Polymer scaffolds fabricated with pore-size gradients as a model for studying the zonal organization within tissue-engineered cartilage constructs

The zonal organization of cells and extracellular matrix (ECM) constituents within articular cartilage is important for its biomechanical function in diarthroidal joints. Tissue-engineering strategies adopting porous three-dimensional (3D) scaffolds offer significant promise for the repair of articular cartilage defects, yet few approaches have accounted for the zonal structural organization as in native articular cartilage. In this study, the ability of anisotropic pore architectures to influence the zonal organization of chondrocytes and ECM components was investigated. Using a novel 3D fiber deposition (3DF) technique, we designed and produced 100% interconnecting scaffolds containing either homogeneously spaced pores (fiber spacing, 1 mm; pore size, about 680 microm in diameter) or pore-size gradients (fiber spacing, 0.5-2.0 mm; pore size range, about 200-1650 microm in diameter), but with similar overall porosity (about 80%) and volume fraction available for cell attachment and ECM formation. In vitro cell seeding showed that pore-size gradients promoted anisotropic cell distribution like that in the superficial, middle, and lower zones of immature bovine articular cartilage, irrespective of dynamic or static seeding methods. There was a direct correlation between zonal scaffold volume fraction and both DNA and glycosaminoglycan (GAG) content. Prolonged tissue culture in vitro showed similar inhomogeneous distributions of zonal GAG and collagen type II accumulation but not of GAG:DNA content, and levels were an order of magnitude less than in native cartilage. In this model system, we illustrated how scaffold design and novel processing techniques can be used to develop anisotropic pore architectures for instructing zonal cell and tissue distribution in tissue-engineered cartilage constructs.     

Administration of the insulin into the scaffold atelocollagen for tissue-engineered cartilage

Three-dimensional culture of the tissue-engineered cartilage constructs may increase the matrix production, but central necrosis must occur if the construct becomes large. To increase the cell viability in the middle part of constructs and to enhance the in vivo cartilage regeneration, we attempted to administer the insulin into the scaffold. Insulin is known to strongly enhance the matrix production in the chondrocytes. The pellets of human auricular chondrocytes with atelocollagen hydrogel were 3D-cultured in the medium. The comparison among three groups (insulin mixed in the atelocollagen, insulin added to the medium, and control group, i.e.; insulin in neither atelocollagen nor medium) revealed that both insulin mixed in the atelocollagen and that in the medium could effectively promoted the cell viability and matrix synthesis of the chondrocytes. The daily assay also showed the gradual release of insulin from the atelocollagen hydrogel, suggesting that this material may work as a control release of insulin. We actually transplanted the poly-L-lactide porous scaffolds carrying the chondrocytes and the atelocollagen mixed with or without insulin, into the nude mice, showing that glycosaminoglycan accumulation was evident in the group with insulin although less without insulin. We thus showed the possibility to enhance the in vivo cartilage regeneration, when administered insulin into the atelocollagen hydrogel.     

制备软骨组织工程支架的方法

背景：软骨组织工程支架作为软骨细胞外基质的替代物,其外形和孔结构对实现其作用和功能具有非常重要的意义。 目的：回顾目前若干种常用软骨组织工程中三维多孔支架的制备方法。 方法：由第一作者检索2000至2013年PubMed数据库,ELSEVIER SCIENCEDIRECT、万方数据库、中国知网数据库。英文检索词为“Cartilage tissue engineering;scaffolds;fabrication”,中文检索词为“软骨组织工程;制备方法;支架材料;多孔支架”。 结果与结论：制备软骨组织工程支架的方法有相分离/冷冻干燥法、水凝胶技术、快速成型技术、静电纺丝法、溶剂浇铸/粒子沥滤法及气体发泡法等。目前研究发现,支架中孔径的大小对组织的重建有着直接的影响,孔径为100-250 μm的孔有益于骨及软骨组织的再生。通过溶液浇铸/粒子沥滤法、气体发泡法所制备的支架孔径大小在这一范围内,因此比较适合用于骨、软骨组织工程支架的构建。研究人员通常将多种方法结合起来,以期能制备出生物和力学性能方面更加仿生的组织工程多孔支架。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Cell-based tissue-engineered allogeneic implant for cartilage repair

The potential for using of allogeneic cartilage chips, transplanted in a biologic polymer with articular chondrocytes, as a tool for articular cartilage repair was studied. Small lyophilized articular cartilage chips were mixed with a cell/fibrinogen solution and thrombin to obtain implantable constructs made of fibrin glue, chondrocytes, and cartilage chips. Specimens were implanted in the subcutaneous tissue on the backs of nude mice (experimental group A). Three groups of controls (groups B, C, and D) were also prepared. Group B consisted of fibrin glue and cartilage chips without chondrocytes. Group C consisted of fibrin glue and chondrocytes without cartilage chips, and group D was composed solely of fibrin glue. All samples were carefully weighed before implantation in the mice. The constructs were harvested from the animals at 6, 9, and 12 weeks, examined grossly, and weighed. The samples were then processed and stained with hematoxylin and eosin for histological examination. Gross evaluation and weight analysis of the constructs at the time of retrieval showed retention of the original mass in the samples made of fibrin glue, chondrocytes, and cartilage chips (group A) and demonstrated a cartilaginous consistency upon probing. Specimens from constructs of fibrin glue and cartilage chips without chondrocytes (control group B) retained most of their volume, but were statistically lighter than specimens from group A and were much softer and more pliable than those in group A. Samples of specimens from constructs of fibrin glue and chondrocytes (groups C) and fibrin glue alone (group D) both showed a substantial reduction of their original masses over the experimental time periods when compared to the samples in groups A and B, although specimens from group C demonstrated new cartilage matrix formation. Histological analysis of specimens in experimental group A demonstrated the presence of cartilage chips surrounded by newly formed cartilaginous matrix, while specimens of control group B showed only fibrotic tissue surrounding the devitalized cartilage pieces. Cartilaginous matrix was also observed in control group C, in which cartilage chips were absent, whereas only fibrin glue debris was observed in control group D. This study demonstrated that a composite of fibrin glue and devitalized cartilage can serve as a scaffold for chondrocyte transplantation, preserve the original phenotype of the chondrocytes, and maintain the original mass of the implant. This may represent a valid option for addressing the problem of articular cartilage repair.     

组织工程化软骨构建的研究进展

背景：组织工程学方法为关节软骨缺损的修复提供了新的治疗模式,具有广阔的应用前景。 目的：探究组织工程化软骨的构建方法、研究方向和应用前景。 方法：检索中国期刊全文数据库(CNKI:1991至2011年)和Web of Science(1991至2011年)数据库,检索词分别为“组织工程,软骨损伤,种子细胞,支架”和“Tissue Engineering,Cartilage Defects,Seed Cell,Scaffolds”,语言分别设为中文和英文。阅读文题和摘要进行筛选,选择具有原创性,论点论据可靠且分析全面,密切相关的文章,排除重复性研究以及质量较差文章。按纳入排除标准筛选后,共纳入30篇文章。 结果与结论：组织工程化软骨多以各种种子细胞与不同的支架材料进行复合,且在软骨缺损修复中体现了较好的应用价值,但应用于临床还有许多具体问题需要解决。     

Differential effects of growth factors on tissue-engineered cartilage

The effects of four regulatory factors on tissue-engineered cartilage were examined with specific focus on the ability to increase construct growth rate and concentrations of glycosaminoglycans (GAG) and collagen, the major extracellular matrix (ECM) components. Bovine calf articular chondrocytes were seeded onto biodegradable polyglycolic acid (PGA) scaffolds and cultured in medium with or without supplemental insulin-like growth factor (IGF-I), interleukin-4 (IL-4), transforming growth factor-beta1 (TGF-beta1) or platelet-derived growth factor (PDGF). IGF-I, IL-4, and TGF-beta1 increased construct wet weights by 1.5-2.9-fold over 4 weeks of culture and increased amounts of cartilaginous ECM components. IGF-I (10-300 ng/mL) maintained wet weight fractions of GAG in constructs seeded at high cell density and increased by up to fivefold GAG fractions in constructs seeded at lower cell density. TGF-beta1 (30 ng/mL) increased wet weight fractions of total collagen by up to 1.4-fold while maintaining a high fraction of type II collagen (79 plus minus 11% of the total collagen). IL-4 (1-100 ng/mL) minimized the thickness of the GAG-depleted region at the construct surfaces. PDGF (1-100 ng/mL) decreased construct growth rate and ECM fractions. Different regulatory factors thus elicit significantly different chondrogenic responses and can be used to selectively control the growth rate and improve the composition of engineered cartilage.     

Combinatorial scaffold morphologies for zonal articular cartilage engineering

Articular cartilage lesions are a particular challenge for regenerative medicine strategies as cartilage function stems from a complex depth-dependent organization. Tissue engineering scaffolds that vary in morphology and function offer a template for zone-specific cartilage extracellular matrix (ECM) production and mechanical properties. We fabricated multi-zone cartilage scaffolds by the electrostatic deposition of polymer microfibres onto particulate-templated scaffolds produced with 0.03 or 1.0 mm³ porogens. The scaffolds allowed ample space for chondrocyte ECM production within the bulk while also mimicking the structural organization and functional interface of cartilage’s superficial zone. Addition of aligned fibre membranes enhanced the mechanical and surface properties of particulate-templated scaffolds. Zonal analysis of scaffolds demonstrated region-specific variations in chondrocyte number, sulfated GAG-rich ECM, and chondrocytic gene expression. Specifically, smaller porogens (0.03 mm³) yielded significantly higher sGAG accumulation and aggrecan gene expression. Our results demonstrate that bilayered scaffolds mimic some key structural characteristics of native cartilage, support in vitro cartilage formation, and have superior features to homogeneous particulate-templated scaffolds. We propose that these scaffolds offer promise for regenerative medicine strategies to repair articular cartilage lesions.     

Hyalograft C: hyaluronan-based scaffolds in tissue-engineered cartilage

Articular cartilage injuries have poor reparative capability and, if left untreated, may progress to osteo-arthritis. Unsatisfactory results with conventional treatment methods have prompted the development of innovative solutions including the use of cell transplantations, with or without a supporting scaffold. Tissue engineering combines cells, scaffolds and bio-active factors, which represents one of the most promising approaches for the restoration of damaged tissues. Available today, hyaluronan, also known as hyaluronic acid, is a natural glycosaminoglycan present in all soft tissues of higher organisms and in particularly high concentrations in the extracellular matrix of articular cartilage and in the mesenchyme during embryonic development in which it plays a number of biological functions, not only as a structural component but as an informational molecule as well. Moreover, hyaluronan can be manufactured in a variety of physical forms including hydrogels, sponges, fibres and fabrics allowing to develop a variety of hyaluronan-based scaffolds. This review will present both theoretical and experimental evidences that led to the development of Hyalograft C, an exploitation of hyaluronic acid technology and a tissue engineering approach for the resolution of articular cartilage defects.     

Mechanical properties of natural cartilage and tissue-engineered constructs

There has been much research over the past two decades with the aim of engineering cartilage constructs for repairing or restoring damaged cartilage. To engineer healthy neocartilage, the constructs must have mechanical properties matching those of native cartilage as well as appropriate for the loading conditions of the joint. This article discusses the mechanical behavior of native cartilage and surveys different types of tensile, compressive, and shear tests with their limitations. It also comprehensively reviews recent work and achievements in developing the mathematical models representing the mechanical properties of both native and engineered cartilage. Different methods for enhancing the mechanical properties of engineered cartilage are also discussed, including scaffold design, mechanical stimulation, and chemical stimulation. This article concludes with recommendations for future research aimed at achieving engineered cartilage with mechanical properties matching those found in native cartilage.     

Characteristics of tissue-engineered cartilage from human auricular chondrocytes

This study was done to define the mechanical and histological properties of tissue-engineered cartilage (TEC) derived from human chondrocytes and to compare these findings with those of native cartilage. Chondrocytes were obtained from 10 human auricular cartilages and seeded onto a biodegradable template of polyglycolic acid and poly L-lactic acid. Each template was shaped into a 1 cm x 2 cm rectangle. The templates were implanted in athymic mice for 8 weeks. Eight human auricular cartilages were used for comparison. Mechanical analysis with a tensile testing device provided values of ultimate tensile strength (UTS), stiffness, and resilience. Statistical analysis was performed with the Student's t-test. Histological assessment was done with hematoxylin-eosin staining along with other special stains. The TEC had UTS of 2.07 MPa, stiffness of 3.7 MPa, and resilience of 0.37 J/m3. The control specimens had UTS of 2.18 MPa, stiffness of 5.11 MPa, and resilience of 0.42 J/m3. No statistical difference was found between the experimental and control groups for each of the three parameters. Histological analysis showed mature cartilage with characteristic collagen, glycosaminoglycans, and elastin in the TEC. The neo-cartilage showed slightly smaller size and more irregular distribution of chondrocytes and unique fibrous capsule formation with peripheral infiltration of fibrous tissue. This study showed that the mechanical qualities of TEC from human chondrocytes are similar to those of native auricular cartilage. It suggests that the engineered cartilage from human chondrocytes may have sufficient strength and durability for clinical uses. The histological findings revealed some differences with neo-cartilage.     

组织工程软骨构建中不同支架材料的特征

BACKGROUND: Most scholars believe that the cartilage tissue engineering is a new direction for the treatment of cartilage defects. It has made partial progress in the basic research, and to construct a scaffold is essential in cartilage tissue engineering. OBJECTIVE: To systematically review and summarize the application of different tissue-engineered scaffolds in articular cartilage repair. METHODS: A computer-based search of CNKI, VIP and PubMed databases was performed for relevant basic research literatures published from January 2005 to January 2015 using the keywords of “tissue engineering,  scaffold, cartilage” in Chinese and English, respectively. Meanwhile, references in the retrieved articles were retrieved manually. RESULTS AND CONCLUSION: For selection and preparation of tissue-engineered cartilage scaffolds, it is necessary to fully take into account the advantages and disadvantages of natural hydrogel materials, synthetic scaffolds, composite scaffolds, nano-scaffolds, and injectable scaffolds. Currently, there is still no normal transition between all tissue-engineered cartilage scaffolds and the natural cartilage. Therefore, to develop nano-multilayer integrated scaffolds with the calcified cartilage layer is expected to become one of the research focuses of bone and cartilage tissue engineering.      

Differential effects of natriuretic peptide stimulation on tissue-engineered cartilage

Tissue engineering is a promising approach for articular cartilage repair; however, it still has proven a challenge to produce substantial quantities of tissue from the limited number of cells that can be extracted from a single individual. Although several approaches have been investigated to enhance the production of cartilaginous tissue in vitro, relatively few techniques exist to reliably increase the population of cells needed for this approach. Alternatively, a single modulator of chondrocyte function, such as the C-type natriuretic peptide (CNP), may serve to address both of these issues. CNP is expressed in the growth plate and regulates cartilage growth through chondrocyte proliferation and differentiation. Thus, the purpose of this study was to determine the effects of CNP stimulation on tissue-engineered cartilage. Isolated bovine articular chondrocytes were seeded on Millicell filters and cultured in the presence of CNP (10 pM to 10 nM) for 4 weeks. Stimulation with CNP resulted in differential effects depending on the dose of the peptide. Low doses of CNP (10 to 100 pM) elicited chondrocyte proliferation with a maximal response observed at 100 pM (43% increase in cellularity). However, high doses of CNP (10 nM) stimulated matrix deposition (36% and 137% increase in proteoglycans and collagen) without an associated change in tissue cellularity. CNP stimulation also downregulated the expression of type X collagen, an early hypertrophic marker associated with endochondral ossification. Thus, by regulating the dose of CNP, it may be possible to produce engineered tissue from the limited number of cells that can be reasonably extracted from a single individual for therapeutic purposes.     

制备软骨组织工程支架的材料和方法

背景：软骨组织工程的研究为修复软骨缺损提供了新的思路和方法,其中如何获得理想的组织工程支架是这一研究的核心和难点。 目的：回顾性分析软骨组织工程支架的材料选择和制备方法。 方法：由第一作者检索2000至2012年 PubMed数据库、ELSEVIER SCIENCEDIRECT、万方数据库、中国知网库有关制备软骨组织工程支架的材料选择和方法等方面的文献。 结果与结论：软骨支架材料分为天然生物材料、人工合成高分子材料和复合材料。可采用相分离法、溶剂浇铸/粒子沥滤技术、气体发泡技术、快速成型技术及静电纺丝法制备支架材料。由于胶原、琼脂糖和藻酸盐等水凝胶类天然材料可提供足够的生物相容性、增殖和黏附能力及亲水性,电纺的人工合成高分子材料复合支架又可以保证支架的力学强度、塑形要求、孔隙率、可降解性等,将天然材料利用包埋技术和表面修饰技术复合于电纺的高分子复合材料支架上将更有利于支架性能的发挥。     

FT-IR imaging of native and tissue-engineered bone and cartilage

Fourier transform infrared (FT-IR) imaging and microspectroscopy have been extensively applied to the analyses of tissues in health and disease. Spatially resolved mid-IR data has provided insights into molecular changes that occur in diseases of connective or collagen-based tissues, including, osteoporosis, osteogenesis imperfecta, osteopetrosis and pathologic calcifications. These techniques have also been used to probe chemical changes associated with load, disuse, and micro-damage in bone, and with degradation and repair in cartilage. This review summarizes the applications of FT-IR microscopy and imaging for analyses of bone and cartilage in healthy and diseased tissues, and illustrates the application of these techniques for the characterization of tissue-engineered bone and cartilage.     

生物反应器中的力学刺激促进组织工程软骨再生

BACKGROUND: Cartilage tissue engineering has been widely used to achieve cartilage regeneration in vitro and repair cartilage defects. Tissue-engineered cartilage mainly consists of chondrocytes, cartilage scaffold and in vitro environment. OBJECTIVE: To mimic the environment of articular cartilage development in vivo, in order to increase the bionic features of tissue-engineered cartilage scaffold and effectiveness of cartilage repair. METHODS: Knee joint chondrocytes were isolated from New Zealand white rabbits, 2 months old, and expanded in vitro. The chondrocytes at passage 2 were seeded onto a scaffold of articular cartilage extracellular matrix in the concentration of 1×106/L to prepare cell-scaffold composites. Cell-scaffold composites were cultivated in an Instron bioreactor with mechanical compression (1 Hz, 3 hours per day, 10% compression) as experimental group for 7, 14, 24, 28 days or cultured statically for 1 day as control group. RESULTS AND CONCLUSION: Morphological observations demonstrated that the thickness, elastic modulus and maximum load of the composite in the experimental group were significantly higher than those in the control group, which were positively related to time (P < 0.05). Histological staining showed the proliferation of chondrocytes, formation of cartilage lacuna and synthesis of proteoglycan in the experimental group through hematoxylin-eosin staining and safranin-O staining, which were increased gradually with mechanical stimulation time. These results were consistent with the findings of proteoglycan kit. Real-time quantitative PCR revealed that mRNA expressions of collagen type I and collagen type II were significantly higher in the experimental group than the control group (P < 0.05). The experimental group showed the highest mRNA expression of collagen type I and collagen type II at 21 and 28 days of mechanical stimulation, respectively (P < 0.05). With the mechanical stimulation of bioreactor, the cell-scaffold composite can produce more extracellular matrix, such as collagen and proteoglycan, strengthen the mechanical properties to be more coincident with the in vivo environment of cartilage development, and increase the bionic features. With the progress of tissue engineering, the clinical bioregeneration of damaged cartilage will be achieved.   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     

Cultured chondrocytes produce injectable tissue-engineered cartilage in hydrogel polymer.

The purpose of this study was to determine if chondrocytes cultured through several subcultures at very low plating density would produce new cartilage matrix after being reimplanted in vivo with or without a hydrogel polymer scaffold. Chondrocytes were initially plated in low-density monolayer culture, grown to confluence, and passaged four times. After each passage cells were suspended in purified porcine fibrinogen and injected into the subcutaneous space of nude mice while simultaneously polymerizing with thrombin to reach a final concentration of 40 million cells/cc. Controls were made by injecting fresh, uncultured cells with fibrin polymer and by injecting the cultured cells in saline (without polymer). All samples were harvested at 6 weeks. When injected in polymer, both fresh cells and cells that had undergone only one passage in culture produced cartilaginous nodules. Cultured cells did not produce cartilage, regardless of length of time spent in culture, when injected without polymer. Cartilage was also not recovered from samples with cells kept in culture for longer than one passage, even when provided with a polymer matrix. All samples harvested were subjected to histological analysis and assayed for total DNA, glycosaminoglycan (GAG), and type II collagen. There was histological evidence of cartilage in the groups that used fresh cells and cultured cells suspended in fibrin polymer that only underwent one passage. No other group contained areas that would be consistent with cartilage histologically. All experimental samples had a higher percent of DNA than native swine cartilage, and there was no statistical difference between the DNA content of the groups containing cultured or fresh cells in fibrin polymer. Whereas the GAG content of native cartilage was 8.39% of dry weight and fresh cells in fibrin polymer was 12.85%, cultured cells in fibrin polymer never exceded the 2.48% noted from first passage cells. In conclusion, this study demonstrates that porcine chondrocytes that have been cultured in monolayer for one passage will produce cartilage in vivo when suspended in fibrin polymer.     

The effects of mycotoxins and selenium deficiency on tissue-engineered cartilage

Objective: To investigate the effects of 3 mycotoxins, deoxynivalenol (DON), nivalenol (NIV) and T-2 toxin, in the presence and absence of selenium (Se) on the metabolism of tissue-engineered cartilage to mimic conditions found in Kashin-Beck disease (KBD) environments. Materials and Methods: Chondrocytes were seeded onto bone matrix gelatin (BMG) to construct engineered cartilage. The 3 toxins were added to the culture media for 3 weeks followed by immunhistochemical analyses of collagens type II and X, aggrecan, matrix metalloproteinases 1 and 3 (MMP-1 and MMP-3), MMP inhibitors 1 and 3 (TIMP-1 and TIMP-3) and α(2) macroglobulin (α2M). Results: Type II collagen was decreased while type X collagen was increased in response to DON, NIV and T-2 toxin. Aggrecan was reduced by all 3 mycotoxins. Compared with the control, the 3 toxins decreased the expression of α2M, TIMP-1 and TIMP-3, and increased the expression of MMP-1 and MMP-3. Se could partially inhibit the effects of DON, NIV and T-2 toxins. Conclusion: Under the low Se condition, the 3 mycotoxins produced procatabolic changes in cartilage resulting in the loss of aggrecan and type II collagen and promoted a hypertrophic phenotype of chondrocytes characterized by increasing type-X-collagen expression, enhancing the expression of MMPs, while weakening the TIMPs. Se could partially block the effects mentioned above. These results support the hypothesis that the combination of mycotoxin stress and Se deficiency would be the causative factors for KBD.     

不同材料构建组织工程软骨及支架的应用

背景：组织工程技术的发展为软骨的再生和修复提供了新的途径,根据软骨自身的结构和特点,作为人工软骨的替代材料和支架材料应具有良好的生物力学性能。 目的：总结运动性关节软骨损伤修复材料及其支架材料的应用进展及其生物替代材料的生物力学特征,评价目前组织工程软骨材料应用的性能及发展前景。 方法：以“组织工程;软骨组织;支架材料;生物相容性”为关键词,应用计算机检索维普数据库和PubMed数据库中1990-01/2011-04关于组织工程软骨应用研究的文章,纳入与有关生物材料与组织工程软骨相关的文章;排除重复研究或Meta分析类文章。以24篇文献为主重点进行了讨论组织工程软骨材料的种类、性能及其应用效果和前景。 结果与结论：目前关节软骨修复领域以自体软骨移植效果为最佳,骨髓基质干细胞在离体试验及动物实验中研究较多,在临床应用中较少,尚在探索阶段。支架材料的应用比较繁复,天然材料、人工合成材料以及复合材料都存在一定的不足,虽然复合材料成为研究的热点,但是某些性能并不能很好地符合支架要求,并且在机体内这些材料所带来的长期影响还不能预见,这就迫切需要新材料的出现,来更好地满足组织软骨织支架的要求,达到修复和重建的目的。 关键词：软骨;组织工程;软骨组织;种子细胞;支架材料 doi:10.3969/j.issn.1673-8225.2012.08.036     

Effect of chondrocyte passage number on histological aspects of tissue-engineered cartilage

Transplantation of cultured chondrocytes can regenerate cartilage tissue in cartilage defects. This method requires serial cell passages to expand chondrocytes to a large number of cells for transplantation. However, as chondrocytes are expanded in number in monolayer culture, the cells gradually lose their differentiated phenotype and may not form cartilage tissue. This study investigated whether chondrocytes cultured through various passages maintain their potential to reexpress a chondrogenic phenotype in three-dimensional scaffolds and form cartilage tissue in vitro and in vivo. The growth rate, viability, synthesis of collagen type I and II, and apoptotic activity of chondrocytes with passage number of 1, 2 and 5 were compared during in vitro culture. As the passage number increased, the cell growth rate and viability decreased and apoptotic cell increased. Passage 2 chondrocytes exhibited a high expression of collagen type II and a low expression of collagen type I. In contrast, passage 5 chondrocytes exhibited a low expression of collagen type II and a high expression of collagen type I, indicating chondrocyte dedifferentiation. To examine the ability of chondrocytes to regenerate cartilage tissues in vitro and in vivo, chondrocytes were expanded in vitro to passage number of 1 or 5, seeded onto biodegradable polymer scaffolds, and maintained in vitro or implanted into subcutaneous spaces of athymic mice for 1 month. Histological and immunohistochemical analyses of cartilage tissues engineered in vitro and in vivo with passage 1 chondrocytes showed mature and well-formed cartilage and the presence of highly sulfated glycosaminoglycans and type II collagen, a collagen type produced by differentiated chondrocytes. In contrast, tissues engineered in vitro and in vivo with passage 5 chondrocytes did not have chondrocyte morphology or cartilage-specific extracellular matrices (i.e., glycosaminoglycans and type II collagen). The results of this study show that chondrocyte passage number is an important factor affecting the quality of cartilage tissue-engineered with the chondrocytes, and that chondrocytes.     

Beneficial effect of hydrophilized porous polymer scaffolds in tissue-engineered cartilage formation

Three dimensional (3D) porous poly(L-lactic acid) (PLLA) scaffolds were fabricated using a modified gas foaming method whose effervescent porogens were a mixture of sodium bicarbonate and citric acid. To improve chondrocyte adhesion, the scaffolds were then hydrophilized through oxygen plasma treatment and in situ graft polymerization of acrylic acid (AA). When the physical properties of AA-grafted scaffolds were examined, the porosity and pore size were 87 approximately 93% and 100 approximately 300 microm, respectively. The pore sizes were highly dependent on the varying ratios (w/w) between porogen and polymer solution. Influenced by their pore sizes, the compressive moduli of scaffolds significantly decreased with increasing pore size. The altered surface characteristics were clearly reflected in the reduced water contact angles that meant a significant hydrophilization with the modified polymer surface. Electron spectroscopy for chemical analysis (ESCA) and time-of-flight secondary ion mass spectrometer (ToF-SIMS) also confirmed the altered surface chemistry. When chondrocytes were seeded onto the AA-grafted PLLA scaffolds, cell adhesion and proliferation were substantially improved as compared to the unmodified scaffolds. The benefit of the modified scaffolds was clear in the gene expressions of collagen type II that was significantly upregulated after 4-week culture. Safranin-O staining also identified greater glycosaminoglycan (GAG) deposition in the modified scaffold. The AA-grafted porous polymer scaffolds were effective for cell adhesion and differentiation, making them a suitable platform for tissue-engineered cartilage.