工程化骨软骨生理微环境构建方法及研究进展

骨组织工程是通过在体外构建有正常组织功能或疾病生理特点的临床模型,用以药物筛选,或研究疾病发生发展过程。骨骼肌肉系统是载重系统,其功能与组织结构、细胞外基质等密切相关。在构建骨组织体外模型时,需要结合骨、软骨及其他构成成分的生理微环境,表现关节骨软骨接合处的生理特点及作用机制,进而模拟正常及病理状态下骨组织系统对刺激的反应。本综述从骨软骨组织的生理构造入手,阐述了骨软骨连接处在退行性关节病变发生发展过程中的作用,并系统的论述了体外构建三维骨软骨组织的方法及这些方法的优势和局限性,为体外构建骨软骨组织工程在临床上应用提供支持。     

碱性成纤维细胞生长因子与胶原对组织工程软骨体外构建的影响

目的：以三维成团培养为培养系统,探讨bFGF与胶原对组织工程软骨体外构建的影响。方法：成团培养兔生长板软骨细胞,设bFGF、胶原及联合作用组。HE染色观察新生组织形态;免疫组化检测Ⅰ、Ⅱ型胶原表达以观察细胞表型;Hoechst 33258法检测细胞DNA含量;羟脯氨酸法与阿新蓝法测定基质中胶原与蛋白多糖的合成。结果：新生软骨的组织学形态近似自然软骨;各实验组软骨细胞DNA含量明显上升;胶原可以显著促进基质的合成;各实验组Ⅰ型胶原的表达少于对照组,Ⅱ型胶原的表达则高于对照组;联合作用组效果更加明显。结论：三维的成团培养可以促进基质合成,有效维持软骨细胞表型;bFGF与胶原有利于工程化软骨构建,其效果具有协同效应,两者联合应用可进一步促进软骨再生。     

细胞外基质的增龄性变化及分子机制的研究进展

人口老龄化及其伴随的各种疾病已成为全球性健康问题,细胞外基质在老化过程中发生的变化及对机体产生的影响逐渐成为研究衰老的热点。在机体发育和衰老过程中,细胞外基质不仅可以为细胞提供结构支架、组织连接,调节实质细胞的形态、增殖、分化、代谢、迁移等生理活动,并且其本身组成成分、合成、代谢、重构等变化也会对机体各系统的功能产生深刻影响,具体表现为骨骼肌僵硬、左心室功能受损、神经突触传导抑制等。本文通过介绍机体在衰老过程中,运动、循环、神经等系统细胞外基质的变化及相关机制的最新研究进展,从非细胞角度探讨老化的机制,了解衰老的过程。     

细胞联合培养在血管构建中的作用

组织工程三大要素为种子细胞、支架材料和信号分子,干细胞因其多分化潜能成为热门的种子细胞。血管化问题是制约工程化组织应用于临床的问题之一。利用干细胞构建组织工程血管的手段之一是在分离培养得到足够的种子细胞后,通过生长因子、细胞外基质、外力作用、其他细胞等的调控实现内皮向分化。只有实现了成功的血管构建,工程化组织才能正常的发挥作用。近年来不少国内外专家学者通过细胞联合培养的方法,观察细胞间的相互作用对血管构建的影响,结果表明,细胞联合培养在血管的形成、存活、稳定方面起到了重要的作用,为组织工程血管化提供了有效的途径,本文就部分细胞联合培养在血管构建中的作用作一综述。     

TGF-β在骨关节炎发生中的可能作用

转化生长因子TGF-β具有广泛的生理效应,对于维持关节软骨正常有重要意义。软骨细胞外基质降解和关节软骨损坏是骨关节炎的重要特征,研究表明TGF-β一方面通过调节细胞外基质成分含量维持基质的正常更新,另一方面通过调节关节软骨细胞的形成、增殖和分化来保持正常数量和正常生理状态的软骨细胞。本文就这两个方面综述了最新研究进展,以探讨TGF-β在骨关节炎发生中的可能调节机制。     

利用大孔微载体体外制备工程化组织

组织工程从诞生至今已有二十年的历史.微载体在组织工程领域的应用研究近年来才日趋升温.利用大孔微载体Cytopore体外构建工程化微构组织.并基于其聚团特性,进行灌注再装配大组织的尝试.结果20 d内微构组织达最高细胞密度16.4×107 cells/cm3;32 d后.所形成的微构组织整体均质性好、活性高、基质生成丰富;培养后期微构组织发生明显聚团,经灌注再装配得到细胞和基质分布均匀的厘米级(12 mm×6 mm)大组织.说明利用大孔微载体构建工程化组织是一种行之有效、富有潜力的体外组织构建方案,有望构建尺寸>5 mm、细胞和基质分布均匀的大组织.为体外组织构建提供新的思路.     

TGF—β在骨关节炎发生中的可能作用

转化生长因子TGF－β具有广泛的生理效应,对于维持关节软骨正常有重要意义。软骨细胞外基质降解和关节软骨损坏是骨关节炎的重要特征,研究表明TGF－β－方面通过调节细胞外基质成分含量维持基质的正常更新,另一方面通过调节关节软骨细胞的形成,增殖和分化来保持正常数量和正常生理状态的软骨细胞,本文就这两个方面综述了最新研究进展,以探讨TGF－β在骨关节炎发生中的可能调节机制。     

运动负荷对骨基质有机成分的影响及其可能机制

骨基质的有机成分主要为骨Ⅰ型胶原基质金属蛋白酶,该酶是细胞外基质降解的重要酶类;基质金属蛋白酶抑制因子则是基质金属蛋白酶活性的抑制剂,它们均为骨代谢过程中的重要标志性物质。本文通过查阅文献资料,对生理、部分病理状态及运动干预条件下,骨基质的Ⅰ型胶原和基质金属蛋白酶及其抑制因子的变化情况进行综述,并对其变化的机制予以阐释。     

间充质干细胞来源外泌体在膝骨关节炎微环境中的作用

骨关节炎是一种涉及所有关节成分（包括关节软骨、软骨下骨、滑膜、韧带、关节囊和关节周围肌肉）的关节退行性疾病，会导致严重的残疾，其中最常见的是膝骨关节炎（knee osteoarthritis，KOA）。外泌体是一种由不同细胞分泌的直径为40~100 nm的胞外囊泡，可以传递DNA、微小RNA、mRNA、蛋白质等多种物质，并通过多种方式进行细胞间的信息传递和功能调节。间充质干细胞（mesenchymal stem cells，MSCs）可以从骨髓、脂肪、滑膜及外周血等组织分离，是一类具有多向分化潜能的祖细胞，以干细胞为基础的疗法可以修复软骨损伤，对抗KOA的发展，间充质干细胞能够分泌多种营养因子来调节受损的微环境，其中间充质干细胞来源的外泌体被认为在KOA炎症反应及软骨细胞代谢中发挥着重要的作用，其能够调节膝骨关节微环境中B细胞、T细胞、滑膜细胞、软骨细胞代谢及其细胞外基质的分解与合成平衡，维持软骨稳态。近期有多项研究表明，不同组织来源的间充质干细胞外泌体对骨关节炎均有明确的治疗作用，本文就MSCs来源的外泌体治疗KOA的具体机制进行综述，以期对干细胞治疗KOA提供理论依据。     

软骨基质来源定向结构支架复合软骨细胞体外构建组织工程软骨的研究

目的：探讨采用软骨细胞外基质材料制备的定向结构软骨支架复合软骨细胞,在体外静态培养条件下生成组织工程软骨的可能性。方法：制备牛关节软骨细胞外基质材料,利用温度梯度热诱导相分离技术构建具备垂直定向孔道结构的软骨支架,同时采用传统冷冻干燥方法制备非定向支架,检测两组支架的力学性能;提取兔关节软骨细胞,分别接种两组支架,体外静态培养2周及4周后取材,对构建的组织工程软骨进行组织切片染色、生物化学分析及生物力学检测。结果：定向软骨支架的压缩弹性模量数值明显高于非定向软骨支架,体外培养时定向支架上种子细胞在3-9d内增殖高于非定向支架,差异有统计学意义（P〈0．05）;体外静态培养4周后形成的两组新生组织工程软骨进行软骨特异性染色均呈阳性,在定向组新生软骨切片中在垂直方向上可见大量呈规则平行排列的粗大胶原纤维,两组新生软骨的生物化学检测包括总DNA、总GAG及总胶原含量差异无统计学意义（P〉0．05）。定向组织工程软骨压缩弹性模量在2周及4周时均高于非定向组织工程软骨,差异有统计学意义（P〈0．05）。但两组组织工程软骨上述指标均显著低于正常关节软骨（P〈0．05）。结论：软骨细胞外基质材料制备的定向结构软骨支架复合软骨细胞,在体外静态培养条件下能够成功生成具有定向纤维结构的组织工程软骨,并可以有效促进新生软骨组织力学性能的提升,在软骨组织工程中具有良好的应用前景。     

Influence of tissue- and cell-scale extracellular matrix distribution on the mechanical properties of tissue-engineered cartilage

The insufficient load-bearing capacity of today’s tissue- engineered (TE) cartilage limits its clinical application. Generally, cartilage TE studies aim to increase the extracellular matrix (ECM) content, as this is thought to determine the load-bearing properties of the cartilage. However, there are apparent inconsistencies in the literature regarding the correlation between ECM content and mechanical properties of TE constructs. In addition to the amount of ECM, the spatial inhomogeneities in ECM distribution at the tissue scale as well as at the cell scale may affect the mechanical properties of TE cartilage. The relative importance of such structural inhomogeneities on mechanical behavior of TE cartilage is unknown. The aim of the present study was, therefore, to theoretically elucidate the influence of these inhomogeneities on the mechanical behavior of chondrocyte-agarose TE constructs. A validated non-linear fiber-reinforced poro-elastic swelling cartilage model that can accommodate for effects of collagen reinforcement and swelling by proteoglycans was used. At the tissue scale, ECM was gradually varied from predominantly localized in the periphery of the TE construct toward an ECM-rich inner core. The effect of these inhomogeneities in relation to the total amount of ECM was also evaluated. At the cell scale, ECM was gradually varied from localized in the pericellular area, toward equally distributed throughout the interterritorial area. Results from the tissue-scale model indicated that localization of ECM in either the construct periphery or in the inner core may reduce construct stiffness compared with that of constructs with homogeneous ECM. Such effects are more significant at high ECM amounts. At the cell scale, localization of ECM around the cells significantly reduced the overall stiffness, even at low ECM amounts. The compressive stiffness gradually increased when ECM distribution became more homogeneous and the osmotic swelling pressure in the interterritorial area increased. We conclude that for the same amount of ECM content in TE cartilage constructs, superior mechanical properties can be achieved with more homogeneous ECM distribution at both tissue and cell scale. Inhomogeneities at the cell scale are more important than those at the tissue scale.     

A fully coupled poroelastic reactive-transport model of cartilage

Cartilage maintains its integrity in a hostile mechanical environment. This task is made more difficult because cartilage has no blood supply, and so nutrients and growth factors need to be transported greater distances than normal to reach cells several millimetres from the cartilage surface. The chondrocytes embedded within the extracellular matrix (ECM) are essential for maintaining the mechanical integrity of the ECM, through a balance of degradation and synthesis of collagen and proteoglycans. A chondrocyte senses various chemical and mechanical signals in its local microenvironment, responding by appropriate adaption of the local ECM. Clearly a 'systems understanding' of cartilage behaviour is of critical importance in developing an integrated understanding of both normal and abnormal physiology of cartilage. In a series of papers, we have developed a reactive-transport porous-media model to investigate the coupled processes of growth factor transport, mechanical deformation and fluid flow, and in this paper, we extend the model to include biosynthesis and degradation of matrix molecules. The model is validated using three independent experimental data sets, it being found that a single set of parameters described the experimental results remarkably well. The model is then employed to make predictions about changes in proteoglycan content under a variety of conditions. This model may prove useful in predicting the behaviour of tissue engineering constructs, or predicting the outcome of repair processes in cartilage.     

l-glutamine is a key parameter in the immunosuppression phenomenon

The present work describes the influence of both vitamin C (VC) and mechanical stimulation on development of the extracellular matrix (ECM) and improvement in mechanical properties of a chondrocyte-agarose construct in a regenerating tissue disease model of hyaline cartilage. We used primary bovine chondrocytes and two types of VC, ascorbic acid (AsA) as an acidic form and ascorbic acid 2-phosphate (A2P) as a non-acidic form, and applied uniaxial compressive strain to the tissue model using a purpose-built bioreactor. When added to the medium in free-swelling culture conditions, A2P downregulated development of ECM and suppressed improvement of the tangent modulus more than AsA. By contrast, application of mechanical stimulation to the construct both increased the tangent modulus more than the free-swelling group containing A2P and enhanced the ECM network of inner tissue to levels nearly as high as the free-swelling group containing AsA. Thus, mechanical stimulation and strain appears to enhance the supply of nutrients and improve the synthesis of ECM via mechanotransduction pathways of chondrocytes. Therefore, we suggest that mechanical stimulation is necessary for homogenous development of ECM in a cell-associated construct with a view to implantation of a large-sized articular cartilage defect.     

Cyclic compression of articular cartilage explants is associated with progressive consolidation and altered expression pattern of extracellular matrix proteins.

In this study, we investigated the biosynthetic response of full thickness, adult bovine articular cartilage explants to 45 h of static and cyclic unconfined compression. The cyclic compression of articular cartilage resulted in a progressive consolidation of the cartilage matrix. The oscillatory loading increased protein synthesis ([35S]methionine incorporation) by as much as 50% above free swelling control values, but had an inhibitory influence on proteoglycan synthesis ([35SO4] incorporation). As expected, static compression was associated with a dose-dependent decrease in biosynthetic activity. ECM oligomeric proteins which were most affected by mechanical loading were fibronectin and cartilage oligomeric matrix protein (COMP). Static compression at all amplitudes caused a significant increase in fibronectin synthesis over free swelling control levels. Cyclic compression of articular cartilage at 0.1 Hz and higher was consistently associated with a dramatic increase in the synthesis of COMP as well as fibronectin. The biosynthetic activity of chondrocytes appears to be sensitive to both the frequency and amplitude of the applied load. The results of this study support the hypothesis that cartilage tissue can remodel its extracellular matrix in response to alterations in functional demand.     

Flow characterization of a wavy-walled bioreactor for cartilage tissue engineering

Cartilage tissue engineering requires the use of bioreactors in order to enhance nutrient transport and to provide sufficient mechanical stimuli to promote extracellular matrix (ECM) synthesis by chondrocytes. The amount and quality of ECM components is a large determinant of the biochemical and mechanical properties of engineered cartilage constructs. Mechanical forces created by the hydrodynamic environment within the bioreactors are known to influence ECM synthesis. The present study characterizes the hydrodynamic environment within a novel wavy-walled bioreactor (WWB) used for the development of tissue-engineered cartilage. The geometry of this bioreactor provides a unique hydrodynamic environment for mammalian cell and tissue culture, and investigation of hydrodynamic effects on tissue growth and function. The flow field within the WWB was characterized using two-dimensional particle-image velocimetry (PIV). The flow in the WWB differed significantly from that in the traditional spinner flask both qualitatively and quantitatively, and was influenced by the positioning of constructs within the bioreactor. Measurements of velocity fields were used to estimate the mean-shear stress, Reynolds stress, and turbulent kinetic energy components in the vicinity of the constructs within the WWB. The mean-shear stress experienced by the tissue-engineered constructs in the WWB calculated using PIV measurements was in the range of 0-0.6 dynes/cm2. Quantification of the shear stress experienced by cartilage constructs, in this case through PIV, is essential for the development of tissue-growth models relating hydrodynamic parameters to tissue properties.     

A reaction-diffusion model to predict the influence of neo-matrix on the subsequent development of tissue-engineered cartilage

Extracellular matrix (ECM) in chondrocytes-seeded agarose aggregates to form islands of matrix. These islands need to coalesce to develop functional cartilage. Hence, macroscopic properties are determined by transport and aggregation of macromolecules at the microscale, which varies temporally and spatially. This study evaluates the importance of the mutual interaction between matrix components and matrix development. Fluorescence recovery after photobleaching measurements demonstrates that diffusivity depends on the presence and density of ECM. A reaction-diffusion model describing synthesis, transport and immobilisation of ECM predicts steep gradients in ECM around chondrocytes, resembling histology. Steric hindrance of diffusion by ECM is essential for the formation of these gradients. Finally, microscopic ECM concentration is linked with macroscopic mechanical properties. Construct softening is predicted when temporal and spatial variations in diffusivity are considered. In conclusion, non-constant diffusion renders significant effects on both the microscopic ECM development and the macroscopic mechanical properties of developing tissue-engineered cartilage.     

Methods in studying ECM degradation

Almost all tissues in our body contain specific cells associated with the tissue itself, and an extracellular matrix (ECM) that consists of a variety of proteins of which the bulk is formed by different types of collagens, glycoproteins and proteoglycans. The ECM plays a pivotal role in numerous processes not only related to the mechanical properties of a tissue, but also in modulating cellular activity. For a proper functioning of a tissue remodeling of the ECM is essential. Some connective tissues are characterized by a very rapid turnover (e.g. periodontal ligament) whereas others hardly show signs of turnover (e.g. cartilage). In all situations degradation of the ECM constituents occur. Under certain conditions, especially during a pathological situation, a high level of degradation may take place. In other situations matrix synthesis and deposition outstrips breakdown, leading to a fibrosis. In order to obtain information on the level of degradation of the different ECM components, various methods have been employed. A number of these methods will be discussed in this article.     

Mathematical model of the role of degradation on matrix development in hydrogel scaffold

Despite tremendous advances in the field of tissue engineering, a number of obstacles are still hindering its successful translation to the clinic. One of these challenges has been to design cell-laden scaffolds that can provide an appropriate environment for cells to successfully synthesize new tissue while providing a mechanical support that can resist physiological loads at the early stage of in situ implementation. A solution to this problem has been to balance tissue growth and scaffold degradation by creating new hydrogel systems that possess both hydrolytic and enzymatic degradation behaviors. Very little is known, however, about the complex behavior of these systems, emphasizing the need for a rigorous mathematical approach that can eventually assist and guide experimental advances. This paper introduces a mathematical and numerical formulation based on mixture theory, to describe the degradation, swelling, and transport of extracellular matrix (ECM) molecules released by cartilage cells (chondrocytes) within a hydrogel scaffold. The model particularly investigates the relative roles of hydrolytic and enzymatic degradations on ECM diffusion and their impacts on two important outcomes: the extent of ECM transport (and deposition) and the evolution of the scaffold’s mechanical integrity. Numerical results based on finite element show that if properly tuned, enzymatic degradation differs from hydrolytic degradation in that it can create a degradation front that is key to maintaining scaffold stiffness while allowing ECM deposition. These results therefore suggest a hydrogel design that could enable successful in situ cartilage tissue engineering.     

Tensile and compressive properties of healthy and osteoarthritic human articular cartilage

Osteoarthritis (OA) is a disease affecting articular cartilage and the underlying bone, resulting from many biological and mechanical interacting factors which change the extracellular matrix (ECM) and cells and lead to increasing levels of cartilage degeneration, like softening, fibrillation, ulceration and cartilage loss. The early diagnosis of the disease is fundamental to prevent pain, further tissue degeneration and reduce hospital costs. Although morphological modifications can be detected by modern non-invasive diagnostic techniques, they may not be evident in the early stages of OA. The mechanical properties of articular cartilage are related to its composition and structure and are sensitive to even small changes in the ECM that could occur in early OA. The aim of the present study was to compare the mechanical properties of healthy and OA cartilage using a combined experimental-numerical approach. Experimental assessments consisted of step wise confined and unconfined compression and tension stress relaxation tests on disks (for compression) or strips (for tension) of cartilage obtained from human femoral heads discarded from the operating room after total hip replacement. The numerical model was based on the biphasic theory and included the tension-compression non-linearity. Considering OA samples vs normal samples, the static compressive modulus was 55-68% lower, the permeability was 60-80% higher, the dynamic compressive modulus was 59-64% lower, the static tension modulus was 72-83% lower. The model successfully simulated the experimental tests performed on healthy and OA cartilage and was used in combination with the experimental tests to evaluate the role of different ECM components in the mechanical response of normal and OA cartilage.     

Detection of cartilage matrix degradation by autofluorescence lifetime

Cartilage is a vital organ to maintain joint function. Upon arthritis, proteolytic enzymes initiate degradation of cartilage extracellular matrix (ECM) resulting in eventual loss of joint function. However, there are only limited ways of non-invasively monitoring early chemical changes in cartilage matrix. Here we report that the autofluorescence decay profiles of cartilage tissue are significantly affected by proteolytic degradation of cartilage ECM and can be characterised by measurements of the autofluorescence lifetime (AFL). A compact multidimensional fluorometer coupled to a fibre-optic probe was developed for single point measurements of AFL and applied to cartilage that was treated with different proteinases. Upon treating cartilage with bacterial collagenase, trypsin or matrix metalloproteinase 1, a significant dose and time dependent decrease of AFL was observed. Our data suggest that AFL of cartilage tissue is a potential non-invasive readout to monitor cartilage matrix integrity that may contribute to future diagnosis of cartilage defects as well as monitoring the efficacy of anti-joint therapeutic agents.