不同弹性模量人工软骨对缺损软骨修复区细胞力学环境影响的研究

应用有限元软件COMSOL建立关节软骨固液双相模型和细胞微观模型,跨尺度研究在生理载荷作用下不同弹性模量的人工软骨修复缺损时,宿主软骨各层细胞的力学环境和液相流场。模拟结果表明,均一弹性模量的人工软骨对不同层区细胞微环境的影响规律不同。随着人工软骨弹性模量增大,浅表层、中间层细胞应力增大,深层细胞应力减小。人工软骨植入改变了中间层、底层软骨的流场方向和营养供给方式,可能会造成软骨细胞营养供应障碍。上述影响可造成修复结果不确定。通过对跨尺度软骨细胞有限元模型进行仿真分析,可定量地评价宿主软骨各层细胞的力学环境,有助于更准确地评估软骨缺损修复的临床效果。     

述评：关节软骨的细胞与分子生物力学

关节软骨是覆盖在动关节骨端表面的一层低摩擦、可承载负荷的水合软组织，其力学性能通过软骨细胞的新陈代谢来维持。物理因素，如关节载荷对软骨细胞的新陈代谢有很大的调节作用。本文对关节软骨和软骨细胞的力学性能和理论模型进行了综述，重点强调物理调节在软骨细胞生物合成和组织维护中的作用，以及在软骨功能性组织工程修复与再生医学中的应用。文中首先描述了关节软骨的分子构成、超微结构及其张力、压力、剪切力等力学性能，着重强调其相互关系。然后，通过介绍已广泛使用的结构模型，即双相和三相混合理论，对组织的力学－电化学行为进行阐述，并着重介绍简化复杂的三相理论的最新研究进展。最后，对软骨细胞和软骨素的机械性能和理论模型进行回顾总结，从而加深对细胞外基质中软骨细胞内及周围力学－电化学信号的认识。根据混合相理论，关节软骨的流动依赖性和非流动依赖性的粘弹性、溶胀行为和电动特性已经在理论上得到了成功阐述。文献中混合相理论用于软骨细胞以及周围细胞外基质的推广也为体内细胞行为物理调节机制的研究提供了新的思路。总之，应用强大的混合相理论、新的实验技术、新的或常规的办法来研究软骨细胞和基质之间的相互作用，可能有助于关节软骨工程学研究的成功。     

关节软骨细胞和细胞周基质的压缩特性及其对软骨细胞代谢的影响

软骨细胞和细胞周基质(pericellular matrix, PCM)的力学特性对于关节软针的生理功能具有重要的意义。软骨细胞在压缩应力下表现出黏弹性固体材料特性，其压缩特性具有各向异性和多相性。PCM对软骨细胞具有明显的力学保护作用。压缩应力影响软骨细胞的代谢活动。软骨细胞和PCM的力学特性有许多仍未澄清，尚需进一步的研究。     

软骨细胞力学信号转导在骨性关节炎中的作用

异常力学负荷是骨关节炎发生的主要危险因素,可导致胶原降解、糖胺聚糖丢失和软骨细胞凋亡,引起软骨和软骨下骨破坏。然而,由于对软骨细胞力学传导认识不足,以及各种软骨修复再生手段的效果并不理想,故迫切需要了解软骨细胞力学传导过程以及软骨机械性损伤发生机制,以期望为研究软骨损伤修复和再生提供参考。详细介绍力学信号如何从细胞外经由细胞膜传至细胞内力学感受器,并着重讨论相关力学传导的信号通路在骨性关节炎中的作用。     

组织工程软骨植入缺损后修复区力学特性分析

目的利用组织工程技术建立体外软骨缺损实验模型,研究修复区人工软骨和宿主软骨的力学特性。方法采用一种琼脂糖凝胶作为人工软骨,制作猪软骨深层缺损,在缺损处仿临床植入人工软骨,用生物胶黏接,建立组织工程修复膝关节软骨缺损的体外模型;在压缩载荷作用下,通过数字图像相关技术研究组织工程软骨植入缺损后修复区即刻力学行为。结果压缩过程中界面处没有出现开裂现象,压缩分别为软骨层厚度的3.5%、5.6%、7.04%和9.0%时获得了修复区中间层应变分布图和应变变化曲线。压缩量从3.5%增加到9%时,在垂直软骨面方向上宿主软骨最大压应变增加75.9%,人工软骨最大拉应变增加226.99%;在平行软骨表面方向,交界面处最大拉应变增加116.9%,增加量远高于宿主软骨区和人工软骨区;对于修复区剪应变,随着压缩量增加交界处剪应变方向发生相反的改变。结论软骨组织工程修复缺损效果有很大的不确定性,这与修复区的力学环境有关。组织工程软骨植入缺损后,修复区受到复杂应变状态,随着压缩量增加,界面处、宿主软骨、人工软骨都发生较大的应变变化,界面处垂直软骨面方向的应变由压应变可转化为拉应变,平行软骨表面方向的拉应变有显著增加,交界处剪应变方向甚至发生了相反的改变,而且剪应力数值迅速增加。这种复杂应变状态造成修复区细胞力学环境的较大变化,还可能引起界面的开裂,影响缺损修复过程,这些力学环境变化应受到临床治疗的重视。     

脱钙骨基质与骨髓间充质干细胞共培养关节腔内的成软骨活性

背景：将骨髓间充质干细胞附着到支架材料上再植入关节软骨缺损处,细胞不但不消失,而且可形成新的软骨。 目的：观察同种异体脱钙骨基质与骨髓间充质干细胞共培养在关节内的成软骨活性。 方法：在54只青紫蓝兔单侧膝关节制作关节软骨全层缺损模型,随机分组：实验组在缺损处植入自体骨髓间充质干细胞与同种异体脱钙骨基质复合物,对照组缺损处仅植入同种异体脱钙骨基质,空白对照组未植入任何物质。 结果与结论：植入后12周,实验组缺损处修复组织呈软骨样,表面光滑平坦,与周围软骨整合的软骨细胞更为成熟,修复组织与软骨下骨结合牢固;修复组织的细胞为透明软骨样细胞,柱状排列,Ⅱ型胶原染色阳性,与周围软骨及软骨下骨整合良好,且实验组组织学评分优于对照组和空白对照组 (P < 0.01)。对照组缺损处修复组织呈纤维样,与周围软骨未结合,空白对照组缺损区无修复组织,两组均无Ⅱ型胶原染色阳性表达。表明同种异体脱钙骨基质与骨髓间充质干细胞共培养后植入膝关节可形成软骨样组织,有效修复关节软骨缺损。     

力学参数随深度变化的关节软骨数值模拟

目的在软骨数值模拟中,对由于设置均匀和随深度变化的力学参数而导致的结果差异进行评估。方法利用COMSOL多孔介质模块建立软骨非线性两相多孔介质模型。在静载荷下,分别用均匀和随深度变化的两种软骨力学参数对模型进行了计算,并对两者的计算结果差异进行了分析。结果对于软骨总应力,两种参数设置的结果之间差异很小。但在分析软骨的固相应力、液体压力和流动等较深入细致的问题时,两种参数设置结果之间的差异不能忽略。结论不同的软骨力学参数设置对软骨总应力的结果几乎没有影响,但对软骨内流速场则影响很大。所以均匀的力学参数设置可用来简化计算软骨总应力的问题,而其他一些更细致的分析需要立足于随深度变化的软骨力学参数。这些结论可以为今后的软骨建模和数值计算提供参考,为人工关节的设计和计算奠定基础。     

力学刺激对体外保存软骨活力影响的实验研究

目的对体外保存猪膝关节软骨施加滚压力学刺激,探究力学刺激对软骨组织活力的影响。方法利用滚压力学加载装置可以为骨软骨组织提供含有组织培养液的环境,并进行仿生的力学刺激。用骨软骨取材器械体外无菌获取猪膝关节软骨,于培养液保存过程中施加力学刺激(1.5和4.0 MPa)后在第2周检测软骨的细胞存活率、蛋白多糖表达、组织形态学、杨氏模量。结果 1.5 MPa组显示最高的软骨细胞存活率、蛋白多糖含量与杨氏模量,组织形态表现良好,差异有统计学意义(P0.05);与静态对照组相比,4.0 MPa组上述指标下降(P0.05),形态学表现较差。结论对体外保存软骨施加适宜的滚压力学刺激,能在2周时间提高软骨细胞存活率,增强细胞外基质表达,并提升生物力学性能,为组织库软骨保存技术提供一种新方法。     

骨性关节炎软骨下骨成骨细胞的分离培养与增殖活性的观察

目的 探讨骨性关节炎软骨下骨成骨细胞的分离、培养、鉴定方法及生长特性.方法 复制改良Hulth兔膝关节不稳模型;采用Ⅰ型胶原酶消化联合组织块贴附法获得软骨下骨成骨细胞;应用倒置显微镜、Ⅰ型胶原免疫化学染色以及瑞氏-姬姆萨染色来进行形态学观察和生物学鉴定;利用软骨细胞与滑膜细胞分别于软骨下骨成骨细胞共培养,应用四甲基偶氮唑蓝检测软骨下骨成骨细胞的增殖活性;检测细胞的Ⅰ型胶原在基因水平的表达.结果 Ⅰ型胶原酶消化后在组织块贴附第11天有细胞开始从组织块周围爬出;Ⅰ型胶原酶免疫化学染色后可见胞浆内出现黄褐色颗粒,为阳性反应.瑞氏-姬姆萨染色显示细胞染成蓝紫色,胞核饱满,核仁清晰;四甲基偶氮唑蓝检测显示在3d时,滑膜细胞和软骨细胞对于软骨下骨成骨细胞的增殖有着明显的促进作用,在第6、10、14天时这种促进作用变得不明显,而软骨下骨成骨细胞的增殖活性超过2个共培养组.Ⅰ型胶原表达水平检测显示在第10天时,软骨细胞对于软骨下骨成骨细胞分泌Ⅰ型胶原的促进作用最强,而滑膜细胞对其的促进作用不明显.结论 酶消化联合组织块贴附法可获得理想的软骨下骨成骨细胞;利用Ⅰ型胶原酶免疫化学染色鉴别软骨下骨成骨细胞方法简便易行;利用软骨下骨成骨细胞与软骨细胞和滑膜细胞共培养的体系适用于骨性关节炎(0A)微环境的研究,且该体系可以模拟类OA软骨下骨成骨细胞、滑膜细胞和软骨细胞相互影响作用的进程.     

张应力对骺软骨生长的形态学和生物力学研究

目的：为临床骨延长术及运动医学提供理论依据。方法：选用新西兰幼兔在股骨远端骺软骨两侧安装外固定支架和力传感器并施加不同张力,观察组织细胞形态变化。另用幼兔新鲜离体股骨,作力学性质测试。结果：４周后张应力使兔股骨增长,骺软骨厚度增加,增殖层和肥大层细胞增生,粗面内质网和毛细血管增生。离体兔股骨随牵张力的增大或间隙时间的增加塑性变形亦增大。结论：在体内较小的张应力能使骺软骨细胞活性增强,实现肢体增长而     

The biomechanical role of the chondrocyte pericellular matrix in articular cartilage

The pericellular matrix (PCM) is a narrow tissue region that surrounds chondrocytes in articular cartilage. Previous parametric studies of cell-matrix interactions suggest that the mechanical properties of the PCM relative to those of the extracellular matrix (ECM) can significantly affect the micromechanical environment of the chondrocyte. The goal of this study was to use recently quantified mechanical properties of the PCM in a biphasic finite element model of the cell-PCM-ECM structure to determine the potential influence of the PCM on the mechanical environment of the chondrocyte under normal and osteoarthritic conditions. Our findings suggest that the mismatch between the Young's moduli of PCM and ECM amplifies chondrocyte compressive strains and exhibits a significant stress shielding effect in a zone-dependent manner. Furthermore, the lower permeability of PCM relative to the ECM inhibits fluid flux near the cell by a factor of 30, and thus may have a significant effect on convective transport to and from the chondrocyte. Osteoarthritic changes in the PCM and ECM properties significantly altered the mechanical environment of the chondrocyte, leading to approximately 66% higher compressive strains and higher fluid flux near the cell. These findings provide further support for a potential biomechanical role for the chondrocyte PCM, and suggest that changes in the properties of the PCM with osteoarthritis may alter the stress-strain and fluid flow environment of the chondrocytes.     

Zonal Uniformity in Mechanical Properties of the Chondrocyte Pericellular Matrix: Micropipette Aspiration of Canine Chondrons Isolated by Cartilage Homogenization

The pericellular matrix (PCM) is a region of tissue that surrounds chondrocytes in articular cartilage and together with the enclosed cells is termed the chondron. Previous studies suggest that the mechanical properties of the PCM, relative to those of the chondrocyte and the extracellular matrix (ECM), may significantly influence the stress–strain, physicochemical, and fluid-flow environments of the cell. The aim of this study was to measure the biomechanical properties of the PCM of mechanically isolated chondrons and to test the hypothesis that the Young's modulus of the PCM varies with zone of origin in articular cartilage (surface vs. middle/deep). Chondrons were extracted from articular cartilage of the canine knee using mechanical homogenization, and the elastic properties of the PCM were determined using micropipette aspiration in combination with theoretical models of the chondron as an elastic incompressible half-space, an elastic compressible bilayer, or an elastic compressible shell. The Young's modulus of the PCM was significantly higher than that reported for isolated chondrocytes but over an order of magnitude lower than that of the cartilage ECM. No significant differences were observed in the Young's modulus of the PCM between surface zone (24.0 ± 8.9 kPa) and middle/deep zone cartilage (23.2 ± 7.1 kPa). In combination with previous theoretical biomechanical models of the chondron, these findings suggest that the PCM significantly influences the mechanical environment of the chondrocyte in articular cartilage and therefore may play a role in modulating cellular responses to micromechanical factors.     

Use of a biphasic graft constructed with chondrocytes overlying a beta-tricalcium phosphate block in the treatment of rabbit osteochondral defects

To evaluate the ability of a biphasic construct to repair osteochondral defects in articular cartilage, plugs made of chondrocytes in collagen gel overlying a resorbable porous beta-tricalcium phosphate (TCP) block were implanted into defects in rabbit knees. The repair tissue was evaluated at 8, 12, and 30 weeks. Eight weeks after implantation of the biphasic construct, histologic examination showed hyaline-like cartilage formation that was positive for safranin O and type II collagen. At 12 weeks, most of the beta-TCP was replaced by bone, with a small amount remaining in the underlying cartilage. In the cell-seeded layer, the newly formed middle and deep cartilage adjacent to the subchondral bone stained with safranin O, but no staining was observed in the superficial layer. In addition, cell morphology was distinctly different from the deep levels of the reparative cartilage, with hypertrophic cells at the bottom of the cartilaginous layer. At 30 weeks, beta-TCP had completely resorbed and a tidemark was observed in some areas. In contrast, controls (defects filled with a beta-TCP block alone) showed no cartilage formation but instead had subchondral bone formation. These findings indicate that beta-TCP-supported chondrocytes in collagen gel can partially repair isolated articular cartilage osteochondral defects.     

Orientation of Primary Cilia of Articular Chondrocytes in Three‐Dimensional Space

Primary cilia have functions as sensory organelles integral to signal transduction and establishment of cell polarity. In articular cartilage the primary cilium has been hypothesized to function as an antenna to sense the biomechanical environment, regulate the secretion of extracellular matrix components, and maintain cellular positional information, leading to high tissue anisotropy. We used analysis of electron microscopy serial sections to demonstrate positional attributes of the primary cilium of adult equine articular chondrocytes in situ. Data for ～500 axonemes, comparing superficial to radiate chondrocytes from both load‐bearing and non‐load‐bearing regions, were graphed using spherical co‐ordinates θ, φ. The data demonstrate the axoneme has a definable orientation in 3D space differing in superficial and radiate zone chondrocytes, cells that differ by 90° in the orientation of their major axes to the articular surface. Axonemal orientation is more definable in load‐bearing than in non‐load‐bearing areas. The position of emergence of the axoneme from the cell also is variable. In load‐bearing regions of the superficial zone, extension of the axoneme is from the cellular side facing the subchondral bone. In radiate zone cells, axonemes extend from either face of the chondrocyte, that is, both toward the articular surface or toward the subchondral bone. In non‐load‐bearing regions this consistency is lost. These observations relate to current hypotheses concerning establishment of tissue anisotropy in articular cartilage during development, involving both migration of cells from the joint periphery and a restricted zone of division within the tissue resulting in the columnar arrangement of radiate zone cells. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

Relationship among biomechanical, biochemical, and cellular changes associated with osteoarthritis

Articular cartilage that lines the surface of long bones is a multilayered material. The superficial layer consists of collagen fibrils and chondrocytes that run parallel to the joint surface. In the deeper layers, the collagen fibrils are more randomly arranged and support vertical units termed chondrons containing rows of chondrocytes. In the deepest layers, the collagen fibrils run almost vertically and ultimately insert into the underlying subchondral bone. Osteoarthritis (OA) is a disease that affects articular cartilage and is characterized by enzymatic and mechanical breakdown of the extracellular matrix, leading to cartilage degeneration, exposure of subchondral bone, pain, and limited joint motion. Changes in mechanical properties of articular cartilage associated with OA include decreases in modulus and ultimate tensile strength. These changes parallel the changes observed after enzymatic degradation of either collagen or proteoglycans in cartilage. Results of recent viscoelastic studies on articular cartilage suggest that the elastic modulus of collagen and fibril lengths decrease in OA and are associated with a loss of the superficial zone and a decreased ability of articular cartilage to store elastic energy during locomotion. It is suggested that osteoarthritic changes to cartilage involve enzymatic degradation of matrix components and fibril fragmentation that is promoted by subsequent mechanical loading.     

Formation of biphasic constructs containing cartilage with a calcified zone interface

The zone of calcified cartilage is the mineralized region of articular cartilage that anchors the hyaline cartilage to the subchondral bone and serves to disperse mechanical forces across this interface. In an attempt to mimic this zonal organization, we have developed the methodology to form biphasic constructs composed of cartilaginous tissue anchored to the top surface of a bone substitute (porous calcium polyphosphate, CPP) with a calcified interface. To accomplish this, chondrocytes were selectively isolated from the deep zone of bovine articular cartilage, placed on top of the CPP substrate, and grown in the presence of beta-glycerophosphate (10 mM, beta-GP). By 8 weeks, cartilage tissue had formed with two zones: a calcified region adjacent to the CPP substrate and a hyaline-like zone above. Little or no mineralization occurred in the absence of beta-GP. The mineral that formed in vitro was identified as hydroxyapatite, similar in composition and crystal size to that found in vivo. The tissue stiffness was seven times greater, and the interfacial shear properties at the cartilage-CPP interface were at least two times greater in the presence of this mineralized zone within the in vitro-formed cartilage than in tissue lacking a mineral zone. In conclusion, developing a biphasic construct with a calcified zone at the tissue-biomaterial interface resulted in significantly better cartilage load-bearing (compressive) properties and interfacial shear strength, emphasizing the importance of the presence of a mineralized zone in bioengineered cartilage. Because failure due to shear occurred at the cartilage-CPP interface instead of the tidemark, as occurs with osteochondral tissue, further study is required to optimize this system so that it more closely mimics the native tissue.     

A hydrogel-mineral composite scaffold for osteochondral interface tissue engineering

Osteoarthritis is the leading cause of physical disability among Americans, and tissue engineered cartilage grafts have emerged as a promising treatment option for this debilitating condition. Currently, the formation of a stable interface between the cartilage graft and subchondral bone remains a significant challenge. This study evaluates the potential of a hybrid scaffold of hydroxyapatite (HA) and alginate hydrogel for the regeneration of the osteochondral interface. Specifically, the effects of HA on the response of chondrocytes were determined, focusing on changes in matrix production and mineralization, as well as scaffold mechanical properties over time. Additionally, the optimal chondrocyte population for interface tissue engineering was evaluated. It was observed that the HA phase of the composite scaffold promoted the formation of a proteoglycan- and type II collagen-rich matrix when seeded with deep zone chondrocytes. More importantly, the elevated biosynthesis translated into significant increases in both compressive and shear moduli relative to the mineral-free control. Presence of HA also promoted chondrocyte hypertrophy and type X collagen deposition. These results demonstrate that the hydrogel-calcium phosphate composite supported the formation of a calcified cartilage-like matrix and is a promising scaffold design for osteochondral interface tissue engineering.     

Immunolocalisation of vascular endothelial growth factor (VEGF) in human neonatal growth plate cartilage

Angiogenesis is essential for the replacement of cartilage by bone during growth and repair. In order to obtain a better understanding of the mechanisms regulating vascular invasion at sites of endochondral ossification we have investigated the expression of the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), by chondrocytes in human neonatal growth plates. VEGF was absent from chondrocytes in the resting zone and only weakly expressed by occasional chondrocytes in the proliferating region. In the hypertrophic zone the number of chondrocytes stained and the intensity of staining for VEGF increased with chondrocyte hypertrophy, maximum expression of VEGF being observed in chondrocytes in the lower hypertrophic and mineralised regions of the cartilage. These observations provide the first demonstration of the presence of VEGF in situ in developing human bone and are consistent with in vitro observations demonstrating the upregulation of proangiogenic growth factor production with increasing chondrocyte hypertrophy. The presence of numerous small blood vessels and vascular structures in the subchondral region where VEGF expression was maximal indicates that VEGF produced by hypertrophic chondrocytes may play a key role in the regulation of vascular invasion of the growth plate.