径的骨移植物中骨细胞转归的实验研究

目的观察不同直径的骨移植物修复骨缺损过程中,供体骨内细胞转归的情况。方法近交系DA大鼠174只,其中雄性大鼠58只,作为供体;雌性大鼠116只,作为受体。将受体随机分为块状骨组、颗粒骨组和空白对照组,建立大鼠桡骨骨缺损模型,取雄性大鼠髂骨为供体骨,分别制成直径为2mm的骨块和直径为0．3～0．5mm的颗粒骨,植入骨缺损,分别于术后1、4d和1、2、4、6、10周取材,采用聚合酶链反应技术观察受体骨组织中Y染色体性别决定基因（Sry）的表达情况并观察各组的组织学改变。结果块状骨组在移植早期Sry的表达逐渐减少,至1周消失。4周后再次出现,并且随时间延长表达逐渐增多;颗粒骨组Sty的表达随时间延长逐渐减少,但各时间段均有Sty的表达。在同一时间点,颗粒骨较块状骨有更多的骨细胞存活,其成骨效果优于块状骨。结论不同直径的骨移植物修复骨缺损时均有供体骨细胞参与,但颗粒骨内有更多的骨细胞存活,这些骨细胞参与骨缺损修复的各个阶段,并能加速骨缺损的修复,为临床上修复骨缺损和脊柱融合提供了新的有效的方法。     

Osteocyte Apoptosis and Osteoclast Presence in Chicken Radii 0–4 Days Following Osteotomy

Osteocyte apoptosis caused by load-induced microdamage is followed by osteoclastic bone remodeling, and a causal link between apoptosis and repair has been suggested. The objectives of the present study were to use a chick model to examine the incidence of osteocyte apoptosis and the presence of osteoclasts during the first 96 hours following an osteotomy, prior to extensive callus mineralization. Osteotomies were performed on the right radii of 24 chicks at 23–24 days of age. The left radii served as controls. Radii were collected and processed at six time points following surgery (0, 12, 24, 48, 72, and 96 hours). Decalcified bone tissue sections were stained either for apoptosis using a modified TUNEL procedure or for tartrate-resistant acid phosphatase to identify osteoclasts in the intracortical and periosteal envelopes. The percentage of apoptotic osteocytes, as well as osteoclast counts (n/mm or n/mm²) were quantified in four regions (0–1, 1–2, 2–4, and 4–8 mm from the site of the osteotomy; regions 1–4, respectively) in the osteotomized radii and in the same measured areas in the control radii. Data for osteocyte apoptosis and osteoclasts in the control limb were subtracted from the osteotomized limb data to identify differences due to surgical influence. The incidence of osteocyte apoptosis was significantly higher at 12, 24, 48, and 72 hours versus 0 hours following osteotomy, and the response was highest in region 1; however, there was no interaction between time and region. Intracortical osteoclast counts (n/mm²) were elevated after 48 hours, and the response was similar in all regions. The data demonstrate that osteocyte apoptosis occurs within 24 hours in response to an osteotomy and temporally precedes an increase in osteoclast presence. Hence, osteocyte apoptosis may play a role in signaling during the bone healing process.