Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged in monolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.     

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged in monolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.     

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged inmonolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.     

Effects of serial expansion of septal chondrocytes on tissue-engineered neocartilage composition.

OBJECTIVES: Cartilage grafts for reconstructive surgery may someday be created from harvested autologous chondrocytes that are expanded and seeded onto biodegradable scaffolds in vitro. This study sought to quantify the biochemical composition of neocartilage engineered from human septal chondrocytes and to examine the effects of cell multiplication in monolayer culture on the ultimate composition of the neocartilage. METHODS: Human septal chondrocytes from 10 donors were either seeded immediately after harvest (passage 0 [P(0)]) onto polyglycolic acid (PGA) scaffolds or underwent multiplication in monolayer culture before scaffold seeding at passage 1 (P(1)) and passage 2 (P(2)). Cell/scaffold constructs were grown in vitro for 7, 14, and 28 days. Neocartilage constructs underwent histologic analysis for matrix sulfated glycosaminoglycan (S-GAG) and type II collagen as well as quantitative assessment of cellularity (Hoescht 33258 assay), S-GAG content (dimethylmethylene blue assay), and collagen content (hydroxyproline assay). RESULTS: Histologic sections of constructs seeded with P(0) cells stained strongly for S-GAG and type II collagen, whereas decreased staining for both matrix components was observed in constructs derived from P(1) and P(2) cells. Cellularity, S-GAG content, and total collagen content of constructs increased significantly from day 7 to day 28. S-GAG accumulation in P(0) constructs was higher than in either P(1) (P < 0.05) or P(2) (P < 0.01) constructs, whereas cellularity and total collagen content showed no difference between passages. CONCLUSION: Neocartilage created from chondrocytes that have undergone serial passages in monolayer culture exhibited decreased matrix S-GAG and type II collagen, indicative of cellular dedifferentiation. SIGNIFICANCE: The alterations of matrix composition produced by dedifferentiated chondrocytes may limit the mechanical stability of neocartilage constructs.     

Low oxygen tension stimulates the redifferentiation of dedifferentiated adult human nasal chondrocytes

OBJECTIVE: To determine the effect of dissolved oxygen tension (DO) on the redifferentiation of dedifferentiated adult human nasal septum chondrocytes cultured as pellets. DESIGN: After isolation, human nasal chondrocytes were expanded in monolayer culture, which resulted in their dedifferentiation. Dedifferentiated cells were pelleted, transferred to a bioreactor and maintained for up to 21 days at 100% DO (21% oxygen), 25% DO (5.25% oxygen) or 5% DO (1% oxygen), which was controlled in the liquid phase. Redifferentiation was assessed by staining the extracellular matrix with safranin-O and by the immunolocalization of collagen types I, II, IX and of a fibroblast membrane marker (11-fibrau). In addition, glycosaminoglycans (GAG) and DNA content were determined spectrophotometrically. RESULTS: In monolayer culture, cells dedifferentiated and multiplied 90- to 100-fold. Cell pellets cultured in a bioreactor under conditions of low oxygen tension (25% DO or 5% DO) stained intensely for GAGs and for collagen type II, but very weakly for collagen type I. After 14 days of culturing, cell pellets maintained at 5% DO stained more intensely for collagen IX and more weakly for 11-fibrau than did those incubated at 25% DO. After 21 days of culturing the GAG content of cell pellets maintained at 5% DO was significantly greater than that of those incubated at 25% DO. Under air-saturated conditions (100% DO), the DNA and GAG contents of cell pellets decreased with time in culture. After 21 days of culturing, both parameters were substantially lower in cell pellets maintained at 100% DO than in those incubated at low oxygen tensions. The staining signals for collagen types II and IX were much weaker, and those for the markers of dedifferentiation (collagen type I and 11-fibrau) much stronger under air-saturated conditions than at low oxygen tensions. CONCLUSION: These observations demonstrate that using the present set-up, low oxygen tension stimulates the redifferentiation of dedifferentiated adult human nasal chondrocytes in pellet cultures.     

Bone morphogenetic protein (BMP)-2 enhances the expression of type II collagen and aggrecan in chondrocytes embedded in alginate beads

OBJECTIVE: For autologous chondrocyte transplantation (ACT) chondrocytes are expanded in vitro. During expansion these cells may dedifferentiate. This change in phenotype is characterized by a raised expression of type I collagen and a decrease in type II collagen expression. Since high expression of type II collagen is of central importance for the properties of hyaline cartilage, we investigated if the growth factor bone morphogenetic protein-2 (BMP-2) may modulate the chondrogenic phenotype in monolayer cell cultures and in three-dimensional culture systems. DESIGN: Chondrocytes from articular knee cartilage of 11 individuals (average age: 39.8 years) with no history of joint disease were isolated and seeded either in monolayer cultures or embedded in alginate beads in presence or absence of human recombinant BMP-2 (hr-BMP-2). Then, cells were harvested and analysis of the chondrogenic phenotype was performed using quantitative RT-PCR, immunocytochemistry and ELISA. RESULTS: Addition of BMP-2 to chondrocytes expanded in two-dimensional (2D) cultures during the first subculture (P1) had no effect on mRNA amounts encoding type II collagen and interleukin-1beta (IL-1beta). In contrast, seeding chondrocytes in three-dimensional (3D) alginate cultures raised type II collagen expression significantly and addition of BMP-2 enhanced this effect. CONCLUSIONS: We conclude that chondrocytes during expansion for ACT may benefit from BMP-2 activation only when seeded in an appropriate 3D culture system.     

人转化生长因子β1重组腺病毒转染传代髓核细胞对细胞外基质合成的影响

目的探讨人转化生长因子融（transforming growthfactorβ，TGF-β1）对传代羊髓核细胞的细胞外基质（extracellular matrix，ECM）和DNA合成调节因子的作用。方法取1岁龄成年山羊腰椎间盘，体外分离培养羊髓核细胞，传至第3代后以携人TGF-β1（humanTGF-β1，hTGF-β1）或lacZ基因的复制缺陷型腺病毒（Ad／hTGF—β1及Ad／lacZ）感染，分别为实验组和阴性对照组；未加病毒液的细胞为空白对照组；原代髓核细胞为原代组。然后继续单层或藻酸钙凝胶三维（3-D）培养10d。对两种系统培养的细胞分别行DNA荧光定量、Westernblot分析和蛋白多糖（glycosaminoglycan，GAG）定量检测。结果DNA荧光定量显示，单层培养时实验组细胞的DNA合成显著高于两对照组（P〈0．05），藻酸钙凝胶3-D培养各组间比较差异无统计学意义（P〉0．05）。Western blot检测hTGF—β1、Ⅱ型胶原、Ⅰ型胶原和Aggrecan的表达显示，两种培养系统中，实验组hTGF—β1、Ⅱ型胶原和Aggrecan的表达均显著高于两对照组（P〈0．05），Ⅰ型胶原的表达显著低于两对照组（P〈0．05），实验组Ⅱ型胶原／Ⅰ型胶原比值较两对照组显著增高（P〈0．05）。GAG定量结果显示，两种培养系统中实验组细胞的GAG合成均显著高于两对照组（P〈0．05）。结论hTGF-β1在很大程度上可起到维持髓核细胞表型，并在细胞传代后仍发挥表型的调节作用。通过基因工程方法使髓核细胞表达hTGF—β1，有望遏制、甚至逆转椎间盘退变；而以Ad／hTGF—β1感染过的髓核细胞，在藻酸钙凝胶3-D培养系统中培养则表现出原始表型。     

Culture of human septal chondrocytes in a rotary bioreactor

Objectives (1) To show that extracellular matrix deposition in 3-dimensional culture of human septal chondrocytes cultured in a rotary bioreactor is comparable to the deposition achieved under static culture conditions. (2) To demonstrate that the biomechanical properties of human septal chondrocytes cultured in a bioreactor are enhanced with time and are analogous to beads cultured under static culture. Study Design Prospective, basic science. Setting Research laboratory. Methods Human septal chondrocytes from 9 donors were expanded in monolayer and seeded in alginate beads. The beads were cultured in a rotary bioreactor for 21 days in media supplemented with growth factors and human serum, using static culture as the control. Biochemical and biomechanical properties of the beads were measured. Results Glycosaminoglycan (GAG) accumulation significantly increased during 2 measured time intervals, 0 to 21 days and 10 to 21 days (P < .01). No significant difference was seen between the static and bioreactor conditions. Substantial type II collagen production was demonstrated in the beads terminated at day 21 of culture in both conditions. In addition, the biomechanical properties of the beads were significantly improved at 21 days in comparison to beads from day 0. Conclusion Human septal chondrocytes cultured in alginate beads exhibit significant matrix deposition and improved biomechanical properties after 21 days. Alginate bead diameter and stiffness positively correlated with GAG and type II collagen accretion. Matrix production in beads is supported by the use of a rotary bioreactor.     

Quantitative analysis of gene expression in human articular chondrocytes assigned for autologous implantation

Autologous chondrocyte implantation (ACI) relies on the implantation of in vitro expanded cells. The aim was to study the dedifferentiation of human articular chondrocytes under different cultivating conditions [days 0–10 in the primary culture (P0); passages in a monolayer from P0 to P3; monolayer vs. alginate and monolayer vs. alginate/agarose hydrogels] using real‐time PCR analysis. The relative gene expressions for collagen type I and II, aggrecan and versican were quantified and the corresponding differentiation indexes (Col2/Col1, Agr/Ver) were calculated. The values of both differentiation indexes decreased exponentially with time in the P0 monolayer culture, and continued with a significant decrease over the subsequent monolayer passages. On the contrary, the chondrocytes seeded in either of the hydrogels significantly increased the indexes compared to their parallel monolayer cultures. These results indicate that alginate and alginate/agarose hydrogels offer an appropriate environment for human articular chondrocytes to redifferentiate after being expanded in vitro. Therefore the three‐dimensional (3D) hydrogel chondrocyte cultures present not only surgical, but also biological advantage over the classic suspension–periosteum chondrocyte implantation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:847–853, 2008     

Characterisation of human knee meniscus cell phenotype

OBJECTIVE: Studies on the biology of the human meniscus cell are scarce. The objective of our studies was to assess survival/proliferation of human meniscus cells in different culture conditions and to characterize the extracellular matrix (ECM) produced by these cells in these artificial environments. The composition of this ECM offers a variable to define the distinct meniscus cell phenotype. MATERIALS AND METHODS: Human meniscus cells were isolated enzymatically from visually intact lateral and medial knee menisci. Cells were cultured in monolayer conditions or in alginate gel. The composition of the cell-associated matrix (CAM) accumulated by the isolated cells during culture was investigated and compared to the CAM of articular chondrocytes cultured in alginate using flow cytometry with fluorescein isothiocyanate-conjugated monoclonal antibodies against type I collagen, type II collagen and aggrecan. Additional cell membrane markers analysis was performed to further identify the different meniscus cell populations in the alginate culture conditions and meniscus tissue sections. Proliferation was analyzed using the Hoechst 33258 dye method. In some experiments, the effect of TGFbeta1 on some of these variables was investigated. RESULTS: The CAM of monolayer cultured meniscus cells is composed of high amounts of type I and II collagen and low amounts of aggrecan. A major population of alginate cultured meniscus cells on the other hand synthesized a CAM containing high amounts of type I collagen, low amounts of type II collagen and high amounts of aggrecan. This population is CD44+CD105+CD34-CD31-. In contrast, a minor cell population in the alginate culture did not accumulate ECM and was mainly CD34+. The CAM of alginate cultured articular chondrocytes is composed of low amounts of type I collagen, high amounts of type II collagen and aggrecan. The expression of aggrecan and of type II collagen was increased by the addition of TGFbeta1 to the culture medium. The proliferation of meniscus cells is increased in the monolayer culture conditions. Cell numbers decrease slightly in the alginate culture, but can be increased after the addition of TGFbeta1. CONCLUSION: These results demonstrate that the human meniscus is populated by different cell types which can be identified by a distinct CAM composition and membrane marker expression. Unlike the monolayer culture conditions, the alginate culture conditions appear to favor a more fibrochondrocyte-like cell accumulating a CAM resembling the native tissue composition. This CAM composition is distinctly different from the CAM composition of phenotypically stable articular cartilage chondrocytes cultured in the same alginate matrix.     

去分化关节软骨细胞生物反应器培养反分化的实验研究

目的 观察体外经传代培养去分化的成人关节软骨细胞，在生物反应器培养后生物学性状的变化，探索去分化软骨细胞反分化的手段，为软骨细胞移植修复关节软骨缺损建立合适的体外培养方法。方法 无菌条件下取成人关节软骨组织，Ⅱ型胶原酶消化法（0．2％，37C，3h）分离软骨细胞，分成两组：一组常规单层传代培养，另一组添加重组人的生长因子（1ng／ml转化生长因子β1＋5ng／ml成纤维细胞生长因子2）体外培养传代大量扩增后，无微载体生物反应器内培养3周。血小板计数器行细胞计数，计算各代细胞倍增时间；细胞爬片和石蜡、冰冻切片进行HE、蕃红O、阿利新蓝染色，Ⅰ、Ⅱ型胶原和aggrecan免疫组织化学检测，观察细胞表型变化。结果 成人关节软骨细胞体外培养3代后迅速去分化，增殖缓慢。添加生长因子培养细胞去分化速度减缓；传10代，细胞扩增2000倍以上，部分去分化，但细胞扩增增殖能力仍很强；传20代软骨细胞表型基本丢失，但仍有增殖能力；置于生物反应器继续培养3周，细胞番红O染色强阳性、aggrecan和Ⅱ型胶原阳性，Ⅰ型胶原阴性，表型恢复良好。结论 软骨细胞在体外大量扩增后，在生物反应器培养，可恢复其表型，可望用于在体外培养时去分化软骨细胞的再分化。     

Redifferentiation of dedifferentiated bovine articular chondrocytes in alginate culture under low oxygen tension.

OBJECTIVE: To determine the influence of low oxygen tension on the redifferentiation and matrix production of dedifferentiated articular chondrocytes in monolayer and alginate bead culture. METHODS: Bovine articular chondrocytes were isolated enzymatically. After multiplication and dedifferentiation in a 2-week monolayer culture under 21% oxygen, the cells were subcultured in monolayer or alginate bead culture and subjected to 21% or 5% O(2)for 2 or 3 weeks in order to redifferentiate. Controls consisted of primary cultures in alginate. Matrix production was monitored immunocytochemically [collagen types I, II, IX, and GAGs (keratan sulfate, chondroitin-4- and -6-sulfate)] and collagen type II additionally assayed by Western blotting. Biosynthetic activity was measured by [(3)H]-proline incorporation and cell-viability by the trypan blue exclusion method. RESULTS: The cell number increased more than four-fold during dedifferentiation. Collagen type II was not produced by dedifferentiated chondrocytes under 5% or 21% oxygen in the monolayers or under 21% in alginate. However, dedifferentiated cells in alginate subjected to 5% oxygen exhibited a strong collagen type II expression indicating a redifferentiation. Additionally, collagen type IX and GAGs were also higher and [(3)H]-proline incorporation increased significantly. Primary cultures in alginate displayed a stronger collagen type II expression under 5% but no significant differences for other extracellular matrix components, or [(3)H]-proline incorporation. Viability was approximately 90% for all alginate cultures. CONCLUSION: A combination of alginate and high oxygen tension might not be suitable for redifferentiation or culturing of dedifferentiated chondrocytes. However, low oxygen tension promotes or induces a redifferentiation of dedifferentiated cells in alginate, stimulates their biosynthetic activity, and increases collagen type II production in primary alginate cultures.     

Loss of chondrogenic potential in dedifferentiated chondrocytes correlates with deficient Shc-Erk interaction and apoptosis

OBJECTIVE: If dedifferentiated chondrocytes could be induced to redifferentiate in vitro, then we might thereby be furnished with a population of phenotypically stable cells for autologous implantation in reconstructive surgery. We therefore investigated the redifferentiation capabilities of chondrocytes which, having migrated from alginate beads to form a monolayer, were subsequently passaged. We also characterized the molecular traits of irreversibly dedifferentiated cells. METHODS: Human chondrocytes that had migrated from alginate beads to form a monolayer (passage 1) were passaged seven times (passages 2-8). Cells from each passage were then recultivated in alginate beads. We assessed the synthesis of type-II collagen, cartilage-specific proteoglycans, adhesion molecules (integrins), signaling proteins (Src-homology collagen [Shc] and extracellular-signal-regulated kinase [Erk]) and the apoptosis marker 'activated' caspase-3 in monolayer or secondary alginate cultures. RESULTS: The synthesis of cartilage-specific type-II collagen, alpha 3-integrin, Shc and activated Erk1/2 decreased rapidly after four passages in monolayer culture. Up to passage 4, cells redifferentiated in alginate culture. However, between passages 5 and 8, cells began to produce activated caspase-3; these cells not only failed to redifferentiate when recultivated in alginate, but underwent apoptosis. CONCLUSION: We conclude that the loss of chondrogenic potential by chondrocytes maintained in monolayer culture is associated with a decrease in the synthesis of cartilage markers and with a suppressed activation of key signaling proteins in the Ras-mitogen-activated protein kinase pathway (Shc and Erk1/2). These events lead to apoptosis. A decrease in Shc/Erk expression/interaction could serve as a recognition marker for irreversibly dedifferentiated chondrocytes in tissue engineering.     

Human serum for tissue engineering of human nasal septal cartilage.

OBJECTIVE: To compare the chondrogenic and proliferative effects of pooled human serum (HS) and fetal bovine serum (FBS) on tissue-engineered human nasal septal chondrocytes. STUDY DESIGN AND SETTING: Human chondrocytes were expanded for one passage in monolayer in medium supplemented with 10% FBS, 2% HS, 10% HS, or 20% HS. Cells were then suspended in alginate beads for 3D culture for 2 weeks with 10% FBS, 2% HS, 10% HS, or 20% HS. RESULTS: Monolayer cell yields were greater with HS than FBS. In alginate, cellular proliferation, glycosaminoglycan production per cell, and type II collagen were significantly higher with 10% HS compared to 10% FBS controls. CONCLUSION: HS results in increased proliferation and production of cartilaginous extracellular matrix by tissue-engineered human nasal septal chondrocytes, compared to FBS controls. SIGNIFICANCE: Culture with human serum may facilitate creation of neocartilage constructs that more closely resemble native tissue.     

Flow cytometric analysis of the human articular chondrocyte phenotype in vitro

OBJECTIVE: To develop flow cytometry for the study of human articular cartilage cell phenotype and to validate the method on chondrocytes cultured in different in-vitro systems. METHODS: Chondrocyte phenotype was modulated by culturing the cells under different in-vitro conditions: i.e. in monolayer and in suspension culture in gelled agarose. Monolayer cultured chondrocyte phenotype was assayed by immunohistochemical staining with monoclonal antibodies against chondrocyte-specific aggrecan, type II and I collagen. Flow cytometry was used to quantify the proportions of chondrocytes expressing these extracellular matrix molecules in both culture conditions. To exclude the effects of cell-harvesting methods on the presence of cell-bound ECM molecules, non-proteolytic isolation procedures were used to obtain the chondrocytes for flow cytometry. Subconfluent cells from monolayer cultures were detached with EDTA. Chondrocytes cultured in gelled agarose were obtained after the agarose was enzymatically digested with agarase. RESULTS: Immunohistochemical staining showed that monolayer-cultured chondrocytes, in the presence of serum, gradually lost the expression of chondrocyte-specific aggrecan and type II collagen, while type I collagen was increasingly expressed. Flow cytometry allowed monolayer cultured chondrocyte phenotype to be assessed reproducibly. Chondrocyte phenotype was characterized through the cell membrane-associated extracellular matrix antigens. EDTA, used to obtain single cells from monolayer cultures, did not affect the cell-associated matrix. Where the chondrocytes had been cultured in gelled agarose, flow cytometry allowed quantification of the percentages of chondrocytes maintaining or reexpressing their original phenotype. The agarase digestion procedure used to isolate the cells from the agarose gel did not affect the plasma membrane-associated extracellular matrix antigens. CONCLUSION: Flow cytometry allows quantification of cells expressing aggrecan, type II and I collagen in their cell-associated extracellular matrix. A continuously increasing number of specific monoclonal antibodies will broaden the range of applications offered by this method.     

Effect of expansion medium on ex vivo gene transfer and chondrogenesis in type II collagen-glycosaminoglycan scaffolds in vitro

OBJECTIVE: Growth factors can profoundly affect the behaviour of chondrocytes during expansion and subsequent growth in three-dimensional (3-D) scaffolds. Prolonging such effects has stimulated investigation of the transfer of growth factor genes to chondrocytes. This study evaluated the effects of the monolayer expansion medium on the proliferation and cartilage matrix molecule synthesis of chondrocytes in 3-D pellet culture and in type II collagen-glycosaminoglycan (CG) scaffolds, and on ex vivo insulin-like growth factor-1 (IGF-1) gene transfer to articular chondrocytes in monolayer. The possibility of transfecting cells in 3-D culture using CG scaffolds was also investigated and the resulting effect of IGF-1 overexpression on glycosaminoglycan (GAG) biosynthesis in 3-D culture was assessed. METHODS: Two expansion media were compared-one that has been widely used for growing chondrocytes (Medium 1) and one that has been found to increase chondrocyte proliferation rates and preserve the redifferentiation potential of monolayer-expanded chondrocytes when subsequently placed in pellet cultures (Medium 2). Chondrocytes were expanded in monolayer culture and then 1) redifferentiated in 3-D culture, or 2) infected with the IGF-1 gene in monolayer or in type II CG scaffolds. RESULTS: The cell count for first passage chondrocytes was more than 3-fold higher when using Medium 2. In 3-D culture, cells expanded with Medium 2 and seeded in CG scaffolds produced more total GAG/DNA and displayed more intense immunohistochemical staining for collagen type II. Gene transfer and IGF-1 release kinetics from infected cells in monolayer were significantly affected by the composition of the expansion medium, the gene transfer method and time. IGF-1 gene transfer in CG scaffolds resulted in a 35-fold elevation in accumulated IGF-1 released from transfected Medium 2-expanded chondrocytes over controls, and resulted in a 40% increase in accumulated GAG/DNA. CONCLUSION: The composition of the expansion medium significantly affects monolayer proliferation of adult canine chondrocytes, GAG synthesis when the cells are subsequently grown in CG scaffolds, and ex vivo IGF-1 gene transfer.     

Redifferentiation of dedifferentiated joint cartilage cells in alginate culture. Effect of intermittent hydrostatic pressure and low oxygen partial pressure

One of the goals in the field of tissue engineering is the development of artificial cartilage for the treatment of cartilage defects. Therefore autologous chondrocytes are seeded on different artificial matrices to test their possible use as implants (resorption, antigenicity, toxicity and their integration in the tissue). One of the main problems in these experiments is that usually the amount of available chondrocytes is too low for treating large-scale defects or for comparing different matrices. An in-vitro-multiplication of the cells is needed which causes the chondrocytes to dedifferentiate and become fibroblast-like. Therefore parameters which induce a redifferentiation are of great interest. The objective of this study was to determine the influence of intermittent hydrostatic pressure and low oxygen partial pressure on the redifferentiation of dedifferentiated bovine articular chondrocytes in monolayer and three-dimensional alginate bead culture. The redifferentiation process was monitored by immunocytochemical detection of newly synthesized collagen type II. The viability of the cells was determined by the trypanblue exclusion test. The chondrocytes were dedifferentiated by a two week culture in plastic flasks with an oxygen level of 20%. After this they were subcultured in monolayer or three-dimensional alginate culture and subjected to three different stimuli for three weeks in order to redifferentiate: 1.) 20% O2 (= 20.26 kPa PO2) + 5% CO2 + 75% N2; 2.) 5% O2 (= 5.07 kPa PO2) + 5% CO2 + 90% N2; 3.) 5% O2 (= 5.07 kPa PO2) + 5% CO2 + 90% N2 + 8 h/d of intermittent hydrostatic pressure (frequency: 3 bar absolute for 30 min and 1 bar absolute for 2 min). In the monolayer there was no detectable collagen type II found by immunocytochemistry under either of the three culture conditions. Therefore a redifferentiation of dedifferentiated chondrocytes was not possible in monolayer cultures with the tested parameters. In the three-dimensional alginate culture there was no immunocytochemical staining of collagen type II found in the beads cultured with 20% oxygen. With 5% oxygen we found a strong collagen type II-production by chondrocytes throughout the whole bead. The intermittent hydrostatic pressure combined with 5% oxygen lead to a decreased collagen type II-production compared to cells subjected to 5% oxygen only. Also chondrocytes closer to the edge of these beads were more often immunopositive and seemed to produce more immunoreactive collagen type II. The viability of the chondrocytes in the alginate culture was close to 90% after three weeks. Our experiments showed that oxygen partial pressure is an important parameter in the cultivation of articular chondrocytes. Reduced partial oxygen pressure promoted or induced the redifferentiation of dedifferentiated chondrocytes in alginate culture.