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。     

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。     

载荷作用下骨细胞分泌因子对成骨细胞和破骨细胞的调节

骨细胞是骨组织主要的力学感受及转导细胞,它们通过众多突触结构相互连接,形成庞大的骨稳态细胞调控网络,联系着成骨细胞、破骨细胞等骨基质表面细胞。骨细胞通过旁分泌途径影响成骨细胞骨形成和破骨细胞骨吸收来调节骨代谢,维持骨更新。针对骨细胞在受到力学刺激后分泌或释放的一些信号分子或蛋白因子对成骨细胞和破骨细胞生长分化的影响,本文综述近年来关于受力学刺激的骨细胞如何与成骨/破骨细胞进行通讯,为骨细胞生物力学研究提供新思路。     

骨内液体流动生物力学的研究进展

骨重建是以骨形成和骨吸收为特征的重要生理过程,人们早已发现骨组织受到力学载荷作用后,会通过骨重建过程优化其结构以适应变化的载荷环境。目前已有大量研究证实,载荷作用下骨内孔隙结构中的液体会发生流动,所产生的流体剪切力是使骨组织细胞产生生物学响应的主要因素。本文综述了近年来骨内液体流动方面的相关研究进展和成果,主要包括流体刺激下骨组织细胞的生物学响应,骨孔隙中的压力及其对液体流动的影响,以及骨内液体流动的实验、理论及数值模拟研究,并对骨内液体流动研究未来的发展趋势加以分析和展望。     

力学刺激对成骨细胞作用机制的研究进展

力学环境在维持骨组织正常形态和功能活动中发挥着重要的影响,目前研究采用的细胞生物力学装置模拟了压应力、张应力及流体剪切力等不同应力模式对培养中细胞的作用.并发现成骨细胞对不同类型的力学刺激有不同的感受和应答机制,甲状旁腺素、应力的频率、大小等也影响着力学刺激对成骨细胞的作用效应.     

成骨细胞的体外剪切应力加载实验的变形分析及信号转导区域探讨

目的 根据已有体外培养鼠成骨细胞的参数实验数据,估算剪切应力加载实验中细胞整体剪切形变,借以研究细胞的主要转导区域.方法 计算过程采用黏弹性力学理论,对细胞运用了标准黏弹性模型,并简化其膜所受剪切力为均匀.结果 细胞剪切力产生的细胞变形大约是引起成骨细胞相同生物学响应的拉伸加载变形的十分之一.结论 从细胞总的力学刺激生物学响应来看,剪切应力加载实验中细胞的整体变形所产生的力学转导是可以忽略的,主要转导区域在承受剪切应力的细胞膜.     

流体剪切力作用下间充质干细胞在三维支架上生长率理论模型的优化

骨组织工程是目前治疗大段骨缺损措施中最有发展前景的途径之一,体外构建骨组织工程移植体时常采用生物反应器对细胞—支架复合体进行灌流培养,以获得具有良好修复效果的骨组织工程移植体。但目前对这一过程中种子细胞(特别是干细胞)生长率的理论建模还不完善,尤其是忽略了干细胞与终末细胞的差异。在本实验室前期研究基础上,本文利用实验室自制的灌流装置对细胞支架复合体施加不同模式和大小的流体剪切力刺激,考察流体剪切力对间充质干细胞(MSCs)增殖和成骨分化的影响,建立了流体剪切力作用对MSCs单个细胞生长率影响的回归分析模型。结果表明,0.022 5 Pa振荡剪切力更能促进MSCs增殖和成骨分化,同时修正了流体剪切力作用下MSCs单个细胞生长率的理论模型。基于以上结果,希望本文研究可为骨组织工程移植体体外灌流培养条件的优化提供理论指导。     

骨细胞间隙连接与物理-生物信号传导研究进展

物理信号特别是力负载在骨转换中起重要作用,而骨组织中的细胞成分是对其周围环境的刺激作出反应的基本结构单位,它们在接受刺激后通过结构和功能的调整从而作出反应.然而力负载通过什么途径对细胞群体产生作用,骨细胞对力负载引起的顺式反应机理至今还令人困惑.本文综合近年来一些最新实验研究,阐述骨细胞间隙连接(gap junction)在组成骨细胞网络,并通过间隙连接的细胞内通讯(GJIC)机制在力学传递中的重要作用,其中对间隙连接的结构、功能,力负载(如应力、底物变形、流体流动、电磁场等)引起的生物物理信号在GJIC的传递,GJIC对成骨细胞分化的调节等方面作了综述,并提出GJIC的进一步研究与组织工程关系的启示.     

骨细胞间隙连接与物理—生物信号传导研究进展

物理信号特别是力负载在骨转换中起重要作用，而骨组织中的细胞成分是对其周围环境的刺激作出反应的基本结构单位，它们在接受刺激后通过结构和功能的调整从而作出反应。然而力负载通过什么途径对细胞群体产生作用，骨细胞对力负载引起的顺式反应机理至今还令人困惑。本综合近年来一些最新实验研究，阐述骨细胞间隙连接(gap junction)在组成骨细胞网络，并通过间隙连接的细胞内通讯(GJIC)机制在力学传递中的重要作用，其中对间隙连接的结构、功能，力负载(如应力、底物变形、流体流动、电磁场等)引起的生物物理信号在GJIC的传递，GJIC对成骨细胞分化的调节等方面作了综述，并提出GJIC的进一步研究与组织工程关系的启示。     

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。     

Mechanotransduction of bone cells<Emphasis Type="Italic">in vitro</Emphasis>: Mechanobiology of bone tissue

Mechanical force plays an important role in the regulation of bone remodelling in intact bone and bone repair. In vitro, bone cells demonstrate a high responsiveness to mechanical stimuli. Much debate exists regarding the critical components in the load profile and whether different components, such as fluid shear, tension or compression, can influence cells in differing ways. During dynamic loading of intact bone, fluid is pressed through the osteocyte canaliculi, and it has been demonstrated that fluid shear stress stimulates osteocytes to produce signalling molecules. It is less clear how mechanical loads act on mature osteoblasts present on the surface of cancellous or trabecular bone. Although tissue strain and fluid shear stress both cause cell deformation, these stimuli could excite different signalling pathways. This is confirmed by our experimental findings, in human bone cells, that strain applied through the substrate and fluid flow stimulate the release of signalling molecules to varying extents. Nitric oxide and prostaglandin E₂ values increased by between two- and nine-fold after treatment with pulsating fluid flow (0.6±0.3 Pa). Cyclic strain (1000 μstrain) stimulated the release of nitric oxide two-fold, but had no effect on prostaglandin E₂. Furthermore, substrate strains enhanced the bone matrix protein collagen I two-fold, whereas fluid shear caused a 50% reduction in collagen I. The relevance of these variations is discussed in relation to bone growth and remodelling. In applications such as tissue engineering, both stimuli offer possibilities for enhancing bone cell growth in vitro.     

一种骨种子细胞体外应力培养系统的建立

目的建立体外应力培养系统，观察应力刺激对骨种子细胞成骨分化的影响。方法选择具有明确成骨分化潜能的间充质干细胞(MSCs)作为种子细胞，以脱细胞骨基质为支架材料，以流体切应力作为对种子细胞的体外应力刺激。建立一种骨种子细胞体外三维应力培养系统——流动腔灌流体系，并利用该系统对MSCs的碱性磷酸酶(ALP)活性和骨钙素产物的影响进行评价。结果该系统可以明显促进ALP活性和骨钙素产物的表达，而细胞计数无明显改变。结论本系统为骨组织工程研究提供了一种有效的体外培养模型。     

Recent progress in studies on osteocytes--osteocytes and mechanical stress

Although osteocytes are of the most abundant cells in bone, our knowledge about the role of osteocytes in bone metabolism is still poor compared with that about osteoblasts and osteoclasts, both being on the surface of bone. Osteocytes are terminally differentiated bone-forming cells. During bone formation, some of the osteoblasts lining the surface of bone are incorporated into the newly formed osteoid matrix and become osteocytes, while the other osteoblasts remain on the surface as lining cells. During this transition from osteoblasts to osteocytes, the cells lose numerous osteoblastic phenotypes and acquire osteocytic characteristics such as high expression of osteocalcin and particularly their specific morphology. Osteocytes are connected with each other in bone and with osteoblasts on the bone surface through canaliculi, forming cellular networks; and gap-junctions present at the contact sites mediate their intercellular communication. Several roles of osteocytes in bone have been proposed so far. Of them, based on the morphological characteristics of osteocytes, sensation of mechanical stress loaded onto bone is suspected to be one of their functions. One of the mechanical stresses on bone is fluid shear stress. Between the osteocyte's plasma membrane and the bone matrix is the periosteocytic space. This space exists both in the lacunae and in the canaliculi, and it is filled with extracellular fluid (ECF). Application of mechanical stress to bone locally deforms the tissue. This periodical deformation subsequently causes an increase in the flow of ECF in the periosteocytic space, resulting in shear stress on the surface of the osteocytes. Experimental studies demonstrated that bone cells were equivalently or more sensitive to the fluid shear stress than epithelial cells. Osteocytic cells cultured enhanced expression of prostaglandin (PG) G/H synthase-2 (COX-2) mRNA in response to shear stress. PGE2 is a potent regulator of proliferation and function of osteoblasts and osteoclasts. Therefore, a metabolic response by osteoblasts and osteoclasts lining the bone surface may be caused by PGE2 produced by osteocytes in response to shear stress when the prostanoid reaches the surface through the canaliculi. In conclusion, osteocytes play an important role in sensing extracellular mechanical stress, and the mechanical signals mediated by osteocytes may regulate the overall metabolism of cells in bone tissue.     

A comparative study of shear stresses in collagen-glycosaminoglycan and calcium phosphate scaffolds in bone tissue-engineering bioreactors

The increasing demand for bone grafts, combined with their limited availability and potential risks, has led to much new research in bone tissue engineering. Current strategies of bone tissue engineering commonly use cell-seeded scaffolds and flow perfusion bioreactors to stimulate the cells to produce bone tissue suitable for implantation into the patient's body. The aim of this study was to quantify and compare the wall shear stresses in two bone tissue engineering scaffold types (collagen-glycosaminoglycan (CG) and calcium phosphate) exposed to fluid flow in a perfusion bioreactor. Based on micro-computed tomography images, three-dimensional numerical computational fluid dynamics (CFD) models of the two scaffold types were developed to calculate the wall shear stresses within the scaffolds. For a given flow rate (normalized according to the cross-sectional area of the scaffolds), shear stress was 2.8 times as high in the CG as in the calcium-phosphate scaffold. This is due to the differences in scaffold geometry, particularly the pore size (CG pore size approximately 96 microm, calcium phosphate pore size approximately 350 microm). The numerically obtained results were compared with those from an analytical method that researchers use widely experimentalists to determine perfusion flow rates in bioreactors. Our CFD simulations revealed that the cells in both scaffold types were exposed to a wide range of wall shear stresses throughout the scaffolds and that the analytical method predicted shear stresses 12% to 21% greater than those predicted using the CFD method. This study demonstrated that the wall shear stresses in calcium phosphate scaffolds (745.2 mPa) are approximately 40 times as high as in CG scaffolds (19.4 mPa) when flow rates are applied that have been experimentally used to stimulate the release of prostaglandin E(2). These findings indicate the importance of using accurate computational models to estimate shear stress and determine experimental conditions in perfusion bioreactors for tissue engineering.     

Adipose tissue-derived mesenchymal stem cells acquire bone cell-like responsiveness to fluid shear stress on osteogenic stimulation

To engineer bone tissue, mechanosensitive cells are needed that are able to perform bone cell-specific functions, such as (re)modeling of bone tissue. In vivo, local bone mass and architecture are affected by mechanical loading, which is thought to provoke a cellular response via loading-induced flow of interstitial fluid. Adipose tissue is an easily accessible source of mesenchymal stem cells for bone tissue engineering, and is available in abundant amounts compared with bone marrow. We studied whether adipose tissue-derived mesenchymal stem cells (AT-MSCs) are responsive to mechanical loading by pulsating fluid flow (PFF) on osteogenic stimulation in vitro. We found that ATMSCs show a bone cell-like response to fluid shear stress as a result of PFF after the stimulation of osteogenic differentiation by 1,25-dihydroxyvitamin D3. PFF increased nitric oxide production, as well as upregulated cyclooxygenase-2, but not cyclooxygenase-1, gene expression in osteogenically stimulated AT-MSCs. These data suggest that AT-MSCs acquire bone cell-like responsiveness to pulsating fluid shear stress on 1,25-dihydroxyvitamin D3-induced osteogenic differentiation. ATMSCs might be able to perform bone cell-specific functions during bone (re)modeling in vivo and, therefore, provide a promising new tool for bone tissue engineering.     

The Mechanical Environment of Bone Marrow: A Review

Bone marrow is a viscous tissue that resides in the confines of bones and houses the vitally important pluripotent stem cells. Due to its confinement by bones, the marrow has a unique mechanical environment which has been shown to be affected from external factors, such as physiological activity and disuse. The mechanical environment of bone marrow can be defined by determining hydrostatic pressure, fluid flow induced shear stress, and viscosity. The hydrostatic pressure values of bone marrow reported in the literature vary in the range of 10.7–120 mmHg for mammals, which is generally accepted to be around one fourth of the systemic blood pressure. Viscosity values of bone marrow have been reported to be between 37.5 and 400 cP for mammals, which is dependent on the marrow composition and temperature. Marrow’s mechanical and compositional properties have been implicated to be changing during common bone diseases, aging or disuse. In vitro experiments have demonstrated that the resident mesenchymal stem and progenitor cells in adult marrow are responsive to hydrostatic pressure, fluid shear or to local compositional factors such as medium viscosity. Therefore, the changes in the mechanical and compositional microenvironment of marrow may affect the fate of resident stem cells in vivo as well, which in turn may alter the homeostasis of bone. The aim of this review is to highlight the marrow tissue within the context of its mechanical environment during normal physiology and underline perturbations during disease.     

Mechanobiology of bone tissue

In order to obtain bones that combine a proper resistance against mechanical failure with a minimum use of material, bone mass and its architecture are continuously being adapted to the prevailing mechanical loads. It is currently believed that mechanical adaptation is governed by the osteocytes, which respond to a loading-induced flow of interstitial fluid through the lacuno-canalicular network by producing signaling molecules. An optimal bone architecture and density may thus not only be determined by the intensity and spatial distribution of mechanical stimuli, but also by the mechanoresponsiveness of osteocytes. Bone cells are highly responsive to mechanical stimuli, but the critical components in the load profile are still unclear. Whether different components such as fluid shear, tension or compression may affect cells differently is also not known. Although both tissue strain and fluid shear stress cause cell deformation, these stimuli might excite different signaling pathways related to bone growth and remodeling. In order to define new approaches for bone tissue engineering in which bioartificial organs capable of functional load bearing are created, it is important to use cells responding to the local forces within the tissue, whereby biophysical stimuli need to be optimized to ensure rapid tissue regeneration and strong tissue repair.     

流体剪应力对成骨细胞的作用研究

研究了生理范围内不同大小的流体剪切力对成骨细胞增殖分化和功能的影响,从细胞水平验证流体剪切力在骨组织力学适应性中发挥的重要作用.运用流室系统提供精确可控的流体剪切力,对原代培养的大鼠颅骨成骨细胞施以5、10、20、30 mN/cm2的剪切力刺激,分别在加载后的3、6、9、12、24、36 h采集样品,检测细胞周期分布,碱性磷酸酶活性,胞外钙质分泌的变化.5 mN/cm2和10 mN/cm2的剪应力促进细胞增殖,进一步增大会有抑制作用,而5、10和20 mN/cm2的剪应力对ALP活性和胞外钙分泌均有促进作用,并且与静态培养相比ALP活性高峰期提前,30 mN/cm2表现出抑制效应.结果显示成骨细胞的增殖、分化、矿化均能受到剪应力的调节,这种调节表现出对剪应力大小的依赖性.     

Initial Stress-Kick Is Required for Fluid Shear Stress-Induced Rate Dependent Activation of Bone Cells

The shear stress induced by the loading-mediated flow of interstitial fluid through the lacuno–canalicular network is a likely stimulus for bone cell adaptive responses. Furthermore, the magnitude of the cellular response is related to the rate of mechanical loading rather than its magnitude. Thus, bone cells might be very sensitive to sudden stress-kicks, as occuring e.g., during impact loading. There is evidence that cells change stiffness under stress, which might make them more sensitive to subsequent loading. We studied the influence of a stress-kick on the mechanosensitivity of MC3T3-E1 osteoblast-like cells under different peak shear rate conditions, as measured by nitric oxide production. MC3T3-E1 bone cells were treated with steady or pulsating fluid shear stress (PFSS) for 5 min with different peak rates (9.70, 17.5, and 22.0 Pa Hz) using varying frequencies (5 and 9 Hz), and amplitudes (0.70 and 0.31 Pa). PFSS treatment was done with or without fluid flow pretreatment phase, which removed the initial stress-kick by first applying a slow fluid flow increase. Nitric oxide production in response to fluid shear stress was rate dependent, but necessitated an initial stress-kick to occur. This suggests that high-rate stimuli condition bone cells to be more sensitive for high-frequency, low-amplitude loads.     

剪切流动下内皮细胞变形的模拟

血液流动和内皮的耦合是重要的生物医学问题 ,引起了学者们的广泛的兴趣。目前已知内皮细胞能感知流场的剪切应力而改变其形态和功能。由于剪切应力被认为是引起内皮细胞重建的始发信号 ,所以了解内皮细胞与流动应力之间的相互作用机制是十分重要的。我们建立了一个理论模型来模拟内皮细胞与流场应力之间的相互作用。根据二维计算流体动力学方法研究了内皮细胞应力、压力的分布以及内皮细胞在剪切应力作用下的变形情况。结果表明 :( 1)内皮细胞的变形随 α(对应于流体作用于细胞表面的切应力 )的变化而变化。当 α>0 .0 2 1时 ,细胞的变形随 α的增大而显著增大 ;( 2 )流动引起了细胞表面应力和压力分布的不均匀 ,从而导致了细胞的变形。但内皮细胞的最大应力总是位于细胞的顶点。同时 ,我们用流室系统提供剪切流动 ,测量了不同剪切应力作用下培养的人主动脉内皮细胞的变形。所得到的实验结果与上述数值模拟结果是吻合的。本文结果提示 ,由于剪切流动引起细胞表面应力和压力分布的不均一 ,可能在细胞激活和细胞功能的调节 (如细胞骨架的调节 ,粘附分子的表达与分布等 )机制上具有特殊的作用。本研究为综合应用动力学方程来建立内皮细胞模型提供了工作框架