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.     

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：     

Bone formation on tissue-engineered cartilage constructs in vivo: effects of chondrocyte viability and mechanical loading

Interactions between bone and cartilage formation are critical during growth and fracture healing and may influence the functional integration of osteochondral repair constructs. In this study, the ability of tissue-engineered cartilage constructs to support bone formation under controlled mechanical loading conditions was evaluated using a lapine hydraulic bone chamber model. Articular chondrocytes were seeded onto polymer disks, cultured for 4 weeks in vitro, and then transferred to empty bone chambers previously implanted into rabbit femoral metaphyses. The effects of chondrocyte viability within the implanted constructs and in vivo mechanical loading on bone formation were tested in separate experiments. After 4 weeks in vivo, biopsies from the chambers consisted of a complex composite of bone, cartilage, and fibrous tissue, with bone forming in direct apposition to the cartilage constructs. Microcomputed tomography imaging of the chamber biopsies revealed that the implantation of viable constructs nearly doubled the bone volume fraction of the chamber tissue from 0.9 to 1.6% as compared with the implantation of devitalized constructs in contralateral control chambers. The application of an intermittent cyclic mechanical load was found to increase the bone volume fraction of the chamber tissue from 0.4 to 3.6% as compared with no-load control biopsies. The results of these experiments demonstrate that tissue-engineered cartilage constructs implanted into a well-vascularized bone defect will support direct appositional bone formation and that bone formation is significantly influenced by the viability of chondrocytes within the constructs and the local mechanical environment in vivo.     

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.     

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。     

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.     

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。     

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.     

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.     

旋转生物反应器培养对组织工程气管软骨力学强度的影响

目的研究旋转生物反应器培养对组织工程气管软骨力学强度的影响，探索适宜的组织工程气管软骨培养方法。 方法分离2周龄Lewis大鼠剑突软骨细胞传代培养，收集第3代软骨细胞种植到DegraPol管状支架上，静态培养7d，然后将软骨细胞-支架复合物分别置于旋转生物反应器内培养（生物反应器组）或继续静态培养培养3周（静态培养组）。取出软骨细胞-支架复合物，以噻唑蓝（MTT）法测定软骨细胞增殖活性，结果以吸光度（A）值表示（每组n=6）；以Zwick1445型材料试验机测定软骨细胞-支架复合物的最大应变值和应力值（每组n=4）；并制备扫描电镜标本，观察软骨细胞在DegraPol支架中培养后的超微结构变化。 结果不同条件下培养3周，生物反应器组和静态培养组A值分别0.17±0.05、0.12±0.01，最大应力值分别为（0.33±0.04）和（0.26±0.01）MPa，最大应变值分别为（3.53±0.91）和（1.71±0.13）mm/mm，2组间3项指标的差异均有统计学意义（均P〈0.05）。扫描电镜观察显示生物反应器组获得更好的软骨样结构和更多的细胞外基质。 结论旋转生物反应器能够提供更好的体外培养条件，有利于组织工程气管软骨的形成。     

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     

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     

Localization of type VI collagen in tissue-engineered cartilage on polymer scaffolds

Together, the chondrocyte and its pericellular matrix have been collectively termed the chondron. Current opinion is that the pericellular matrix has both protective and signalling functions between chondrocyte and extracellular matrix. Formation of a native chondrocyte pericellular matrix or chondron structure might therefore be advantageous when tissue engineering a functional hyaline cartilage construct. The presence of chondrons has not been previously described in cartilage engineered on a scaffold. In this paper, we describe a modified immunochemical method to detect collagen VI, a key molecular marker for the pericellular matrix, and an investigation of type VI collagen distribution in engineered hyaline cartilage constructs. Cartilage constructs were engineered from adult human or bovine hyaline chondrocytes cultured on sponge or nonwoven fiber based HYAFF 11 scaffolds. Type VI collagen was detected in all constructs, but a distinctive, high-density, chondron-like distribution of collagen VI was present only in constructs exhibiting additional features of hyaline cartilage engineered using nonwoven HYAFF 11. Chondron structures were localized in areas of the extracellular matrix displaying strong collagen II and GAG staining of constructs where type II collagen composed a high percentage (over 65%) of the total collagen.     

Effects of temporal hydrostatic pressure on tissue-engineered bovine articular cartilage constructs

The objective of this study was to determine the effects of temporal hydrostatic pressure (HP) on the properties of scaffoldless bovine articular cartilage constructs. The study was organized in three phases: First, a suitable control for HP application was identified. Second, 10 MPa static HP was applied at three different timepoints (6-10 days, 10-14 days, and 14-18 days) to identify a window in construct development when HP application would be most beneficial. Third, the temporal effects of 10-14-day static HP application, as determined in phase II, were assessed at 2, 4, and 8 weeks. Compressive and tensile mechanical properties, GAG and collagen content, histology for GAG and collagen, and immunohistochemistry for collagen types I and II were assessed. When a culture control identified in phase I was used in phase II, HP application from 10 to 14 days resulted in a significant 1.4-fold increase in aggregate modulus, accompanied by an increase in GAG content, while HP application at all timepoints enhanced tensile properties and collagen content. In phase III, HP had an immediate effect on GAG content, collagen content, and compressive stiffness, while there was a delayed increase in tensile stiffness. The enhanced tensile stiffness was still present at 8 weeks. For the first time, this study examined the immediate and long-term effects of HP on biomechanical properties, and demonstrated that HP has an optimal application time in construct development. These findings are exciting as HP stimulation allowed for the formation of robust tissue-engineered cartilage; for example, 10 MPa static HP resulted in an aggregate modulus of 273 +/- 123 kPa, a Young's modulus of 1.6 +/- 0.4 MPa, a GAG/wet weight of 6.1 +/- 1.4%, and a collagen/wet weight of 10.6 +/- 2.4% at 4 weeks.     

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.      

Molecular diffusion in tissue-engineered cartilage constructs: effects of scaffold material, time, and culture conditions

Diffusion is likely to be the primary mechanism for macromolecular transport in tissue-engineered cartilage, and providing an adequate nutrient supply via diffusion may be necessary for cell proliferation and extracellular matrix production. The goal of this study was to measure the diffusivity of tissue-engineered cartilage constructs as a function of scaffold material, culture conditions, and time in culture. Diffusion coefficients of four different-sized fluorescent dextrans were measured by fluorescence recovery after photobleaching in tissue-engineered cartilage constructs seeded with human adipose-derived stem cells or acellular constructs on scaffolds of alginate, agarose, gelatin, or fibrin that were cultured for 1 or 28 days in either chondrogenic or control conditions. Diffusivities in the constructs were much greater than those of native cartilage. The diffusivity of acellular constructs increased 62% from Day 1 to Day 28, whereas diffusivity of cellular constructs decreased 42% and 27% in chondrogenic and control cultures, respectively. The decrease in diffusivity in cellular constructs is likely due to new matrix synthesis, which may be enhanced with chondrogenic media, and matrix contraction by the cells in the fibrin and gelatin scaffolds. The increase in diffusivity in the acellular constructs is probably due to scaffold degradation and swelling.     

The maturity of tissue-engineered cartilage in vitro affects the repairability for osteochondral defect

Cartilage tissue engineering using cells and biocompatible scaffolds has emerged as a promising approach to repair of cartilage damage. To date, however, no engineered cartilage has proven to be equivalent to native cartilage in terms of biochemical and compression properties, as well as histological features. An alternative strategy for cartilage engineering is to focus on the in vivo regeneration potential of immature engineered cartilage. Here, we used a rabbit model to evaluate the extent to which the maturity of engineered cartilage influenced the remodeling and integration of implanted extracellular matrix scaffolds containing allogenous chondrocytes. Full-thickness osteochondral defects were created in the trochlear groove of New Zealand white rabbits. Left knee defects were left untreated as a control (group 1), and right knee defects were implanted with tissue-engineered cartilage cultured in vitro for 2 days (group 2), 2 weeks (group 3), or 4 weeks (group 4). Histological, chemical, and compression assays of engineered cartilage in vitro showed that biochemical composition became more cartilagenous, and biomechanical property for compression gradually increased with culture time. In an in vivo study, gross imaging and histological observation at 1 and 3 months after implanting in vitro-cultured engineered cartilage showed that defects in groups 3 and 4 were repaired with hyaline cartilage-like tissue, whereas defects were only partially filled with fibrocartilage after 1 month in groups 1 and 2. At 3 months, group 4 showed striking features of hyaline cartilage tissue, with a mature matrix and a columnar arrangement of chondrocytes. Zonal distribution of type II collagen was most prominent, and the International Cartilage Repair Society score was also highest at this time. In addition, the subchondral bone was well ossified. In conclusion, in vivo engineered cartilage was remodeled when implanted; however, its extent to maturity varied with cultivation period. Our results showed that the more matured the engineered cartilage was, the better repaired the osteochondral defect was, highlighting the importance of the in vitro cultivation period.     

The effect of fibroblast growth factor and transforming growth factor-beta on porcine chondrocytes and tissue-engineered autologous elastic cartilage

Elastic cartilage responds mitogenically in vitro to transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (basic FGF). We studied the effects of these growth factors separately or in a combination on porcine auricular chondrocytes in vitro and on the autologous elastic cartilage produced. Cells were harvested from the elastic auricular cartilage of 16- to 18-kg Yorkshire swine. Viability and quantification of the cells was determined. Cells were plated at equal concentration and studied in vitro in one of four identical media environments except for the growth factors: Group I contained Ham's F-12 with supplements but no growth factors, Group II also contained basic-FGF, Group III also contained TGF-beta, and Group IV also contained a combination of both growth factors. After 3 weeks in vitro, the cells were chemically dissociated with 0.25% trypsin. Cell suspensions composed of 3 x 10(7) cells/cc in 30% Pluronic F-127/Ham's F-12 were injected subcutaneously. Implants were harvested at 6, 8, 10, and 12 weeks of in vivo culture and then were examined with histologic stains. After 3 weeks of in vitro culture the total number of cells was as follows: Group I, 1.8 x 10(8); Group II, 3.5 x 10(8); Group III, 1.3 x 10(8); Group IV, 2.5 x 10(8). After 8 weeks of in vivo autologous implantation, the average weight (g) and volume (cm3) of each group was as follows: Group I, 0.7 g/0.15 cm3; Group II, 1.5 g/0.8 cm3; Group III, 0.6 g/0.1 cm3; Group IV, 1.2 g/0.3 cm3. Histologically, Groups I, II, and IV generated cartilage similar to native elastic cartilage, but Group III specimens demonstrated fibrous tissue ingrowth. Basic FGF produced the most positive enhancement on the quantity and quality of autologous tissue engineered elastic cartilage produced in this porcine model both in vitro and in vivo.     

Effect of seeding and bioreactor culture conditions on the development of human tissue-engineered cartilage

Human cartilage was produced using fetal chondrocytes seeded into polyglycolic acid (PGA) mesh scaffolds and cultured in recirculation bioreactors. The effect of scaffold thickness, seeding cell density, and bioreactor operating conditions on the quality of the engineered cartilage was investigated. Thin (2.15-mm-thick) PGA scaffolds lost their structural integrity during bioreactor culture and the resulting constructs were small and misshapen compared with tissues generated using 4.75-mm-thick scaffolds. Increasing the seeding cell number from 1.2 x 10(7) to 2.2 x 10(7 )per 4.75-mm-thick scaffold resulted in a doubling of the construct wet weight, a 4.4-fold increase in glycosaminoglycan (GAG) concentration, and a 2.9-fold increase in total collagen concentration in the tissues. Levels of GAG and total collagen were also improved significantly when 100 mL or 50% v/v of the culture medium was replaced periodically during operation of the bioreactors compared with 50, 25, or 5 mL. The proportion of GAG lost from the tissues into the medium was reduced by increasing the seeding cell number and replaced medium volume. This work demonstrates that the quality of tissue-engineered cartilage can be manipulated substantially depending on the cell seeding and bioreactor culture conditions employed.