破骨细胞在骨组织工程中的意义及其研究策略

破骨细胞是骨吸收细胞,在骨塑造和重塑中发挥重要作用。目前在破骨细胞对成骨细胞调节方面的影响了解很少。最近的研究表明在骨发育过程中破骨细胞的缺乏会导致骨基质排列紊乱、矿化减少、成骨细胞行为异常。破骨细胞在骨组织工程的引入将会解决骨组织工程面临的许多问题。破骨细胞前体在组织工程化骨上生成破骨细胞是一条生理、实际的破骨细胞引入途径。     

流体剪切力在骨生长、重建中的重要作用

骨组织具有最优化结构,是一个典型的结构-功能受生物力学控制的例子。力学因素在骨的生长、重建和成形中起着十分重要的作用,但力学刺激的本质形式至今无法确认。骨组织存在多孔结构,力学负荷引起的变形会促使流体流动对骨细胞产生作用,离体实验也证实骨细胞能对流体产生响应,因此流体剪切力是研究骨组织受力学调控时考虑的一个重要方面。现有的研究主要体现在流体的有效作用形式,细胞的生物学反应,以及流体剪切力在细胞中的力学转导;各个方面的研究都提示,流体剪切力至少部分的参与了骨组织内的力转导。作者就这方面的研究进展作一综述。     

快速成形技术在骨组织工程领域的应用进展

在骨组织工程中 ,具有高孔隙率的人工细胞外基质或支架是骨组织细胞 (成骨细胞、破骨细胞和骨细胞 )黏附、增殖、分化和骨组织的形成必不可少的条件 ,传统的支架材料由于缺乏应有的机械强度、相互连通的孔道和可控的孔隙率或通道尺度而缺乏实际应用价值 ,因此寻找理想的支架材料是目前骨组织工程研究的热点之一 ,本文对目前正在不断发展的快速成形技术在骨组织丁程中的应用现状进行了综述。     

锶盐与模拟失重大鼠骨细胞的凋亡

背景:模拟失重促进成骨细胞、骨细胞凋亡,而锶能降低失重状态下骨组织中成骨细胞、骨细胞凋亡率,促进骨形成。目的:观察锶盐对失重状态下骨组织中细胞凋亡的防治效应。方法:5周龄SD大鼠建立失重动物模型,在悬吊前3d或悬吊时开始以锶盐灌胃。建模后7d,使用全自动生化仪检测大鼠血清中钙、磷、碱性磷酸酶水平,放射免疫分析法测定骨钙素质量浓度,原位细胞凋亡检测技术检测骨组织中细胞凋亡,免疫组化方法检测骨组织中Fas蛋白水平。结果与结论:模型组大鼠血清中碱性磷酸酶、骨钙素水平均显著低于相应对照组(P0.05),但血钙,磷浓度均较相应对照组升高(P0.05),骨组织中成骨细胞、骨细胞及骨髓间充质细胞凋亡指数、骨组织中Fas抗原水平均高于相应对照组(P0.01)。悬吊前3d始及悬吊时始锶盐灌胃组碱性磷酸酶,骨钙素水平均显著高于模型组(P0.05),而血钙、骨组织中成骨细胞、骨细胞及骨髓间充质细胞凋亡指数、骨组织中Fas抗原水平均显著低于模型组(P0.05)。说明尾部悬吊模拟失重增加骨组织中Fas蛋白表达,从而增加大鼠骨组织中成骨细胞、骨细胞及骨髓间充质细胞凋亡,而锶盐可降低失重状态下大鼠骨组织Fas蛋白表达,从而降低成骨细胞、骨细胞及骨髓间充质细胞凋亡。     

骨细胞的力学感受器

骨细胞是骨骼中最丰富和寿命最长的细胞,是骨重建的调节器。骨细胞在内分泌调节和钙磷酸盐代谢中发挥重要作用,也是力学刺激的主要响应者,感知力学刺激以直接或间接的方式对刺激做出反应。骨细胞中的力学转导是一个复杂而精细的调节过程,涉及细胞与其周围环境、相邻细胞以及细胞内部不同功能的力学感受器之间的相互作用。目前已知的骨细胞主要力学感受器包括初级纤毛、Piezo离子通道、整合素、细胞外基质以及基于连接蛋白的细胞间连接。这些力学感受器在骨细胞中发挥着至关重要的作用,它们能够感知并转导力学信号,进而调节骨稳态。本文对5种力学感受器进行系统的介绍,以期为理解骨细胞如何响应力学刺激和维持骨组织稳态提供新的视角和认识。     

骨组织疲劳损伤的生物力学研究概述

骨组织是人体重要的承重器官,具有力学适应性,在疲劳载荷作用下会出现疲劳损伤,常见于运动员长跑、新兵训练以及老年人的日常活动,主要表现为骨组织显微裂纹的萌生、扩展,力学性能下降甚至是应力性骨折,危害很大。骨组织的疲劳损伤存在于包括超显微结构、显微结构和组织宏观的各个层面,皮质骨中的抗疲劳结构(骨单元)及内部的细胞成分(主要为骨细胞)对抵抗疲劳损伤、识别显微裂纹以及诱导后续定向骨重建过程具有重要作用。总结相关研究结果与结论有助于系统了解骨组织疲劳损伤行为及相应识别修复过程,同时也为后续骨组织疲劳损伤预防及治疗等临床研究工作的开展提供参考和方向。     

流体剪切力对骨组织细胞作用的研究进展

骨组织细胞包括成骨细胞、破骨细胞、骨细胞和骨衬细胞。在体内生理条件下,流体剪切力可能是这些细胞受到的最主要的力学刺激。近年来对流体剪切力影响骨组织细胞的研究取得了较大的进展,相关研究主要集中在流体剪切力引起骨组织细胞的细胞内信号分子、细胞内钙信号、细胞间隙连接和细胞骨架系统改变这几个方面,同时,流体剪切力会引起骨组织细胞间的相互作用的改变。本文就这些方面的重要研究内容做简要综述。     

抑制剂BYL719防治卵巢切除致骨质疏松症的研究

目的 PI3K信号通路在破骨细胞前体细胞增殖、破骨细胞分化中均发挥重要功能。BYL719是经典的PI3K通路抑制剂,但其对骨代谢的影响,目前未见报道。方法通过构建卵巢切除后小鼠骨质疏松症模型,运用microCT进行骨组织形态学分析,研究BYL719对骨质疏松症的治疗作用。在此基础上,通过体外研究BYL719对破骨细胞前体细胞活性、破骨细胞分化、破骨细胞特异性基因表达以及破骨细胞骨吸收功能,明确BYL719对破骨细胞的功能。结果 BYL719在5 mg/kg和10 mg/kg浓度下,均可有效防治小鼠卵巢切除导致的骨丢失,可以抑制破骨细胞前体细胞活性,抑制破骨细胞分化以及破骨细胞特异基因表达。结论 PI3K-mTOR抑制剂BYL719可通过影响破骨细胞前体细胞活性、破骨细胞分化,最终达到治疗骨质疏松症的作用。     

人体骨组织数值与形态特点

目的 ：研究确立骨组织数据的基准参照点，为医学、生物学和人类学等相关研究提供参考。方法 ：利用昆明市区10例，汉族、30岁±1岁男性的正常额骨和股骨新鲜解剖学教学标本作为研究材料，制作骨磨片。获取额骨鳞部垂直位、水平位骨磨片和股骨中段水平位骨磨片。在光学显微镜下，观察骨组织的类型和各自的细胞数量（代表骨组织所占百分比例）；并测量骨细胞的长径、宽径和厚径。分别直接测量与应用生物力学测量仪测量“额鳞中部”新鲜骨、脱脂脱水骨和煅烧骨的密度与抗压强度。结果 ：额骨鳞部骨组织分为编织骨（62.0%）、板层骨（28.8%）和束状骨（9.2%）3种类型；骨细胞亦呈扁椭圆形，但体积大于股骨骨细胞；其中编织骨骨细胞椭圆面平行于颅骨表面。股骨中份骨组织分为骨单位（68.5%）、间骨板（27.3%）和环骨板（4.2%）3种类型；股骨多数骨细胞长径与骨单位中央管平行。“额鳞中部”新鲜骨、脱脂脱水骨和煅烧骨的密度与抗压强度的相关系数为0.6074。结论 ：获得一特定人群中额骨与股骨的骨细胞的方向和径值、骨组织的类型和占比，确立了骨组织结构数据的一个基准参照点。额鳞中部骨的密度与抗压强度呈线性正相关关系。     

骨组织力学信号转导的研究

力学因素对骨组织的生长和重建有着很大的影响 ,成骨细胞和骨细胞在这一过程中起着非常重要的作用。本文概述了骨骼对力学刺激的感受、信号转导、基因表达改变以及效应细胞在此反应中所起作用的新进展     

Nano–Microscale Models of Periosteocytic Flow Show Differences in Stresses Imparted to Cell Body and Processes

In order to understand how local changes in mechanical environment are translated into cellular activity underlying tissue level bone adaptation, there is a need to explore fluid flow regimes at small scales such as the osteocyte. Recent developments in computational fluid dynamics (CFD) provide impetus to elucidate periosteocytic flow through development of a nano–microscale model to study local effects of fluid flow on the osteocyte cell body, which contains the cellular organelles, and on the osteocyte processes, which connect the cell to the entire cellular network distributed throughout bone tissue. For each model, fluid flow was induced via a pressure gradient and the velocity profile and wall shear stress at the cell-fluid interface were calculated using a CFD software package designed for nano/micro-electro-mechanical-systems device development. Periosteocytic flow was modeled, taking into consideration the nanoscale dimensions of the annular channels and the flow pathways of the periosteocytic flow volume, to analyze the local effects of fluid flow on the osteocyte cell body (within the lacuna) and its processes (within the canaliculi). Based on the idealized model presented in this article, the osteocyte cell body is exposed primarily to effects of hydrodynamic pressure and the cell processes (CP) are exposed primarily to fluid shear stress, with highest stress gradients at sites where the process meets the cell body and where two CP link at the gap junction. Hence, this model simulates subcellular effects of fluid flow and suggests, for the first time to our knowledge, major differences in modes of loading between the domain of the cell body and that of the cell process.     

Sexual dimorphism and age dependence of osteocyte lacunar density for human vertebral cancellous bone

The sexual dimorphism in age-related loss of human vertebral cancellous bone is not fully understood and could be related to dimorphism in the bone cell populations. The objective of this study was to investigate age- and gender-related differences in the osteocyte population and its relationship with bone volume fraction for human vertebral cancellous bone. Histomorphometric techniques were used to quantify osteocyte lacunae (a measure of osteocyte population) and bone volume fraction in male and female human T12 vertebrae, the most common site of vertebral fracture. Two measures of osteocyte population [number of osteocytes per bone area (OtLcDn) and number of osteocytes per total area (OtLcN/TA)] and their relationships with age and bone volume fraction were found to be sexually dimorphic. Dimorphism in osteocyte density may explain the dimorphic patterns of bone loss in human vertebrae due to the sensory and signal communication functions that osteocytes perform.     

Mechanosensation and Transduction in Osteocytes

There may be no single mechanoreceptor in osteocytes, but instead a combination of events that has to be triggered for mechanosensation and transduction of signal to occur. Possibilities include shear stress along dendritic processes and/or the cell body, cell deformation in response to strain, and primary cilia. These events could occur independently or simultaneously to activate mechanotransduction. Signal initiators include calcium channel activation and ATP, nitric oxide, and prostaglandin release. Means of signal transfer include gap junctions and hemichannels, and the release of signaling molecules into the bone fluid. Questions remain regarding the magnitude of strain necessary to induce an osteocyte response, how the response propagates within the osteocyte network, and the timing involved in the initiation of bone resorption and/or formation on the bone surface. Mechanical loading in the form of shear stress is clearly involved not only in mechanosensation and transduction, but also in osteocyte viability. It remains to be determined if mechanical loading can also affect mineral homeostasis and mineralization, which are newly recognized functions of osteocytes.     

Osteoblast‐osteocyte transformation. A SEM densitometric analysis of endosteal apposition in rabbit femur

Transformation of osteoblasts into osteocytes is marked by changes in volume and cell shape. The reduction of volume and the entrapment process are correlated with the synthesis activity of the cell which decreases consequently. This transformation process has been extensively investigated by transmission electron microscopy (TEM) but no data have yet been published regarding osteoblast-osteocyte dynamic histomorphometry. Scanning electron microscope (SEM) densitometric analysis was carried out to determine the osteoblast and open osteocyte lacunae density in corresponding areas of a rabbit femur endosteal surface. The lining cell density was 4900.1 ± 30.03 n mm⁻², the one of open osteocyte lacunae 72.89 ± 22.55 n mm⁻². This corresponds to an index of entrapment of one cell every 67.23 osteoblasts (approximated by defect). The entrapment sequence begins with flattening of the osteoblast and spreading of equatorial processes. At first these are covered by the new apposed matrix and then also the whole cellular body of the osteocyte undergoing entrapment. The dorsal aspect of the cell membrane suggests that closure of the osteocyte lacuna may be partially carried out by the same osteoblast-osteocyte which developed a dorsal secretory territory. A significant proportion of the endosteal surface was analysed by SEM, without observing any evidence of osteoblast mitotic figures. This indicates that recruitment of the pool of osteogenic cells in cortical bone lamellar systems occurs prior to the entrapment process. No further additions occurred once osteoblasts were positioned on the bone surface and began lamellar apposition. The number of active osteoblasts on the endosteal surface exceeded that of the cells which become incorporated as osteocytes (whose number was indicated by the number of osteocyte lacunae). Therefore such a balance must be equilibrated by the osteoblasts'' transformation in resting lining cells or by apoptosis. The current work characterised osteoblast shape changes throughout the entrapment process, allowing approximate calculation of an osteoblast entrapment index in the rabbit endosteal cortex.     

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.     

Determination of bone volume by osteocyte population

During development and growth, biological tissues and organisms can control their size and mass by regulating cell number (Raff, 1992; Conlon and Raff, 1999). Later in life both cell number and organ mass decrease (Buetow, 1985). We demonstrate that the number density of bone cells buried in the calcified matrix (osteocyte lacunar density) predicts extracellular matrix volume for both cancellous and cortical bone in a broad cross-section of the population (males and females, age range 23-91 years, r(2) = 0.98). Our hypothesis is that bone mass is determined by the control of osteocyte number, and that this is a particular instance of the control of organ size through the social controls on cell survival and death (Raff, 1992; Conlon and Raff, 1999).     

Attachment of Osteocyte Cell Processes to the Bone Matrix

In order for osteocytes to perceive mechanical information and regulate bone remodeling accordingly they must be anchored to their extracellular matrix (ECM). To date the nature of this attachment is not understood. Osteocytes are embedded in mineralized bone matrix, but maintain a pericellular space (50–80 nm) to facilitate fluid flow and transport of metabolites. This provides a spatial limit for their attachment to bone matrix. Integrins are cell adhesion proteins that may play a role in osteocyte attachment. However, integrin attachments require proximity between the ECM, cell membrane, and cytoskeleton, which conflicts with the osteocytes requirement for a pericellular fluid space. In this study, we hypothesize that the challenge for osteocytes to attach to surrounding bone matrix, while also maintaining fluid‐filled pericellular space, requires different “engineering” solutions than in other tissues that are not similarly constrained. Using novel rapid fixation techniques, to improve cell membrane and matrix protein preservation, and transmission electron microscopy, the attachment of osteocyte processes to their canalicular boundaries are quantified. We report that the canalicular wall is wave‐like with periodic conical protrusions extending into the pericellular space. By immunohistochemistry we identify that the integrin αvβ3 may play a role in attachment at these complexes; a punctate pattern of staining of β3 along the canalicular wall was consistent with observations of periodic protrusions extending into the pericellular space. We propose that during osteocyte attachment the pericellular space is periodically interrupted by underlying collagen fibrils that attach directly to the cell process membrane via integrin‐attachments. Anat Rec, 292:355–363, 2009. © 2009 Wiley‐Liss, Inc.     

Bcl-2, tissue transglutaminase and p53 protein expression in the apoptotic cascade in ribs of premature infants

Apoptotic cells of the human growth plate have not previously been demonstrated in situ. We have investigated the distribution of apoptotic cells in costosternal growth plates and bone of premature infants aged 4–11 d with a gestational age of ∼ 26 wk. In addition, we investigated the immunolocalisation of apoptosis-related proteins within the growth plates and associated bone. A proportion of late hypertrophic chondrocytes and osteocytes within newly formed primary spongiosa showed evidence of highly fragmented DNA. The incidence of osteocyte apoptosis decreased as the distance from the chondroosseous junction increased. Tissue transglutaminase (tTG) expression was associated with apoptosis of osteocytes and hypertrophic chondrocytes. In contrast the presence of tTG was demonstrated in osteoblasts and bone lining cells but it did not colocalise with evidence of apoptosis. The anti-apoptotic gene product Bcl-2 was absent from the growth plate but was present in osteocytes. Visual assessment indicated a greater occurrence of the protein in cells occupying regions of low apoptosis. P53 was not demonstrated in the growth plate or bone. These findings would indicate that human growth plate chondrocytes appear to show little provision for ensuring cell longevity. In contrast osteocyte apoptosis appears negatively correlated with the skeletal distribution of Bcl-2 protein in the human infant, implying a potential selective vulnerability in individual cells. Lack of Bcl-2 and the high incidence of osteocyte apoptosis in the more rapidly remodelling bone of the human infant suggest a potential role of osteocyte apoptosis in the remodelling process.     

A model of osteoblast-osteocyte kinetics in the development of secondary osteons in rabbits

The kinetics of osteogenic cells within secondary osteons have been examined within a 2-D model. The linear osteoblast density of the osteons and the osteocyte lacunae density were compared with other endosteal lamellar systems of different geometries. The cell density was significantly greater in the endosteal appositional zone and was always flatter than the central osteonal canals. Fully structured osteons compared with early structuring (cutting cones) did not show any significant differences in density. The osteoblast density may remain constant because some of them leave the row and become embedded within matrix. The overall shape of the Haversian system represented a geometrical restraint and it was thought to be related to osteoblast-osteocyte transformation. To test this hypothesis of an early differentiation and recruitment of the osteoblast pool which completes the lamellar structure of the osteon, the number and density of osteoblasts and osteocyte lacunae were evaluated. In the central canal area, the mean osteoblast linear density and the osteocyte lacunae planar density were not significantly different among sub-classes (with the exclusion of the osteocyte lacunae of the 300-1000 μm(2) sub-class). The mean number of osteoblasts compared with osteocyte lacunae resulted in significantly higher numbers in the two sub-classes, no significant difference was seen in the two middle sub-classes with the larger canals, and there were significantly lower levels in the smallest central canal sub-class. The TUNEL technique was used to identify the morphological features of apoptosis within osteoblasts. It was found that apoptosis occurred during the late phase of osteon formation but not in osteocytes. This suggests a regulatory role of apoptosis in balancing the osteoblast-osteocyte equilibrium within secondary osteon development. The position of the osteocytic lacunae did not correlate with the lamellar pattern and the lacunae density in osteonal radial sectors was not significantly different. These findings support the hypothesis of an early differentiation of the osteoblast pool and the independence of the fibrillar lamellation from osteoblast-osteocyte transformation.     

机体衰老对骨细胞力学响应的影响

骨骼是一个动态变化的器官,骨细胞的形态、结构和功能随力学刺激大小、方向、形式的不同而发生变化。适当的力学刺激是维持骨形成和骨吸收动态平衡的关键。随着年龄的增加,骨组织衰老会引起包括骨组织微环境、骨细胞形态、骨细胞内信号通路等在内的一系列变化,使骨骼力学响应能力减弱,进而引起骨质疏松等多种疾病。因此,研究衰老如何影响骨细胞的力学响应具有重要意义。重点讨论机体衰老对骨细胞力学响应的影响。