骨细胞及其功能研究进展

骨细胞(osteocyte)是位于矿化的骨基质中骨的细胞,是骨组织中含量最丰富、分布最广泛的细胞.骨细胞通过细胞突触保持了彼此之间以及骨基质表面的成骨细胞的联系.骨细胞作为骨机械应力的直接感受器,可将机械应力转变为骨形成或骨吸收的信号,并将这些生化信号传递给效应细胞,在骨重建过程中起重要的作用.此外,骨细胞还具有调整体内矿物质平衡作用.对骨细胞的研究,可以为代谢性骨病的治疗探索新途径.     

机械应力、骨细胞凋亡和骨重建

骨细胞是骨中数量最丰富的细胞,有诸多重要作用。其作为骨组织的机械应力感受细胞,将机械载荷转化为体内生化反应过程,如细胞凋亡,同时产生一些调节成骨细胞和破骨细胞活性的因子和激素,共同参与骨组织的重建。骨细胞凋亡是一些骨代谢疾病的发病机制,可导致骨组织缺失或障碍性。本文简述了外界机械应力通过对成骨细胞和破骨细胞活性的调节,并刺激机体产生激素调节骨细胞凋亡,从而影响骨塑型和骨重建。通过机械应力对骨细胞凋亡调节的研究,为预防、治疗、诊断骨代谢疾病提供新的理论依据。     

骨组织应力转导过程中主要信号通路的研究进展

细胞感受机械改变,将其转化为生化信号,最终引起细胞变化的过程,称为应力转导[1].其过程受到一系列信号通路的影响和调节,机制尚不十分明确.应力转导对活体组织的结构和功能具有重要意义,特别是在骨组织中.骨组织在不断的更新重建中,应力刺激可以引发骨组织表达特殊的生化信号,调节其生长和功能.机械刺激增强时,刺激成骨及成软骨细胞分化、增殖和凋亡,造成骨生成[2];机械刺激减弱或缺失时,骨组织合成减少,破骨细胞造成骨基质吸收增加[3].对临床应用有实际意义的是,应力减少时皮质骨及小梁骨丢失增多,造成骨质疏松[4],例如宇航员、四肢瘫及长期卧床患者.     

骨细胞在骨重建和维持矿物质稳态中的作用

骨细胞是骨组织中含量最为丰富、分布最广泛的细胞。既往认为骨细胞是相对静止和休眠的细胞, 但事实并非如此。在最近几年里, 骨生物学的知识发生了很大的变化。目前认为骨细胞是维持骨骼正常功能所必需的高度活跃的细胞, 其在骨组织微环境中发挥着重要作用。本文重点介绍了骨细胞的研究现状, 以及骨细胞在骨重建中的作用。除此之外, 骨细胞还具有内分泌功能。它能分泌硬化素(Sclerostin)和成纤维生长因子(FGF-23)。硬化素对个体的骨量起负性调节作用, 而FGF-23可以调节磷酸盐代谢。骨细胞还能感受机械应力, 并将机械应力转化为一系列生物化学信号, 从而对效应细胞(包括成骨细胞和破骨细胞)起作用。因此, 骨细胞在骨生物学中起重要作用。深入了解骨细胞调控成骨细胞和破骨细胞功能的机制, 发现新的骨细胞分子, 可能成为治疗骨质疏松症和其他骨骼疾病的潜在药理靶点。     

骨细胞

在过去的10年里，对骨细胞的研究有了爆炸性的突破。过去骨细胞被认为是“被动静止”的。但事实远非如此，骨细胞在许多方面都有着重要的作用。得益于其骨陷窝-小管结构，骨细胞可以感受机械应力并将其转化为生物信号，从而“指导”成骨细胞和破骨细胞根据外界力学环境的变化进行骨骼重建。另外，骨细胞还具有重要的内分泌功能，它不仅能够调节骨骼表面的细胞，同样还可以影响包括肾脏、甲状旁腺在内的其它组织器官，从而在钙磷代谢中起到重要的作用。另外，相比于成骨细胞和破骨细胞，骨细胞的寿命非常长。越来越多的证据显示，凋亡或功能异常的骨细胞可能是某些骨骼疾病的罪魁祸首。通过对骨细胞多方面功能的深人了解，将有机会在骨骼疾病方面找到新的治疗目标。本文将从骨细胞在应力感应、骨重建、内分泌调节、细胞生存等方面对骨细胞进行综述。     

流体剪切力对骨形成及修复的作用

应力包括牵张力、流体剪切力(FSS)和压应力等,是骨形成和骨修复的"指导性"因素.应力成骨及其机制是近几年来骨科学、生物医学工程及康复医学领域的研究热点.骨组织特殊的腔隙-小管网络结构,使得FSS在完整骨重塑和损伤骨修复中扮演着重要角色;体外实验也证明骨细胞对FSS刺激更为敏感,突显出FSS对骨组织的作用.在正常情况下,FSS刺激骨细胞产生不同的信号分子,如前列腺素E2、一氧化氮,并能兴奋不同的信号通路,如细胞外信号调节激酶通路、一氧化氮合酶通路等,从而调节成骨细胞和破骨细胞的多种生理功能,使它们在骨组织的形成和修复中发挥重要作用,保证骨形成和骨吸收的动态平衡.     

流体剪切力对破骨细胞的影响

骨组织受到成骨细胞成骨作用和破骨细胞溶骨作用共同影响。与成骨细胞和骨细胞不同,破骨细胞起源于造血干细胞,由单核巨噬细胞发育而来,具有吞噬功能,能对多种刺激因素产生效应,其中机械刺激是始终存在的影响方式。机械刺激诱导的成骨细胞的正性调节对于骨生成、骨修复有至关重要的作用,而破骨细胞在这些方面的效应也十分关键。流体剪切力作为骨组织内机械刺激的一种常见形式,作用于成骨细胞和骨细胞的时候,通过对Ca2+、前列腺素、NO、RANKL和OPG等信号因子的影响,表现出介导破骨细胞迁移、分化、吸收和生存维持等现象的功能,这承载了骨代谢必不可少的一部分。同时这些因子相互交错,表现出复杂信息网络及阶段特异性。但总的来说,人们对流体剪切力调节破骨细胞生理及病理功能具体信号传导的了解还不是很深入,需要用更多的研究来阐明流体剪切力和破骨细胞之间的关系。本文就流体剪切力对破骨细胞相关分子信号影响做一综述,简要展示流体剪切力影响破骨细胞的相关机理。     

间隙液流体剪切力在骨细胞机械力传导过程中的作用

机械负荷是骨结构和骨量重要调节因素，已经证明增加外源性负荷能促进骨形成而移去负荷能使骨量减少。近年来，对骨细胞感知其周围机械环境变化的分子机制理解有了很大进步，其中细胞间隙液流体剪切力在机械力信号传导中发挥了重要的作用。现将骨细胞感知间隙液流体剪切力可能的分子机制及这种生物物理信号在组织工程中的应用综述如下。     

骨细胞网络结构对骨形成和骨吸收的影响

骨细胞的数量是骨组织中最多的,占骨组织细胞95%以上。骨细胞均匀地分布在矿化的骨基质中,通过细胞膜的多个突触结构互相连接并与骨基质表面的细胞连接形成网状的细胞结构。骨细胞被认为是一种理想的细胞,在骨组织发育和代谢过程中启动细胞的生物化学反应。最新的研究已证实骨细胞通过直接整合骨的矿化基质和其他的多细胞网络结构,感受刺激、调控效应细胞(例如成骨细胞、破骨细胞、骨髓基质干细胞),除此之外骨细胞已被证实作为一种内分泌细胞并且可以调控磷的代谢。随着研究的深入,骨细胞网络结构对骨形成和吸收的影响逐渐明显,在生理条件下骨细胞网络结构通过激活破骨细胞促进骨吸收,通过抑制成骨细胞抑制骨形成。本篇综述将主要讨论骨细胞网络结构对骨生成和骨吸收的影响及其相应的研究和理论依据。     

骨吸收的细胞生物学

近年来的研究发现破骨细胞系属于单核吞噬系统，源于多能造血干细胞，在骨组织的细胞以及激素微环境下，单核细胞，巨噬细胞以及成熟程度较低的造血前体细胞具有分化成有降钙素受体和凹陷性骨吸收能力的破骨细胞，成骨细胞在破骨细胞激活和破细胞性骨吸收的激素调控方面居中心地位，如果细胞以及激素作用对破骨细胞的形成和功能出现异常，可导致破骨细胞的数量以及活性的异常，产生病理性骨吸收，其它细胞如巨噬细胞，肿瘤细胞具有狡     

An ultrastructural and immunogold localization study of proteoglycans associated with the osteocytes of fetal bone in osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a rare, heterogeneous, inherited connective tissue disorder frequently caused by abnormalities of type I collagen. It is characterized by bone fragility, osteopenia, and progressive skeletal deformities. Electron microscopy of three OI type II fetal bone samples revealed numerous large osteocyte lacunae. In addition, there was a perilacunar osteoid-like band of collagen surrounding the osteocytes, which was unmineralized and morphologically unusual. Furthermore, large osteocyte lacunae contained fine particles and filamentous material similar to the expected ultrastructural appearance of proteoglycans. More detailed examination was carried out using histochemical and immunogold localization of proteoglycans at light and ultrastructural levels. These tests and the use of electron probe X-ray microanalysis confirmed that the material in the osteocyte lacunae was proteoglycan. In contrast, in the age- and site-matched normal fetal bone, all the osteocyte lacunae appeared negative for proteoglycan. Proteoglycans are regarded as inhibitors of calcification. Our observation of substantial amounts of proteoglycan in abnormally enlarged osteocytic lacunae of some OI fetal bone suggests association with the abnormal bone of this particular subtype of OI type II.     

The early mouse 3D osteocyte network in the presence and absence of mechanical loading

Osteocytes are considered to act as mechanosensory cells in bone. They form a functional synctia in which their processes become interconnected to constitute a three-dimensional (3D) network. Previous studies reported that in mice, the two-dimensional osteocyte network becomes progressively more regular as they grow, although the key factors governing the arrangement of the osteocyte network during bone growth remain unknown. In this study, we characterized the 3D formation of the osteocyte network during bone growth.Morphological skeletal changes have been reported to occur in response to mechanical loading and unloading. In order to evaluate the effect of mechanical unloading on osteocyte network formation, we subjected newborn mice to sciatic neurectomy in order to immobilize their left hind limb as an unloading model. The osteocyte network was visualized by staining osteocyte cell bodies and processes with fluorescently labeled phalloidin. First, we compared the osteocyte network in the femora of embryonic and 6-week-old mice in order to understand the morphological changes that occur with normal growth and mechanical loading. In embryonic mice, the osteocyte network in the femur cortical bone displayed a random cell body distribution, non-directional orientation of cell processes, and irregularly shaped cells. In 6-week-old mice, the 3D network contained spindle-shaped osteocytes, which were arranged parallel to the longitudinal axis of the femur. In addition, more and longer cell processes radiated from each osteocyte. Second, we compared the cortical osteocyte networks of 6-week-old mice that had or had not undergone sciatic neurectomy in order to evaluate the effect of unloading on osteocyte network formation. The osteocyte network formation in both cortical bone and cancellous bone was affected by mechanical loading. However, there were differences in the extent of network formation between cortical bone and cancellous bone in response to mechanical loading with regard to the orientation, nuclear shape and branch formation.     

Involvement of different ion channels in osteoblasts'' and osteocytes'' early responses to mechanical strain

The involvement of functional ion channels in previously documented early responses of osteocytes and osteoblasts to mechanical strain in bone tissue was investigated in explants of rat ulnae by the use of ion channel blockers. Gadolinium chloride (a blocker of stretch/shear-sensitive cation channels) elevated basal prostaglandin (PG) E₂ and prostacyclin (PGI₂) release and osteocyte glucose-6-phosphate dehydrogenase (G6PD) activity, but was associated with a reduction in basal nitric oxide (NO) production. Gadolinium abolished loading-related increases in the release of PGI₂ and NO and osteocyte G6PD activity. Gadolinium also reduced the loading-related release of PGE₂ assumed to originate from osteoblasts and the magnitude of loading-related increases in G6PD activity in these cells. Nifedipine (a blocker of L-type voltage-dependent calcium channels) had no effect on basal levels of prostanoid or NO release, or G6PD activity in osteocytes or osteoblasts, and did not affect loading-related release of PGI₂ or increase in osteocyte G6PD. However, nifedipine prevented loading-related increases in PGE₂ and NO release and osteoblast G6PD activity. These results are consistent with osteocytes' response to bone loading requiring activatable ion channels sensitive to gadolinium, but not those sensitive to nifedipine. In osteoblasts, the early responses to bone loading appear to be associated with ion channels sensitive to gadolinium and nifedipine; however, the nifedipine-sensitive channels seem to have the dominant effect.     

Bone death in hip fracture in the elderly

Summary We examined femoral head bone from 50 cadavers and from 21 patients who had suffered pathologic fracture of the femoral neck. We used a histochemical technique for lactate dehydrogenase (LDH) activity to demonstrate osteocyte viability. The femoral heads were removed within 36 hours of death or fracture, as LDH activity persists in the cytoplasm of viable cells for this time at 37° after interruption of the blood supply. In the controls, there was an age-related reduction in mean osteocyte viability, from 88±7% (mean±SD) at age 10–29 years to 58±12% at age 70–89 years. In the hip fracture patients, mean osteocyte viability was 58±21% but there was much variability in both osteocyte viability and bone mass. In 5 fracture patients, there was extensive osteocyte death, suggesting that most of the femoral head bone was nonviable; these patients had little microfracture callus. Others had predominantly viable bone which was usually osteoporotic, and their bone frequently showed microfracture callus. Osteomalacia was not seen in any patient. It is suggested that bone death, in addition to osteoporosis, may sometimes contribute to hip fracture in the elderly.     

骨收获室内骨细胞的方向性生长

目的:利用骨收获室内无应力的物理特性,观察无应力时骨重塑是否有方向性。方法:采用骨收获室模型的动物实验方法,5只日本大耳白雌兔。研究中不限制兔的活动。骨收获室是带有1个宽1mm的骨长入孔道的钛制圆柱形植入体,允许孔道内的成分被反复取出,仅伴有少量的对周围骨的干扰。先将骨收获室置放兔胫骨内8周,使其与骨达到较好的整合。8周后开始正式实验,其后每隔1个月收获组织。收获的组织,经固定、脱钙,采用石蜡包埋的组织切片。动物实施4阶段的4次手术:第1次空置;第2、3次收获的组织做纵向切片;第4次做横向切片。方向性分析:以每张切片所有骨细胞核方向的标准差为统计量,分析纵切面与横切面细胞的方向是否有差异。结果:收获室内长入组织,纵切面的细胞排列的方向性明显强于横切面,差异有统计学意义。结论:在无应力情况下,骨重建具有方向性。应力不是骨方向改建的直接指导因素。推测骨重建的方向性与骨收获室的结构特点相关,生理的方向性骨重建与应力的关系可能是通过血管介导,应力可能通过影响血管的方向性分布起作用。     

Alterations in the osteocyte lacunar-canalicular microenvironment due to estrogen deficiency

While reduced estrogen levels have been shown to increase bone turnover and induce bone loss, there has been little analysis of the effects of diminished estrogen levels on the lacunar-canalicular porosity that houses the osteocytes. Alterations in the osteocyte lacunar-canalicular microenvironment may affect the osteocyte's ability to sense and translate mechanical signals, possibly contributing to bone degradation during osteoporosis. To investigate whether reduced estrogen levels affect the osteocyte microenvironment, this study used high-resolution microscopy techniques to assess the lacunar-canalicular microstructure in the rat ovariectomy (OVX) model of postmenopausal osteoporosis. Confocal microscopy analyses indicated that OVX rats had a larger effective lacunar-canalicular porosity surrounding osteocytes in both cortical and cancellous bone from the proximal tibial metaphysis, with little change in cortical bone from the diaphysis or cancellous bone from the epiphysis. The increase in the effective lacunar-canalicular porosity in the tibial metaphysis was not due to changes in osteocyte lacunar density, lacunar size, or the number of canaliculi per lacuna. Instead, the effective canalicular size measured using a small molecular weight tracer was larger in OVX rats compared to controls. Further analysis using scanning and transmission electron microscopy demonstrated that the larger effective canalicular size in the estrogen-deficient state was due to nanostructural matrix-mineral level differences like loose collagen surrounding osteocyte canaliculi. These matrix-mineral differences were also found in osteocyte lacunae in OVX, but the small surface changes did not significantly increase the effective lacunar size. The alterations in the lacunar-canalicular surface mineral or matrix environment appear to make OVX bone tissue more permeable to small molecules, potentially altering interstitial fluid flow around osteocytes during mechanical loading.     

Architecture of the osteocyte network correlates with bone material quality

In biological tissues such as bone, cell function and activity crucially depend on the physical properties of the extracellular matrix which the cells synthesize and condition. During bone formation and remodeling, osteoblasts get embedded into the matrix they deposit and differentiate to osteocytes. These cells form a dense network throughout the entire bone material. Osteocytes are known to orchestrate bone remodeling. However, the precise role of osteocytes during mineral homeostasis and their potential influence on bone material quality remains unclear. To understand the mutual influence of osteocytes and extracellular matrix, it is crucial to reveal their network organization in relation to the properties of their surrounding material. Here we visualize and topologically quantify the osteocyte network in mineralized bone sections with confocal laser scanning microscopy. At the same region of the sample, synchrotron small‐angle X‐ray scattering is used to determine nanoscopic bone mineral particle size and arrangement relative to the cell network. Major findings are that most of the mineral particles reside within less than a micrometer from the nearest cell network channel and that mineral particle characteristics depend on the distance from the cell network. The architecture of the network reveals optimization with respect to transport costs between cells and to blood vessels. In conclusion, these findings quantitatively show that the osteocyte network provides access to a huge mineral reservoir in bone due to its dense organization. The observed correlation between the architecture of osteocyte networks and bone material properties supports the hypothesis that osteocytes interact with their mineralized vicinity and thus, participate in bone mineral homeostasis.     

Mechanotransduction and functional response of the skeleton to physical stress: The mechanisms and mechanics of bone adaptation

The skeleton's primary mechanical function is to provide rigid levers for muscles to act against as they hold the body upright in defiance of gravity. Many bones are exposed to thousands of repetitive loads each day. During growth and development, the skeleton optimizes its architecture by subtle adaptations to these mechanical loads. The mechanisms for adaptation involve a multistep process of cellular mechanotransduction including: mechanocoupling— conversion of mechanical forces into local mechanical signals, such as fluid shear stresses, that initiate a response by bone cells; biochemical coupling— transduction of a mechanical signal to a biochemical response involving pathways within the cell membrane and cytoskeleton; cell-to-cell signaling from the sensor cells (probably osteocytes and bone lining cells) to effector cells (osteoblasts or osteoclasts) using prostaglandins and nitric oxide as signaling molecules; and effector response— either bone formation or resorption to cause appropriate architectural changes. These architectural changes tend to adjust and improve the bone structure to its prevailing mechanical environment. Structural changes can be predicted, to some extent, by mathematical formulas derived from three fundamental rules: (1) bone adaptation is driven by dynamic, rather than static, loading; (2) extending the loading duration has a diminishing effect on further bone adaptation; (3) bone cells accommodate to a mechanical loading environment, making them less responsive to routine or customary loading signals. Received for publication on Dec. 25, 1997; accepted on Feb. 24, 1998