骨组织工程基质材料的现状及展望

通过阐述理想骨组织工程细胞外基质材料的要求,并总结生物类材料、生物陶瓷类材料、聚合物类材料和复合类材料在骨组织工程中用作细胞外基质材料的优缺点,认为理想的骨组织工程细胞外基质材料最好由生物陶瓷类材料、人工合成聚合物类材料或天然聚合物类材料组成,并含有最佳的生长因子缓释系统的多孔三维立体泡沫,这种复合型细胞外基质材料的研制是骨组织工程研究中十分重要而迫切的任务。     

输尿管细胞外基质修复输尿管缺损的实验研究

为了寻求理想的输尿管替代材料 ,采用生化方法制备了一种新型的组织工程材料细胞外基质 (Extra-cellular Matrix ,ECM) ,并用于替代同种异体输尿管缺损 ,结果表明 ECM很好的促进了缺损组织的再生 ,替代术后 16周再生组织的形态与功能已与正常组织无明显差异。异体输尿管细胞外基质是一种理想的输尿管替代材料     

组织工程基质材料研究进展

细胞外基质材料的研究是组织工程中一项重要而紧迫的任务。本文就其材料选择、制备方法、影响因素和表面修饰等方面的研究现状进行了较系统的综述 ,并指出了当前细胞外基质材料研究所面临的问题以及未来的研究方向     

软骨组织工程的研究现状及展望

目前,软骨组织工程的研究内容主要集中在以下几个方面:种子细胞;三维生物材料载体;细胞培养体系;人工软骨性能、实验动物模型和临床试验评价。我们综述了软骨组织工程的研究现状及其进展。     

A Paradigm for Functional Tissue Engineering of Articular Cartilage via Applied Physiologic Deformational Loading

Deformational loading represents a primary component of the chondrocyte physical environment in vivo. This review summarizes our experience with physiologic deformational loading of chondrocyte-seeded agarose hydrogels to promote development of cartilage constructs having mechanical properties matching that of the parent calf tissue, which has a Young's modulus E(Y) = 277 kPa and unconfined dynamic modulus at 1 Hz G* = 7 MPa. Over an 8-week culture period, cartilage-like properties have been achieved for 60 x 10(6) cells/ml seeding density agarose constructs, with E(Y) = 186 kPa, G* = 1.64 MPa. For these constructs, the GAG content reached 1.74% ww and collagen content 2.64% ww compared to 2.4% ww and 21.5% ww for the parent tissue, respectively. Issues regarding the deformational loading protocol, cell-seeding density, nutrient supply, growth factor addition, and construct mechanical characterization are discussed. In anticipation of cartilage repair studies, we also describe early efforts to engineer cylindrical and anatomically shaped bilayered constructs of agarose hydrogel and bone (i.e., osteochondral constructs). The presence of a bony substrate may facilitate integration upon implantation. These efforts will provide an underlying framework from which a functional tissue-engineering approach, as described by Butler and coworkers (2000), may be applied to general cell-scaffold systems adopted for cartilage tissue engineering.     

生物功能表面及其在组织工程中的应用

材料生物功能表面的构筑,从材料学角度讲是通过材料表面工程使生物材料适应并被组织"接纳",赋予其动态整合性;从临床应用的角度,功能化表面接合周围组织与植入材料,起到"人工细胞外基质"的作用。生物功能表面的构筑在组织工程学中占有非常重要的地位。本文在简单介绍生物表面科学的基础上,综述了生物功能表面的构筑及其在组织工程中的应用。     

软骨组织工程用材料进展

对近年来软骨组织工程用材料及其制备技术作了较系统的综述,指出了当前软骨组织工程所面临的问题,并针对此问题对未来软骨组织工程用材料的研究作出了展望。     

细胞外基质材料在骨组织工程中的研究进展

由疾病、外部创伤等原因引起的大骨骼缺损的治疗需要通过骨移植手术,寻找安全易得的替代骨已经成为临床上的重要课题,近年来快速发展的组织工程骨为解决这一难题提供了一种新的途径。支架材料作为组织工程的核心要素,其表面性状、结构,机械性能和生物学性能均能调控细胞的各种生命活动和体内组织的修复再生。细胞外基质由于其天然性、低免疫排斥性和优异的生物相容性等特点,已被广泛用作再生医学的支架材料。通过回顾近些年来细胞外基质材料在骨组织工程中的应用,阐述多种细胞外基质材料的构建修饰方法及其体外、体内的生物学效应,并对其在骨再生领域的应用前景进行展望。     

多层软骨组织工程支架的制备

软骨组织工程为关节软骨损伤的治疗提供了一种理想的治疗途径,但体外构建的软骨如何在临床应用时采用适当的方法短期内在体内进行良好地固定仍然没有得到很好的解决,阻碍了采用组织工程方法治疗关节软骨疾病的临床应用.本研究通过冷冻干燥的方法制备出一个上层为胶原,下层逐层过渡到以羟基磷灰石为主的复合一体化软骨组织工程支架,利用复合支架下层羟基磷灰石良好的骨传导作用,将支架整体通过底部的快速固定达到对上层软骨进行固定的目的.本研究对该复合支架的制备及其形态结构特点进行了初步研究.结果表明胶原层的孔径孔隙率可以通过冷冻温度及胶原溶液浓度进行控制;羟基磷灰石粒度及其粒度分布明显影响其在胶原支架内部的分布;SEM照片表明用该方法制备出了三层具有不同羟基磷灰石含量的复合胶原羟基磷灰石软骨组织工程支架,支架总高度为6cm,每层高度约为2cm,三层之间不存在界面形成了一个均匀的整体结构.为软骨组织工程提供了一种新型的支架.     

组织工程引导骨再生膜的体外构建

采用多孔的 PCL/ PL A共聚膜 ,对材料进行亲水性和细胞亲和性表面加工后 ,用作组织工程载体材料。通过将 5× 10 6 / ml成骨诱导 10 d的 MSCs与 PCL/ PL A共同培养 7d,扫描电镜观察细胞在材料中的生长状态。发现材料作为细胞生长的支持结构与细胞的相容性良好 ,细胞可以黏附在材料的表面和孔隙中。实验证明该材料具有良好的生物相容性 ,可以在体外与骨髓间质干细胞共同构建组织工程化的细胞 -材料复合物用于引导性骨再生。     

Extracellular Matrix Composition of Full-Thickness Defect Repair Tissue is Little Influenced by Exercise in Rat Articular Cartilage

Full-thickness articular cartilage defects in the femoral condyles of adult rats were examined four and eight weeks after injury. Quantitative polarized light microscopic analysis showed that birefringence of the tissue in the central repair area increased more in rats exercised on a treadmill. Glycosaminoglycan content in the repair tissue was also higher than in the intermit-tent active motion group at four weeks after injury, but by eight weeks the levels were similar in both groups. No normal-looking articular cartilage was formed in the lesions, and only in one animal type II collagen was observed in the superficial zone of repair tissue. No 3B3(-) antigenicity of the proteoglycans was seen during repair. In conclusion, exercise minimally modified the repair of full-thickness articular cartilage defects in adult rats. The repair in the exercised group may occur slightly faster in the early stages but no difference was seen at the eight week time interval between the exercised and the intermittently active group.     

组织工程软骨培养的细胞自动机算法模型与优化

建立一个基于细胞自动机算法的软骨细胞和干细胞体外动态培养数学模型,并进行模拟计算与优化,为组织工程软骨的体外培养控制软件的编写提供理论依据。建立细胞自动机算法模型,采用软骨细胞和软骨干细胞作为研究对象进行Matlab仿真实验。实验分为3组,每组各自设置空白对照组、快速扩增组、力学刺激组、扩增与力学刺激复合培养组。空白对照组的结果显示,软骨细胞和干细胞诱导软骨细胞的细胞占空间区域分别为51%和45%;综合考虑细胞生长情况,软骨细胞培养最佳压强施加大小为100 kPa;干细胞诱导培养最佳压强施加大小为15 kPa。运用细胞自动机算法建立的数学模型来动态培养软骨细胞,可以为体外生物反应器控制软件的编写提供理论依据,对于组织工程培养软骨组织及其他器官培养是一种有效的可供参考的方法。     

The Local Matrix Distribution and the Functional Development of Tissue Engineered Cartilage,a Finite Element Study

Assessment of the functionality of tissue engineered cartilage constructs is hampered by the lack of correlation between global measurements of extra cellular matrix constituents and the global mechanical properties. Based on patterns of matrix deposition around individual cells, it has been hypothesized previously, that mechanical functionality arises when contact occurs between zones of matrix associated with individual cells. The objective of this study is to determine whether the local distribution of newly synthesized extracellular matrix components contributes to the evolution of the mechanical properties of tissue engineered cartilage constructs. A computational homogenization approach was adopted, based on the concept of a periodic representative volume element. Local transport and immobilization of newly synthesized matrix components were described. Mechanical properties were taken dependent on the local matrix concentration and subsequently the global aggregate modulus and hydraulic permeability were derived. The transport parameters were varied to assess the effect of the evolving matrix distribution during culture. The results indicate that the overall stiffness and permeability are to a large extent insensitive to differences in local matrix distribution. This emphasizes the need for caution in the visual interpretation of tissue functionality from histology and underlines the importance of complementary measurements of the matrixs intrinsic molecular organization.     

脂肪干细胞在软骨组织工程中的研究进展

近年来脂肪干细胞（ADSC）的成软骨潜能备受瞩目,其作为种子细胞在软骨组织工程应用方面前景广阔,然而目前并没有一种高效的诱导方法被世界公认。外源性生长因子、基因修饰、共培养、生长因子与共培养联合、支架材料等多种手段均可诱导ADSC向软骨细胞方向分化,但各方法的优劣,诱导分化时的信号通路及新生软骨的性状并未见详细的分析和讨论。文中介绍了以上多种诱导方法的研究现状及问题,并讨论了化学因素、供体年龄、组织获取部位等对其的影响,以期为ADSC的临床应用提供一定的理论参考。     

Using Cartilage to Repair Bone: An Alternative Approach in Tissue Engineering

Materials and techniques currently used for bone replacement/repair conform to the current paradigm, relying on bone or bone products to produce bone or induce bone formation. Yet, nature forms and heals most of the skeleton by ossification of a cartilaginous model. In this study, we cultured aggregates of E10.5 or E12 mouse embryonic limb cells in the bioreactor for 3 weeks, determined the stages of cartilage differentiation attained, and assessed the ossification and bone healing potential of the spheroids by implantation adjacent to, or directly in, a skull defect. Cultured spheroids had large cartilaginous areas, sometimes with cellular arrangements characteristic of growth plate zones. Aggregates implanted for 2 weeks adjacent to a defect mineralized and ossified (histology, micro-CT). Defects with implants had a central mass of differentiated and differentiating bone, with osteoclast activity, filling the defect. Controls had considerable remodeling on the bone edges demarcating the still present defect. This study shows that cartilage, grown in the bioreactor for 3 weeks, ossified when implanted adjacent to a bone defect, and when implanted directly in a defect, contributed to its healing. Our ability to grow differentiated bone-forming cartilage for implantation is an alternative approach in the field of bone repair.     

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。     

液氮冷冻保存对主动脉瓣膜细胞活性及细胞外基质的影响

液氮冷冻保存是目前最好的同种心瓣膜保存方法 ,但液氮冷冻保存也可导致瓣膜的结构损害。为了探讨液氮冷冻保存对主动脉瓣膜组织结构的影响情况 ,本实验通过采用XTT比色法、形态学观察、免疫组织化学荧光染色法研究了不同冷冻保存时间对猪主动脉瓣膜细胞活性及细胞外基质的影响。研究结果显示 ,液氮保存后瓣膜细胞活性明显下降 (P <0 .0 5 ) ,细胞超微结构损害增加 ,可溶性细胞外基质纤维连接蛋白与硫酸软骨素不同程度流失 ,间质胶原纤维排列紊乱。但在冻存时间小于 3月时 ,损害的程度与冻存时间无明显关系。     

The Effects of Collagen Fragments on the Extracellular Matrix Metabolism of Bovine and Human Chondrocytes

Cartilage matrix degradation generates collagen type II fragments. The objective of this study is to explore the possibility that these collagen fragments may be part of an endogenous metabolic feedback. Initially, collagen fragments were extracted from normal or osteoarthritic cartilage, as part of a matrix fragment preparation. Later, collagen fragments were generated by digestion of bovine collagen type II with bacterial collagenase (col2f). These fragments were added to cultures of isolated chondrocytes (bovine and human) and cartilage explants (human). In a dose-dependent manner, col2f caused inhibition of cell attachment to collagen, inhibition of collagen synthesis, and induction of matrix degradation. In addition, when col2f were added to human cartilage explants, an induction of gelatinase activity was detected in the media. These data sets present first evidence that degradation products of collagen may be directly involved in the regulation of cartilage homeostasis.     

Synergistic Stimuli by Hydrodynamic Pressure and Hydrophilic Coating on PLGA Scaffolds for Extracellular Matrix Synthesis of Engineered Cartilage

Abstract

Scaffold surface properties and mechanical stimuli have been known to have a critical influence on cell proliferation and extracellular matrix synthesis in cultured cells for tissue engineering. Hydrophilic surface and hydrodynamic pressure (HP) stimulation have been shown to have a beneficial effect on chondrocyte activity. The aim of this study was, thus, to assess the synergic influences of HP and hydrophilic coating on cell activity using primary porcine chondrocytes inoculated in hydrophilic-coated poly(lactide-co-glycolide) (PLGA) sponge scaffolds. The natural materials hyaluronic acid (HA), chitosan and HA/chitosan were cross-linked on porous PLGA as a hydrophilic surface modification. HP was applied to scaffolds at an amplitude of 2.24 MPa and a frequency of 0.1 Hz for 30 min per day, twice a week, over a period of 28 days. Cell activities were determined by the MTS assay and the dimethylmethylene blue assay for glycosaminoglycan (GAG) quantification. Our results displayed that PLGA coated with both HA and chitosan had the best hydrophilicity (contact angle 49.46°) and initial compressive modulus (1.10 ± 0.13 MPa) among the tested scaffold groups. Additionally, HP stimulation enhanced cell proliferation as well as GAG production (up to 3-fold in culture medium and 15-fold in scaffolds at 28 days compared to static culture of PLGA alone in the scaffold group) in the hydrophilic-coated scaffold groups. The synergistic benefit from hydrophilic coating and HP stimulation may be imperative in regenerating engineered cartilage in the long-term.     

Cell density alters matrix accumulation in two distinct fractions and the mechanical integrity of alginate-chondrocyte constructs

Chondrocyte density in articular cartilage is known to change with the development and growth of the tissue and may play an important role in the formation of a functional extracellular matrix (ECM). The objective of this study was to determine how initial chondrocyte density in an alginate hydrogel affects the matrix composition, its distribution between the cell-associated (CM) and further removed matrix (FRM) fractions, and the tensile mechanical properties of the developing engineered cartilage. Alginate constructs containing primary bovine chondrocytes at densities of 0, 4, 16, and 64 million cells/ml were fabricated and cultured for 1 or 2 weeks, at which time structural, biochemical, and mechanical properties were analyzed. Both matrix content and distribution varied with the initial cell density. Increasing cell density resulted in an increasing content of collagen and sulfated-glycosaminoglycan (GAG) and an increasing proportion of these molecules localized in the CM. While the equilibrium tensile modulus of cell-free alginate did not change with time in culture, the constructs with highest cell density were 116% stiffer than cell-free controls after 2 weeks of culture. The equilibrium tensile modulus was positively correlated with total collagen (r² = 0.47, p < 0.001) and GAG content (r² = 0.68, p < 0.001), and these relationships were enhanced when analyzing only those matrix molecules in the CM fraction (r² = 0.60 and 0.72 for collagen and GAG, respectively, each p < 0.001). Overall, the results of this study indicate that initial cell density has a considerable effect on the developing composition, structure, and function of alginate–chondrocyte constructs.