组织工程修复关节软骨缺损的力学状态研究

背景：力学状态对软骨的正常生理有重要影响,若应力集中过大将造成人工软骨退变和原宿主软骨退化,影响治疗效果。目前的各种力学手段很难实现活体软骨力学状态测量,而有限元动态分析能有效地模拟修补后软骨的受力情况。 目的：通过有限元仿真研究组织工程修复膝关节软骨缺损后人工软骨和宿主软骨的力学状态。 方法：以人体膝关节软骨受滚压部分为研究对象,建立滚动运动下关节软骨的有限元模型。根据行走过程中股骨与胫骨间的滚压边界条件,对软骨在取不同弹性模量、不同压缩量、不同载荷速度及不同缺损大小的情况进行了滚压受力分析。 结果与结论：在滚压载荷下,植入人工软骨弹性模量和软骨压缩量的不同都使人工软骨和宿主软骨受到的Mises应力值变化,二者对修复缺损处软骨Mises应力分布的影响比较明显,是临床治疗软骨缺损和术后康复阶段值得注意的因素。模拟中使用的载荷速度和缺损大小对软骨应力值的影响不明显。当人工软骨弹性模量取某个值时,人工软骨和宿主软骨的Mises应力差别可以达到很小值,二者趋于吻合。应力差别还和个体宿主软骨的力学性能有关,据此,应针对不同病例选择最佳弹性模量的人工软骨植入。     

滚压载荷下成年和幼年软骨的棘轮行为

文题释义： 非接触数字相关技术：非接触式测量方法以前主要有光学式和气动式两种,实验采用光学式图像采集系统。图像测量技术作为一种新兴的非接触测量方法有着独特的优越性,它通过把被测对象的图像作为检测和传递信息的手段,从图像中提取有用信息进而获得待测参数,研究通过图像采集点的坐标变化从而计算出受载前后软骨的应变。 棘轮效应：材料受到拉伸或压缩时,如果力大于材料的屈服强度,那么材料就会发生塑性变形。在非对称应力控制循环加载下,材料反向变形大小就会小于初始变形,进而产生了残余应变,如此反复而产生的沿应力方向上塑性变形累积的现象,这种现象即称为棘轮效应。 背景：国内外学者对关节软骨在不同力学环境及循环压缩载荷下的受力情况做了不少研究,但均集中在循环压缩载荷对软骨的作用,有关软骨年龄因素对软骨力学特性影响的研究和软骨在复杂受力环境下的特性研究不深入。                                                      目的：研究不同滚压载荷条件对成年和幼年关节软骨棘轮行为的影响。 方法：以成年猪股骨软骨和幼年猪股骨软骨为实验对象,在不同实验条件下(压缩量：10%,20%,30%;滚压速率：1.66,3.44,6.68 mm/s;缺损宽度：1,2,4 mm)采用滚压加载装置施加载荷,同时使用非接触数字相关技术对加载过程中的试样进行图像采集,通过分析处理图像,研究循环滚压载荷作用下成年及幼年关节软骨的棘轮行为。 结果与结论：①在滚压载荷下,随着滚压循环载荷的进行,成年软骨和幼年软骨的棘轮应变都呈现先快速增加后缓慢增加的趋势;②随着压缩量的增加,成年软骨和幼年软骨的棘轮应变都增加;在相同压缩量下,幼年软骨的棘轮应变大于成年软骨,并且他们的棘轮应变沿着软骨深度从表层到深层逐渐降低;③随着滚压速率的增加,成年软骨和幼年软骨的棘轮应变减小;④1 mm微型缺损关节软骨的棘轮应变数值和趋势与完整无缺损软骨大致相同。在2,4 mm缺损状态下,缺损软骨的棘轮应变值均比同样条件下完整软骨的棘轮应变值要高。 ORCID: 0000-0003-3586-1073(李凯);0000-0002-7288-6686(高丽兰) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

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。     

滑动载荷作用下关节软骨不同层区的法向位移

目的 获得滑动载荷作用下关节软骨不同层区的法向位移分布,探讨压缩应变、滑动速率和滑动次数对不同软骨深度法向位移的影响。方法 以新鲜猪关节软骨为研究对象,采用非接触式数字图像相关技术,对滑动载荷作用下软骨不同层区的法向位移分布进行研究。 结果 滑动载荷作用下,关节软骨表层的法向位移最大,深层的法向位移最小,中间层的位移介于二者之间;随着压缩应变的增大,沿软骨厚度方向的法向位移都增大,并且表层的法向位移增加幅度最大。滑动速率越大,软骨沿厚度方向的法向位移越小。在不同的滑动次数下,法向位移随滑动时间的进行都呈上升趋势;随着滑动次数的增加,不同滑动时间时的法向位移都增大,并且发现从第1次到第2次滑动时法向位移增大最明显。结论 滑动载荷作用下,软骨不同层区的法向变形有差异,不同层区的法向位移随着压缩应变、滑动速率和滑动次数的变化而变化。本研究可以为临床软骨疾病治疗和软骨缺损修复等方面提供依据,同时对人工软骨结构组成、人工构建、力学功能评价有重要意义。     

大鼠牙槽骨理想模型的流固耦合数值模拟研究

载荷作用下松质骨孔隙中的液体流动是刺激骨组织细胞产生生物学响应并调控骨重建的主要因素。因此,阐明牙槽骨内孔隙结构中的液体流动情况对于深入理解力学作用在牙槽骨内的传导过程以及牙齿发育、正畸牙移动等细胞水平的调控机制具有重要意义。此工作首先进行了大鼠牙齿正畸的动物实验,并基于微计算机断层扫描(micro-CT)图像构建了牙齿-牙周韧带-牙槽骨有限元模型,分析了咬合力或正畸力作用下牙槽骨中的应变状态;进而构建了理想模型,应用流固耦合数值模拟方法,分析了动态咬合力加载下无正畸加载、正畸拉伸加载、正畸压缩加载三种情况下骨内液体的流动情况。模拟结果表明,动态咬合力作用下,沿咬合方向排列的骨小梁表面流体剪应力水平高于非咬合方向排列的骨小梁,正畸力对骨内液体的流动没有影响。上述结果说明,临床上通过调整牙齿咬合面形状等方法改变咬合力的方向,会在牙槽骨表面引起不同水平的流体剪应力,进而刺激骨组织表面的细胞产生响应,最终调控牙槽骨的结构重建。     

组织工程化软骨的应力—应变研究

为研究组织工程化软骨的应力－应变特性,采用生物力学方法对体外培养离心管软骨和裸鼠皮下培养胶原海绵构建的软骨在5％变形时的压缩模量进行了测试,结果表明,体外离心管培养软骨在4,8,12,16周时的压缩模量分别为0．3518,0．653,0．233,0．262MPa,其中在8周时达最高,低于天然人胎关节软骨压缩模量2个数量级,体内皮下培养的胶原海绵软骨16周时的压缩模量的动态变化提示它与软骨细胞分泌GAG的活性变化一致,都在8周时达高峰,离心管软骨压缩模量总体偏低的现象提示可能是由于培养液与人工软骨大分子之间缺乏屏障保护,细胞分泌的PGS分子易于流失到培养液中的结果,而体内皮下培养的软骨模量低于天然软骨的原因可能是软骨发育过程中缺乏负荷刺激,因此,从生物力学角度看,组织工程化软骨的体外培养方法尚需改进。     

牛膝关节软骨的力学承载特性及其有限元仿真分析

目的分析软骨的压缩变形行为和液相力学承载特性的关系。方法利用压痕实验测定牛膝关节软骨在不同压头直径、不同载荷下的压缩变形位移,建立有限元模型模拟关节软骨内部液相流动及承载特性。结果模拟压缩位移与实验结果最大相对误差为1.73%,在相同载荷作用下,随着压头直径的增大,软骨的弹性模量与渗透系数随之增大;在相同压头直径作用下,随着载荷的增大,软骨的弹性模量与渗透系数随之减小。载荷作用在软骨上,软骨内部液相主要在软骨内流动,随着载荷的持续,液相逐渐向软骨外流动。软骨表面的孔隙压力、轴向应力、径向应力由于液相的流动呈非线性变化。结论软骨表面的液相流动、孔隙压力及应力分布等影响软骨表面的承载特性;在不同压头、不同载荷下,软骨的承载特性有较大差异。     

关节软骨生物力学与力学生物学 2022 年研究进展

关节软骨是动关节内骨表面具有弹性的负重结缔组织，能提供低磨损润滑、缓冲震荡、传递载荷等支撑作用，具有层级纤维复合结构和优异的力学性能。软骨内没有血管、神经和淋巴，代谢缓慢，损伤后难以实现自我修复。目前，高发的关节炎疾病仍是基础与临床研究的一大热点。关节软骨是力学敏感组织，力学环境影响着组织不同方向的发展。2022年，学者们继续对关节软骨的生物力学与力学生物学开展大量研究；对软骨形态、功能与力学状态，以及不同条件下软骨力学状态的研究报道较多；研究设计了一些软骨相关的动物、组织及细胞水平的加载装置，也开展了体外及在体力学载荷下软骨退变、损伤的修复研究，获得了重要的修复方法及手段。关节软骨的生物力学与力学生物学研究是关节炎、软骨缺损及修复的基础，关节软骨损伤修复定量力学条件的影响还需体内和体外深入研究。     

用于骨力生物学研究的加载装置

背景：关节软骨组织细胞对力学刺激产生反馈来维持它的形态和结构,进而适应环境。加载装置能提供研究关节软骨力生物学的合适的力学环境。 目的：根据组织工程仿生的原理,建立一种用于软骨力学生物学研究的双频加载装置。 方法：该力学环境采用双频加载方式实现,通过控制可调凸轮和压电陶瓷振幅和频率来实现低频-高幅载荷耦合高频-低幅载荷,同时使用有限元法对受载体进行力学分析。 结果与结论：按照仿真的原理,研制出用于软骨力生物学研究的加载装置。在双频加载的条件下,软骨浅表层受到的力学载荷最大,其次是中间层,深层受到的力学应力最小;在高频10 Hz和20 Hz与低频1 Hz和2 Hz叠加时,软骨表现出不同的力学响应,但是二者引起的差异很小。该力学环境的构建可能有助于组织工程的发展和临床医学的应用,未来将采用生物学实验进行检验。     

软骨组织工程生物反应器的研究进展

体外软骨构建是软骨组织工程产业化发展及临床应用的重要手段。然而,采用现有的体外构建技术无法构建功能接近正常的软骨。生物反应器能够在一定程度上模拟体内环境,有望弥补现有体外构建技术的弊端。研究发现,流体剪切力、静态液压力和直接压缩力是体内软骨发育和成熟的重要力学因素,常用软骨生物反应器均据此设计而产生。由于不同类型生物反应器各具特点,研究和开发新型复合式生物反应器将成为未来的发展方向。对目前软骨组织工程生物反应器的研究现状做一综述。     

Direct compression as an appropriately mechanical environment in bone tissue reconstruction in vitro

Determining how to apply an appropriately mechanical environment which can improve the quality and function of bone-like construct in vitro is a required problem to be solved for the current development of bone tissue engineering. A specific mechanical force may be a key determinant of tissue development in vitro in bone tissue engineering. From the standpoint of bionics, the mechanical environments applied on bone tissue engineering should work in three aspects: providing adequately mechanical stimuli to the cells seeded in 3-D scaffold; ensuring the efficient mass-transport of the nutrients and waste products of the cells; promoting the development of functionally extracellular matrix in 3-D scaffold. After the analysis of several differently mechanical environments comparing with that in vivo, the directly dynamical compression environment, instead of hydrostatic pressure or microgravity or direct perfusion, can recreate the in vivo mechanisms of mechanosensation, mechanotransduction and mass-transport during engineered bone-like tissue culturing process in vitro. Therefore, it is hypothesized that the directly dynamic compression will be a specific mechanical environment to bone tissue reconstruction in vitro.     

In vitro cartilage tissue engineering with 3D porous aqueous-derived silk scaffolds and mesenchymal stem cells

Adult cartilage tissue has limited self-repair capacity, especially in the case of severe damages caused by developmental abnormalities, trauma, or aging-related degeneration like osteoarthritis. Adult mesenchymal stem cells (MSCs) have the potential to differentiate into cells of different lineages including bone, cartilage, and fat. In vitro cartilage tissue engineering using autologous MSCs and three-dimensional (3-D) porous scaffolds has the potential for the successful repair of severe cartilage damage. Ideally, scaffolds designed for cartilage tissue engineering should have optimal structural and mechanical properties, excellent biocompatibility, controlled degradation rate, and good handling characteristics. In the present work, a novel, highly porous silk scaffold was developed by an aqueous process according to these criteria and subsequently combined with MSCs for in vitro cartilage tissue engineering. Chondrogenesis of MSCs in the silk scaffold was evident by real-time RT-PCR analysis for cartilage-specific ECM gene markers, histological and immunohistochemical evaluations of cartilage-specific ECM components. Dexamethasone and TGF-beta3 were essential for the survival, proliferation and chondrogenesis of MSCs in the silk scaffolds. The attachment, proliferation, and differentiation of MSCs in the silk scaffold showed unique characteristics. After 3 weeks of cultivation, the spatial cell arrangement and the collagen type-II distribution in the MSCs-silk scaffold constructs resembles those in native articular cartilage tissue, suggesting promise for these novel 3-D degradable silk-based scaffolds in MSC-based cartilage repair. Further in vivo evaluation is necessary to fully recognize the clinical relevance of these observations.     

A comparison of the osteogenic potential of adult rat mesenchymal stem cells cultured in 2-D and on 3-D collagen glycosaminoglycan scaffolds.

Adult mesenchymal stem cells (MSCs) have the capability to differentiate along several lineages including those of bone, cartilage, tendon and muscle, thus offering huge potential for the field of tissue engineering. The purpose of this study was to characterise the differentiation capacity of rat MSCs cultured on standard plastic coverslips in 2 dimensions and on a novel collagen glycosaminoglycan scaffold in the presence of a standard combination of osteoinductive factors. Cells were cultured for 3, 7, 14 and 21 days and several markers of osteogenesis were analysed. While the initial response of the cells in 3-D seemed to be faster than cells cultured in 2-D, as evidenced by collagen type I expression, later markers showed that osteogenic differentiation of MSCs took longer in the 3-D environment of the collagen GAG scaffold compared to standard 2-D culture conditions. Furthermore, it was shown that complete scaffold mineralisation could be evoked within a 6 week timeframe. This study further demonstrates the potential use of MSC-seeded collagen GAG scaffolds for bone tissue engineering applications.     

环境、材料、安全、可控：组织工程技术修复关节软骨的症结*

背景：传统的软骨缺损的修复方法都有其局限性,组织工程技术的出现从根本上改变了“以创伤修复创伤”的传统治疗模式。 目的：总结分析目前组织工程技术修复关节软骨的研究进展。 方法：由第一作者检索1990年至2011年 PubMed数据及中国知网数据库有关应用组织工程技术修复关节软骨方面的文献。共检索中文187 篇,英文211 篇,最终保留49篇进入结果分析。 结果与结论：软骨组织工程的主要方法就是应用工程学和生命科学原理,在体外分离、培养、扩增所需要的种子细胞,然后将之种植于合适的生物支架材料上,将细胞支架复合体植入体内组织缺损部位,并加入一定的诱导条件,逐渐形成新的有功能的软骨组织。文章在种子细胞的选择方面重点叙述了自体软骨细胞、异体软骨细胞、胚胎干细胞、骨髓间充质干细胞的研究进展;在细胞诱导及条件培养方面重点叙述了细胞因子、细胞条件培养、转基因技术的研究进展;并对生物支架材料的选择和研究进行了相关叙述。找到最理想的种子细胞,合理联合应用细胞因子,更加真实的模拟细胞生存的微环境,基因工程安全、高效、可控转染,构建理想的支架材料,将是今后组织工程研究的重点和热点。     

自体脂肪间充质干细胞复合人脐带Wharton胶支架修复兔膝关节软骨缺损****◇

背景：脐带Wharton胶富含透明质酸,糖胺多糖及胶原等,成分与天然软骨细胞外基质类似,因此由人脐带提取的Wharton胶很可能是一种较为理想的软骨组织工程支架材料。 目的：评价自体脂肪间充质干细胞复合人脐带Wharton胶支架修复兔膝关节软骨缺损的效果。 方法：将终浓度为1010 L -1、成软骨方向诱导后的兔自体脂肪间充质干细胞与人脐带Wharton胶支架复合,继续培养1周构建组织工程软骨,对兔膝关节全层软骨缺损进行修复(实验组),并与单纯支架修复的对照组及空白组进行比较。术后3个月对修复组织行大体观察、组织学检测、糖胺多糖、总胶原定量检测及生物力学测定。 结果与结论：实验组的缺损多为透明软骨修复,对照组以纤维组织修复为主,空白组无明显组织修复。提示脂肪间充质干细胞作为软骨组织工程种子细胞具有可行性;实验构建的组织工程软骨能有效的修复关节软骨缺损,人脐带Wharton胶可作为软骨组织工程良好的支架材料。     

Application of microstereolithography in the development of three-dimensional cartilage regeneration scaffolds

Conventional methods for fabricating three-dimensional (3-D) tissue engineering scaffolds have substantial limitations. In this paper, we present a method for applying microstereolithography in the construction of 3-D cartilage scaffolds. The system provides the ability to fabricate scaffolds having a pre-designed internal structure, such as pore size and porosity, by stacking photopolymerized materials. To control scaffold structure, CAD/CAM technology was used to generate a scaffold pattern algorithm. Since tissue scaffolds must be constructed using a biocompatible, biodegradable material, scaffolds were synthesized using liquid photocurable TMC/TMP, followed by acrylation at the terminal ends, and photocured under UV light irradiation. The solidification properties of the TMC/TMP polymer were also assessed. To assess scaffold functionality, chondrocytes were seeded on two types of 3-D scaffold and characterized for cell adhesion. Results indicate that scaffold geometry plays a critical role in chondrocyte adhesion, ultimately affecting the tissue regeneration utility of the scaffolds. These 3-D scaffolds could eventually lead to optimally designed constructs for the regeneration of various tissues, such as cartilage and bone.     

修复运动性肌腱损伤组织工程化肌腱及种子细胞和支架材料

背景：获得大规模、具有再生活力的种子细胞以及具有与正常人体肌腱组织相接近的力学性能的理想支架材料是当前组织工程化肌腱研究面临的最为关键的限制性因素。 目的：总结和分析组织工程肌腱研究中的种子细胞和支架材料的研究进展。 方法：查阅近年来肌腱组织工程研究的相关文献,综合国内外最新研究成果,就肌腱组织工程中合适的种子细胞来源、研究更为理想的支架材料及组织相容性等方面的进展进行概述。 结果与结论：肌腱组织工程中常用的种子细胞有间充质干细胞、肌腱干细胞及胚胎干细胞等,可以向骨、软骨和脂肪分化,修复肌腱损伤的理想细胞。肌腱组织工程支架材料有天然材料及人工合成材料等,肌腱组织工程支架材料应有良好的生物相容性和适度的机械性能,复合材料将是肌腱组织工程支架材料研究的重点。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程      

Modulation of collagen II fiber formation in 3-D porous scaffold environments

Collagen II, a major extracellular matrix component in cartilaginous tissues, undergoes fibrillogenesis under physiological conditions. The present study explored collagen II fiber formation in solution and in two- (coverslip) and three-dimensional (scaffold) environments under different incubation conditions. These conditions include variations in adsorption buffers, the presence of 1-ethyl-3-(3-dimenthylaminopropyl) carbodiimide/N-hydroxysuccinimide crosslinker and the nature of the material surfaces. We extend our observations of collagen II fiber formation in two dimensions to develop an approach for the formation of a fibrillar collagen II network throughout surface-modified polylactide-co-glycolide porous scaffolds. Morphologically, the collagen II network is similar to that present in native articular cartilage. Biological validation of the resultant optimized functional scaffold, using rat bone marrow-derived mesenchymal stem cells, shows appreciable cell infiltration throughout the scaffold with enhanced cell spreading at 24h post-seeding. This economic and versatile approach is thus believed to have significant potential in cartilage tissue engineering applications.     

A cartilage ECM-derived 3-D porous acellular matrix scaffold for in vivo cartilage tissue engineering with PKH26-labeled chondrogenic bone marrow-derived mesenchymal stem cells

We developed a natural, acellular, 3-D interconnected porous scaffold derived from cartilage extracellular matrix (ECM). Human cartilage was physically shattered, then decellularized sequentially with use of hypotonic buffer, TritonX-100, and a nuclease solution and made into a suspension. The scaffold was fabricated by simple freeze-drying and cross-linking techniques. On histology, scaffolds showed most of the ECM components after removal of the cell fragments, and scanning electron microscopy revealed a 3-D interconnected porous structure. Cellular viability assay revealed no cytotoxic effects. In vitro study showed that the novel scaffold could provide a suitable 3-D environment to support the adheration, proliferation and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) to chondrocytes in culture with chondrogenic medium after 21 days. Chondrogenically induced BMSCs labeled with fluorescent dye PKH26 were then grown on scaffolds and implanted subcutaneously into nude mice. Four weeks later, cartilage-like tissue formed, with positive staining for Safranin O, tuoluidine blue and collagen II. Cells in the samples seemed to confirm that they originated from the labeled BMSCs, as confirmed by in vivo fluorescent imaging and immunofluorescence examination. In conclusion, the cartilage ECM-derived porous scaffold shows potential as biomaterial for cartilage tissue engineering, and PKH26 fluorescent labeling and in vivo fluorescent imaging can be useful for cell tracking and analyzing cell-scaffold constructs in vivo.