软骨组织工程种子细胞的来源、培养和评价

可靠而安全的软骨种子细胞来源是保证软骨组织工程持续深入研究的前提。本介绍了目前软骨组织工程种子细胞来源、培养和评价等方面的最新研究进展。     

软骨组织工程种子细胞的基础和应用研究进展

软骨种子细胞的老化和缺乏是限制软骨组织工程在临床应用的瓶颈问题。本文概述了目前对软骨种子细胞研究的两个方面 :扩大种子细胞的来源及预防和延缓种子细胞功能老化 ,并展望软骨组织工程在监床的应用前景。     

软骨组织工程种子细胞的基础和应用研究进展

软骨种子细胞的老化和缺乏是限制软骨组织工程在临床应用的瓶颈问题。本概述了目前对软骨种子细胞研究的两个方面，扩大种子细胞的来源及预防和延缓种子细胞功能老化，并展望软骨组织工程在临床的应用前景。     

中国软骨组织工程研究领域——著名专家介绍

<正>软骨组织工程是组织工程研究领域开展最早,发展最快的学科领域之一,但软骨组织构建的种子细胞来源有限,软骨细胞体外培养扩增困难且极易     

胚胎干细胞向成骨、软骨细胞定向分化研究进展

骨、软骨组织工程的热点之一是寻找合适的种子细胞.胚胎干细胞因具有全能性和无限增殖的能力有望成为组织工程中的种子细胞新来源.主要介绍了胚胎干细胞定向诱导分化为成骨、软骨细胞研究所取得的进展,并展望了胚胎干细胞作为组织工程种子细胞的前景和所面临的困难.     

软骨组织工程种子细胞及预防其老化的研究进展

软骨组织的修复和再生能力较弱,加之软骨种子细胞易于老化,这些因素限制着软骨组织工程在临床的应用.本文就近年来有关软骨组织工程种子细胞的建立及延缓和预防软骨种子细胞老化的研究进展和前景作一综述.     

机械拉伸诱导骨髓间充质干细胞向软骨细胞分化的实验研究

目的软骨组织工程种子细胞的来源问题是当前限制软骨组织工程发展的主要因素之一。骨髓间充质干细胞(mesenchymal stem cell,MSCs)是一种具有多项分化潜能的细胞,已有研究证实,MSCs经诱导能定向分化为软骨细胞,进而形成软骨组织。由于MSCs具有易于取材、特性稳定、可连续传代培养和冷冻保存等特点使得MSCs成为软骨组织工程理想的种子细胞来源。笔者实验拟研究大鼠MSCs的分离、纯化的方法,采用四点弯曲的加载方式产生基底应变,对MSCs施加周期性拉伸,探索间充质干细胞向软骨细胞分化的条件,为修复软骨提供理论依据和技术基础。     

软骨组织工程种子细胞及预防其老化的研究进展

软骨组织的修复和再生能力较弱，加之软骨种子细胞易于老化，这些因素限制着软骨组织工程在临床的应用。本就近年来有关软骨组织工程种子细胞的建立及延缓和预防软骨种子细胞老化的研究进展和前景作一综述。     

骨、软骨组织工程种子细胞及其免疫学相关研究进展

近年来,细胞生物与生物材料学的迅速发展加快了骨、软骨组织工程的研究.骨、软骨组织工程的系列研究中,种子细胞是骨、软骨组织工程研究的核心内容.本文就骨、软骨组织工程的种子细胞成骨细胞、软骨细胞、骨髓基质细胞、间充质干细胞、胚胎干细胞及其免疫学相关研究进展作一讨论.     

软骨组织工程研究进展

软骨组织工程技术,结合生物、工程、医学与科学,以构建有功能的组织替代损伤组织为目标。本文从支架材料、种子细胞的来源及体外培养、生长因子和生物学评价几个方面中综述了软骨组织工程的进展,并提出了一些尚待解决的问题。     

Multiplication of human chondrocytes with low seeding densities accelerates cell yield without losing redifferentiation capacity

To treat a cartilage defect with tissue-engineering techniques, multiplication of donor cells is essential. However, during this multiplication in monolayer expansion culture chondrocytes will lose their phenotype and produce matrix of inferior quality (dedifferentiation). Dedifferentiation occurs more extensively with low seeding densities and passaging. To obtain cartilage of good quality it is important that the multiplicated cells regain their cartilaginous phenotype (redifferentiation capacity). A "gold standard" for the multiplication of chondrocytes in monolayer, with respect to seeding density and passaging, is lacking. In numerous available studies, various cell densities have been used, making comparison of the results of these studies difficult. Therefore, we performed a comparative study to gain insight concerning the effect of seeding density and passaging on the capacity of cells to redifferentiate. From the resulting data we deduced the seeding density in monolayer culture for which cell expansion is both sufficient and fast, while the cells retain a capacity to redifferentiate. As a guideline we calculated that, at minimum, 20-fold multiplication is needed to fill an average cartilage defect of 4 cm(2) with the amount of donor chondrocytes we obtained. For this study we used isolated ear chondrocytes from five children. Four different seeding densities in monolayer culture were used, ranging from 3500 to 30000 cells/cm(2). The cells were cultured for four passages. The capacity of the expanded chondrocytes to redifferentiate (redifferentiation capacity) was studied after an additional 3-week culture in alginate beads and was assessed by glycosaminoglycan production and immunohistochemical stainings for collagen type I, collagen type II, elastin, and a fibroblast marker (11-fibrau). In general, we found that both passaging and decreasing seeding density yielded an increase in expanded chondrocytes, but at the same time decreased the dedifferentiation capacity. In further analyzing our data according to the proposed guidelines we found that with lower seeding densities sufficient multiplication (20 times) was reached in less time and with less passaging than at higher seeding densities. Importantly, the redifferentiation capacity of these chondrocytes was preserved. It was equal to or even surpassed that of chondrocytes multiplied 20 times at higher seeding densities, which required more time and more passages in monolayer culture. Thus, for cartilage tissue-engineering purposes we propose that expansion culture with low seeding densities is preferable.     

Seeding cells into needled felt scaffolds for tissue engineering applications

Tissue engineering methods are under development that will enable the repair or replacement of a variety of tissues, including articular cartilage and bone. To engineer functional tissue it is necessary that scaffolds initially be seeded with a large number of cells distributed evenly throughout the scaffold structure. It previously has been shown that, compared to static seeding conditions, seeding scaffolds under dynamic conditions facilitates high seeding densities and even distributions of cells (Li et al., Biotechnology Progress 2001;17:935-944). The efficiency of seeding HOSTE85 cells and bovine chondrocytes into needled felt scaffolds following agitation at different speeds was determined. Seeding efficiency was determined using the Hoechst 33258 assay, and cell viability was assessed using the Alamar Blue trade mark assay. The distribution of cells within the scaffolds was imaged using scanning electron microscopy. It was found that the optimum seeding conditions varied for HOSTE85 cells and bovine chondrocytes, with different agitation speeds leading to different seeding efficiencies, cell viabilities, and distributions of cells within scaffolds. The optimum agitation speeds for seeding a high number of viable cells into scaffolds so that they were arranged evenly were 300 rpm for HOSTE85 cells and 200 rpm for bovine chondrocytes.     

软骨组织工程研究的新进展

软骨组织的再生能力有限 ,组织工程软骨的构建对修复软骨缺损意义重大。本文从四方面介绍了软骨组织工程的研究的新进展 ,包括软骨种子细胞的研究、软骨细胞与支架的体外培养、细胞支架复合物植入体内的研究及软骨细胞移植的临床应用。     

Repair of large articular cartilage defects with implants of autologous mesenchymal stem cells seeded into beta-tricalcium phosphate in a sheep model

Tissue engineering has long been investigated to repair articular cartilage defects. Successful reports have usually involved the seeding of autologous chondrocytes into polymers. Problems arise because of the scarcity of cartilage tissue biopsy material, and because the in vitro expansion of chondrocytes is difficult; to some extent, these problems limit the clinical application of this promising method. Bone marrow-derived mesenchymal stem cells (MSCs) have been proved a potential cell source because of their in vitro proliferation ability and multilineage differentiation capacity. However, in vitro differentiation will lead to high cost and always results in decreased cell viability. In this study we seeded culture-expanded autologous MSCs into bioceramic scaffold-beta-tricalcium phosphate (beta-TCP) in an attempt to repair articular cartilage defects (8 mm in diameter and 4 mm in depth) in a sheep model. Twenty-four weeks later, the defects were resurfaced with hyaline-like tissue and an ideal interface between the engineered cartilage, the adjacent normal cartilage, and the underlying bone was observed. From 12 to 24 weeks postimplantation, modification of neocartilage was obvious in the rearrangement of surface cartilage and the increase in glycosaminoglycan level. These findings suggest that it is feasible to repair articular cartilage defects with implants generated by seeding autologous MSCs, without in vitro differentiation, into beta-TCP. This approach provides great potential for clinical applications.     

New technique of seeding chondrocytes into microporous poly(L-lactide-co-epsilon-caprolactone) sponge by cyclic compression force-induced suction

The initial requirement for a functional engineered cartilage tissue is the effective and reproducible seeding of chondrocytes into the interior of microporous scaffolds. High seeding efficiency, high cell viability, uniform cell distribution, and short operation time are also essential. We devised a new technique of seeding rabbit chondrocytes into microporous poly(L-lactide-co-epsilon-caprolactone) (PLCL) (porosity, 71- 80%; wall thickness, 2 and 6 mm) sponges under compression force-induced suction using a custom designed loading apparatus. Cell distribution and cell viability were determined using confocal laser scanning microscopy with fluorescent dye-staining techniques. Factors that affect the quality of a cell seeded construct were studied, namely, the porosity and thickness of sponges and suction cycles. Under 1 cycle of suction, an increase in porosity promoted cell seeding efficiency (CSE; defined as the percentage of the number of cells in the sponges relative to the initial number of cells seeded), cell viability (at 1 day post seeding), and a relatively uniform cell distribution, whereas thick sponges exhibited an inhomogeneous cell distribution irrespective of incubation time. Multiple cycles of suction of 5 and 10 at 0.1 Hz significantly improved the CSE, whereas high cell viability was maintained and even spatial cell distribution was achieved in 1 week. This study revealed that our newly developed cell seeding technique with multiple cycles of suction is a promising approach to inoculating cells into microporous sponges with high CSE, high cell viability, and homogeneous cell distribution.     

以壳聚糖-胶原共混膜为三维支架同种异体工程化软骨的构建

目的探讨以壳聚糖-胶原共混膜为三维支架材料的同种异体软骨细胞构建组织工程化软骨的能力。方法将分离、培养、扩传兔软骨细胞，接种在壳聚糖-胶原共混膜上，倒置显微镜下观察细胞在共混膜上的生长情况。体外培养7d后，将细胞-材料复合物种植在新西兰兔皮下，6周取材，对获得的同种异体工程化软骨进行组织学评价。结果兔软骨细胞接种于壳聚糖-胶原共混膜上4h后有贴壁现象出现，细胞呈梭形。培养48h后，软骨细胞分裂增殖越来越多并向周围延伸，培养第7天取材，HE染色示细胞生长良好，呈梭形。体内培养6周取材，HE染色、Masson染色为均一的成熟软骨组织，且共混膜已降解。结论以壳聚糖-胶原共混膜为支架材料同种异体软骨细胞在有免疫力的动物体内可形成工程化软骨。     

Tissue-engineered cartilage using serially passaged articular chondrocytes. Chondrocytes in alginate, combined in vivo with a synthetic (E210) or biologic biodegradable carrier (DBM)

In vitro multiplication of isolated autologous chondrocytes is required to obtain an adequate number of cells to generate neo-cartilage, but is known to induce cell-dedifferentiation. The aim of this study was to investigate whether multiplied chondrocytes can be used to generate neo-cartilage in vivo. Adult bovine articular chondrocytes, of various differentiation stages, were suspended in alginate at densities of 10 or 50 million/ml, either directly after isolation (P0) or after multiplication in monolayer for one (P1) or three passages (P3). Alginate with cells was seeded in demineralized bovine bone matrix (DBM) or a fleece of polylactic/polyglycolic acid (E210) and implanted in nude mice for 8 weeks. The newly formed tissue was evaluated by Alcian Blue and immunohistochemical staining for collagen type-II and type-I. Structural homogeneity of the tissue, composed of freshly isolated as well as serially passaged cells, was found to be enhanced by high-density seeding (50 million/ml) and the use of E210 as a carrier. The percentage of collagen type-II positive staining P3-cells was generally higher when E210 was used as a carrier. Furthermore, seeding P3-chondrocytes at the highest density (50 million/ml) enhanced collagen type-II expression. This study shows promising possibilities to generate structurally regular neo-cartilage using multiplied chondrocytes in alginate in combination with a fleece of polylactic/polyglycolic acid.     

Growth factors for clinical-scale expansion of human articular chondrocytes: relevance for automated bioreactor systems

The expansion of chondrocytes in automated bioreactors for clinical use requires that a relevant number of cells be generated, starting from variable initial seeding densities in one passage and using autologous serum. We investigated whether the growth factor combination transforming growth factor beta 1/fibroblast growth factor 2/platelet-derived growth factor BB (TFP), recently shown to enhance the proliferation capacity of human articular chondrocytes (HACs), allows the efficiency of chondrocyte use to be increased at different seeding densities and percentages of human serum (HS). HACs were seeded at 1,000, 5,000, and 10,000 cells/cm2 in medium containing 10% fetal bovine serum or 10,000 cells/cm2 with 1%, 5%, or 10%HS. The chondrogenic capacity of post-expanded HACs was then assessed in pellet cultures. Expansion with TFP allowed a sufficient number of HACs to be obtained in one passage even at the lowest seeding density and HS percentage and variability in cartilage-forming capacity of HACs expanded under the different conditions to be reduced. Instead, larger variations and insufficient yields were found in the absence of TFP. By allowing large numbers of cells to be obtained, starting from a wide range of initial seeding densities and HS percentages, the use of TFP may represent a viable solution for the efficient expansion of HACs and addresses constraints of automated clinical bioreactor systems.     

羧乙基壳聚糖-纳米羟基磷灰石复合材料细胞相容性和生物力学性能的实验研究

制备羧乙基壳聚糖-纳米羟基磷灰石(NCECS/nHA)复合材料,研究其生物力学性能以及与气管软骨细胞的生物相容性。方法 气管软骨片段取自8周龄大耳白兔,Ⅱ型胶原酶消化,将所获得软骨细胞传代培养。将体外制备的NCECS/nHA复合材料分别进行干态标本和湿态标本的生物力学检测。将第3代软骨细胞种植到NCECS/nHA复合材料,分别计算材料表面软骨细胞在2h、6h、12h细胞贴壁率,并用噻唑蓝(MTT)法测定细胞增殖活性。结果 NCECS/nHA复合材料具有良好的生物力学性能。兔气管软骨细胞在NCECS/nHA复合材料表面上12h的贴壁率达(88.4±2.1)％,与其他组差异无统计学意义(P＞0.05)。同时MTT显示气管软骨细胞在NCECS/nHA复合材料表面生长状态良好。扫描电镜结果显示软骨细胞在NCECS/nHA薄膜上增殖和分化良好。结论 NCECS/nHA复合材料具备良好的细胞相容性和适宜的生物力学强度,作为一种具有开发潜力的生物材料,可用于组织工程气管的体外构建。     

Ex vivo synthesis of articular cartilage

This review discusses modern methods used for the synthesis of articular cartilage ex vivo. The value of culturing articular chondrocytes as a monolayer and in three-dimensional lattices is discussed. Of particular interest are techniques involving seeding of chondrocytes onto synthetic, biodegradable, polymeric scaffolds, and natural materials, such as collagen and agarose. Also discussed is the use of bioreactors to modulate the fluid-flow-induced shear environment of cell-seeded scaffolds. Biodegradable scaffolds are central to the efforts to tissue engineer articular cartilage ex vivo. A review of salient efforts to design and use such scaffolds is presented, along with our thoughts on potential future improvements.