Ⅱ型胶原-硫酸软骨素支架的制备及组织工程软骨的构建

研究经乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC)处理的Ⅱ型胶原-硫酸软骨素支架材料的性能特点,并在体外构建组织工程软骨。从鸡软骨中提取Ⅱ型胶原,以不同浓度的EDC为交联剂通过冷冻干燥的方法制备Ⅱ型胶原与硫酸软骨素复合支架并测定其理化性质。将体外培养的新生兔关节软骨细胞接种在Ⅱ型胶原与硫酸软骨素复合支架上,观察软骨细胞在支架上的生长形态并检测支架上软骨细胞分泌的糖胺聚糖含量及Ⅱ型胶原含量。结果表明:采用EDC与硫酸软骨素交联增加了支架的稳定性,最适的交联剂质量浓度为7 mg/mL。软骨细胞在复合支架上增殖分化良好,并保持软骨细胞特异分化的表型,分泌Ⅱ型胶原与蛋白多糖(GAG)。培养14 d后已有软骨样组织形成。     

低强度周期性静水压力对人膝关节软骨细胞的影响

目的:探讨低强度周期性静水压力对体外培养的人膝关节软骨细胞增殖、凋亡,以及细胞Ⅱ型胶原分泌表达的影响。方法:体外酶消化法分离培养成人膝关节正常软骨细胞,将培养的第3代软骨细胞分为两组:正常对照组、3.0MPa组压力实验组,应用多功能恒温体外细胞培养中高压静水压力加载装置加载低强度周期性压力,共5d,每天2h。Ⅱ型胶原免疫组织化学染色法和甲苯胺蓝染色法鉴定软骨细胞,流式细胞术检测细胞凋亡,四甲基偶氮唑蓝(MTT)法绘制细胞生长曲线,qRT-PCR、Western-Blot检测Ⅱ型胶原的分泌和表达。结果:软骨细胞Ⅱ型胶原免疫组织化学染色和甲苯胺蓝染色均显示为阳性。与正常对照组相比,3.0MPa组表现出促进软骨细胞增殖,抑制细胞凋亡,且Ⅱ型胶原的合成分泌明显升高(P0.05)。结论:通过体外模拟人生理情况下较低强度(3.0MPa)的周期性静水压力对人软骨细胞增殖、凋亡水平及周围基质分泌合成功能的影响,初步证实了较低强度压力有助于软骨自我修复和自身保护作用的发挥。     

周期性张应力对大鼠髁突软骨细胞Ⅱ型胶原表达的影响

目的:在成功构建髁突软骨细胞体外培养-力学刺激模型的基础上,探讨周期性张应力对髁突软骨细胞主要细胞外基质(Ⅱ型胶原)合成的影响.方法:本研究采用FX-5000T应力加载系统对体外培养的第3代大鼠髁突软骨细胞分别施加1h、6h、12h和24 h的周期性张应力,应力刺激强度为10％1 HZ.加力完成后即刻收集加力细胞,提取细胞总RNA反转录成cDNA,应用RT-PCR技术检测髁突软骨细胞主要细胞外基质Ⅱ型胶原(type-Ⅱ collagen,Col-Ⅱ)mRNA的表达变化情况.结果:与对照组(0h组)相比,加力1 h时Col-Ⅱ的表达增加,但无统计学意义;加力6h时Col-Ⅱ表达显著增加(P＜0.05);加力12h时Col-Ⅱ表达开始下降;当加力至24h时表达量显著降低(P＜0.05).结论:周期性张应力可以影响髁突软骨细胞主要细胞外基质的合成,在一定范围内随加力时间的延长基质合成逐渐增强;进一步延长加力时间,基质的合成受到明显抑制.     

软骨基质来源定向结构支架复合软骨细胞体外构建组织工程软骨的研究

目的：探讨采用软骨细胞外基质材料制备的定向结构软骨支架复合软骨细胞,在体外静态培养条件下生成组织工程软骨的可能性。方法：制备牛关节软骨细胞外基质材料,利用温度梯度热诱导相分离技术构建具备垂直定向孔道结构的软骨支架,同时采用传统冷冻干燥方法制备非定向支架,检测两组支架的力学性能;提取兔关节软骨细胞,分别接种两组支架,体外静态培养2周及4周后取材,对构建的组织工程软骨进行组织切片染色、生物化学分析及生物力学检测。结果：定向软骨支架的压缩弹性模量数值明显高于非定向软骨支架,体外培养时定向支架上种子细胞在3-9d内增殖高于非定向支架,差异有统计学意义（P〈0．05）;体外静态培养4周后形成的两组新生组织工程软骨进行软骨特异性染色均呈阳性,在定向组新生软骨切片中在垂直方向上可见大量呈规则平行排列的粗大胶原纤维,两组新生软骨的生物化学检测包括总DNA、总GAG及总胶原含量差异无统计学意义（P〉0．05）。定向组织工程软骨压缩弹性模量在2周及4周时均高于非定向组织工程软骨,差异有统计学意义（P〈0．05）。但两组组织工程软骨上述指标均显著低于正常关节软骨（P〈0．05）。结论：软骨细胞外基质材料制备的定向结构软骨支架复合软骨细胞,在体外静态培养条件下能够成功生成具有定向纤维结构的组织工程软骨,并可以有效促进新生软骨组织力学性能的提升,在软骨组织工程中具有良好的应用前景。     

几丁质支架对间充质干细胞成软骨分化中Ⅱ型胶原表达的影响

目的将间充质干细胞诱导分化为软骨细胞,观察分化后的细胞在单层培养和几丁质支架上培养的差别。方法抽取兔股骨骨髓,密度梯度离心分离BMSCs,用TGF-β1诱导第3代的BMSCs向软骨方向分化,2周后用免疫组化检测Ⅱ型胶原表达情况。将分化后的软骨样细胞传代,用无TGF-β1的培养基分别进行单层培养和接种在几丁质支架上培养2周,再用免疫组化检测Ⅱ型胶原表达情况。结果BMSCs经TGF-β1诱导后,具有软骨细胞特点,然而进行无TGF-β1的继续培养后,单层培养的类软骨细胞很快呈去分化表型,而接种在支架上的细胞仍能较好表达Ⅱ型胶原。结论几丁质支架具有延缓软骨样细胞去分化和老化的作用。     

TGF-β1对体外培养的关节软骨细胞的作用

以原代培养的软骨细胞为材料,研究了转化生长因子IGF－β1对关节软骨细胞的增殖和软骨特异性基质代谢的作用,结果显示TGF－β1可以刺激细胞外基质中蛋白多糖含量的增加,并可以在转录水平上调节Ⅱ型胶原的表达。TGF－β1可以在体外诱导高密度培养的软骨细胞形成透明软骨样结构。没有用TGF－β1处理的高密度培养细胞不能形成透明软骨样结构,在透射电镜下观察发现其内部结构发生了异常变化。这些结果表明TGF－β1对于支持关节软骨细胞的体外培养具有重要的作用。     

BMSCs与PLGA三维生物支架复合修复喉软骨缺损的研究

目的 探讨骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）与聚乳酸/羟基乙酸共聚物（poly (lactide-co-glycolide),PLGA）三维生物支架在软骨源性形态发生蛋白1（cartilage-derived morphogenetic protein 1,CDMP1）和转化生长因子－β1（transforming growth factor-β1,TGF-β1）作用下向软骨细胞表型分化及体内修复喉软骨缺损的能力。方法 在体外高密度细胞悬液与PLGA共同构筑的三维立体培养体系下CDMP1和（或）TGF-β1联合诱导BMSCs向软骨细胞分化,观察诱导后细胞表型的表达;将培养体系移植入动物体内,从大体、组织学方面观察其对喉软骨缺损的修复效果。结果 诱导后的培养体系可表达特异性软骨基质Ⅱ型胶原和GAG;将培养体系移植入动物体内,可有效的修复喉软骨缺损。结论 BMSCs与PLGA三维生物支架在CDMP1和TGF-β1作用下所得组织工程化软骨可以有效的修复喉软骨缺损。     

生长分化因子-5及碱性成纤维细胞生长因子诱导家兔椎间盘髓核细胞成骨作用的研究

目的建立家兔椎间盘髓核细胞的体外培养模型,研究重组人生长分化因子-5(recombinant human growth differentiation factor-5,rhGDF-5)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)对髓核细胞成骨潜能的激发作用。方法将诱导剂rhGDF-5和bFGF分别及联合加入体外培养的髓核细胞中,观察髓核细胞的成骨表型表达和细胞学特性的变化。结果 rhGDF-5和bFGF均能促进钙盐沉积形成钙结节。rhGDF-5抑制髓核细胞增殖同时增加骨钙素表达;bFGF促进髓核细胞增殖及Ⅰ型胶原表达,但对骨钙素表达无显著影响。联合使用rhGDF-5和bFGF对髓核细胞成骨潜能(促进髓核细胞增殖、Ⅰ型胶原及骨钙素表达和钙盐沉积)的激发作用均优于单独使用其中任一细胞因子。结论 rhGDF-5诱导髓核细胞向成骨细胞分化,bFGF加强该诱导作用,联合使用rhGDF-5和bFGF能充分激发髓核细胞的成骨潜能。     

GGCX基因在兔骨关节炎软骨退变中的作用机制

通过将GGCX慢病毒转染软骨细胞,探讨骨关节炎患者软骨组织中γ-谷氨酰羧化酶(GGCX)基因过度表达对兔骨关节炎(OA)软骨细胞分化的影响及其机制。首先从OA兔体内分离软骨细胞,用茜素红染色标记软骨细胞。然后将分离的软骨细胞分为正常对照组、GGCX过表达组和载体组3组。编码GGCX的慢病毒用于过表达GGCX,流式细胞分析检测出病毒转染后细胞凋亡。实时荧光定量PCR和蛋白质印迹法用于检测GGCX、基质金属蛋白酶13 (MMP13)、Ⅹ型胶原、Ⅱ型胶原、肿瘤坏死因子(TNF)和白细胞介素-1β。慢病毒编码GGCX提高了OA软骨细胞中中GGCS的表达水平,mRNA和蛋白水平都显著升高(p0.05)。GGCX过表达显著增加Ⅱ型胶原,而mRNA和蛋白水平均降低MMP13、Ⅹ型胶原、TNF和IL-1β表达(p0.05)。相对于载体,GGCX过表达抑制了OA软骨细胞的细胞凋亡。GGCX过表达可调节细胞外基质的平衡,促进软骨细胞蛋白多糖合成,这与细胞凋亡减少有关。     

Role of pericellular matrix in development of a mechanically functional neocartilage

The role of the chondrocyte pericellular matrix (PCM) was examined in a three-dimensional chondrocyte culture system to determine whether retention of the native pericellular matrix could stimulate collagen and proteoglycan accumulation and also promote the formation of a mechanically functional hyaline-like neocartilage. Porcine chondrocytes and chondrons, consisting of the chondrocyte with its intact pericellular matrix, were maintained in pellet culture for up to 12 weeks. Sulfated glycosaminoclycans and type II collagen were measured biochemically. Immunocytochemistry was used to examine collagen localization as well as cell distribution within the pellets. In addition, the equilibrium compressive moduli of developing pellets were measured to determine whether matrix deposition contributed to the mechanical stiffness of the cartilage constructs. Pellets increased in size and weight over a 6-week period without apparent cell proliferation. Although chondrocytes quickly rebuilt a PCM rich in type VI collagen, chondron pellets accumulated significantly more proteoglycan and type II collagen than did chondrocyte pellets, indicating a greater positive effect of the native PCM. After 5 weeks in chondron pellets, matrix remodeling was evident by microscopy. Cells that had been uniformly distributed throughout the pellets began to cluster between large areas of interterritorial matrix rich in type II collagen. After 12 weeks, clusters were stacked in columns. A rapid increase in compressive strength was observed between 1 and 3 weeks in culture for both chondron and chondrocyte pellets and, by 6 weeks, both had achieved 25% of the equilibrium compressive stiffness of cartilage explants. Retention of the in vivo PCM during chondrocyte isolation promotes the formation of a mechanically functional neocartilage construct, suitable for modeling the responses of articular cartilage to chemical stimuli or mechanical compression.     

The effect of oxygen tension on human articular chondrocyte matrix synthesis: Integration of experimental and computational approaches

Significant oxygen gradients occur within tissue engineered cartilaginous constructs. Although oxygen tension is an important limiting parameter in the development of new cartilage matrix, its precise role in matrix formation by chondrocytes remains controversial, primarily due to discrepancies in the experimental setup applied in different studies. In this study, the specific effects of oxygen tension on the synthesis of cartilaginous matrix by human articular chondrocytes were studied using a combined experimental‐computational approach in a “scaffold‐free” 3D pellet culture model. Key parameters including cellular oxygen uptake rate were determined experimentally and used in conjunction with a mathematical model to estimate oxygen tension profiles in 21‐day cartilaginous pellets. A threshold oxygen tension (pO₂ ≈ 8% atmospheric pressure) for human articular chondrocytes was estimated from these inferred oxygen profiles and histological analysis of pellet sections. Human articular chondrocytes that experienced oxygen tension below this threshold demonstrated enhanced proteoglycan deposition. Conversely, oxygen tension higher than the threshold favored collagen synthesis. This study has demonstrated a close relationship between oxygen tension and matrix synthesis by human articular chondrocytes in a “scaffold‐free” 3D pellet culture model, providing valuable insight into the understanding and optimization of cartilage bioengineering approaches. Biotechnol. Bioeng. 2014;111: 1876–1885. © 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Enhancing Post-Expansion Chondrogenic Potential of Costochondral Cells in Self-Assembled Neocartilage

The insufficient healing capacity of articular cartilage necessitates mechanically functional biologic tissue replacements. Using cells to form biomimetic cartilage implants is met with the challenges of cell scarcity and donor site morbidity, requiring expanded cells that possess the ability to generate robust neocartilage. To address this, this study assesses the effects of expansion medium supplementation (bFGF, TFP, FBS) and self-assembled construct seeding density (2, 3, 4 million cells/5 mm dia. construct) on the ability of costochondral cells to generate biochemically and biomechanically robust neocartilage. Results show TFP (1 ng/mL TGF-β1, 5 ng/mL bFGF, 10 ng/mL PDGF) supplementation of serum-free chondrogenic expansion medium enhances the post-expansion chondrogenic potential of costochondral cells, evidenced by increased glycosaminoglycan content, decreased type I/II collagen ratio, and enhanced compressive properties. Low density (2 million cells/construct) enhances matrix synthesis and tensile and compressive mechanical properties. Combined, TFP and Low density interact to further enhance construct properties. That is, with TFP, Low density increases type II collagen content by over 100%, tensile stiffness by over 300%, and compressive moduli by over 140%, compared with High density. In conclusion, the interaction of TFP and Low density seeding enhances construct material properties, allowing for a mechanically functional, biomimetic cartilage to be formed using clinically relevant costochondral cells.     

P38 mitogen‐activated protein kinase promotes dedifferentiation of primary articular chondrocytes in monolayer culture

Articular cartilage is an avascular tissue with poor regenerative capacity following injury, a contributing factor to joint degenerative disease. Cell‐based therapies for cartilage tissue regeneration have rapidly advanced; however, expansion of autologous chondrocytes in vitro using standard methods causes ‘dedifferentiation’ into fibroblastic cells. Mitogen‐activated protein kinase (MAPK) signalling is crucial for chondrocyte metabolism and matrix production, and changes in MAPK signals can affect the phenotype of cultured cells. We investigated the effects of inhibition of MAPK signalling on chondrocyte dedifferentiation during monolayer culture. Blockade of extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) signalling caused a significant increase in cartilage gene expression, however, also caused up‐regulation of fibrotic gene expression. Inhibition of p38 MAPK (p38) caused a significant up‐regulation of collagen type II while suppressing collagen type I expression. P38 inhibition also resulted in consistently more organized secretion of collagen type II protein deposits on cell culture surfaces. Follow‐on pellet culture of treated cells revealed that MAPK inhibition reduced cell migration from the pellet. ERK and JNK inhibition caused more collagen type I accumulation in pellets versus controls while p38 inhibition strongly promoted collagen type II accumulation with no effect on collagen type I. Blockade of all three MAPKs caused increased GAG content in pellets. These results indicate a role for MAPK signalling in chondrocyte phenotype loss during monolayer culture, with a strong contribution from p38 signalling. Thus, blockade of p38 enhances chondrocyte phenotype in monolayer culture and may promote more efficient cartilage tissue regeneration for cell‐based therapies.     

Retention of the native chondrocyte pericellular matrix results in significantly improved matrix production.

The interaction of the cell with its surrounding extracellular matrix (ECM) has a major effect on cell metabolism. We have previously shown that chondrons, chondrocytes with their in vivo-formed pericellular matrix, can be enzymatically isolated from articular cartilage. To study the effect of the native chondrocyte pericellular matrix on ECM production and assembly, chondrons were compared with chondrocytes isolated without any pericellular matrix. Immediately after isolation from human cartilage, chondrons and chondrocytes were centrifuged into pellets and cultured. Chondron pellets had a greater increase in weight over 8 weeks, were more hyaline appearing, and had more type II collagen deposition and assembly than chondrocyte pellets. Minimal type I procollagen immunofluorescence was detected for both chondron and chondrocyte pellets. Chondron pellets had a 10-fold increase in proteoglycan content compared with a six-fold increase for chondrocyte pellets over 8 weeks (P<0.0001). There was no significant cell division for either chondron or chondrocyte pellets. The majority of cells within both chondron and chondrocyte pellets maintained their polygonal or rounded shape except for a thin, superficial edging of flattened cells. This edging was similar to a perichondrium with abundant type I collagen and fibronectin, and decreased type II collagen and proteoglycan content compared with the remainder of the pellet. This study demonstrates that the native pericellular matrix promotes matrix production and assembly in vitro. Further, the continued matrix production and assembly throughout the 8-week culture period make chondron pellet cultures valuable as a hyaline-like cartilage model in vitro.     

Enhanced chondrogenic differentiation of dental pulp-derived mesenchymal stem cells in 3D pellet culture system: effect of mimicking hypoxia

High incidence of articular cartilage defects resulting from age-related degeneration or trauma injuries is a major problem worldwide. Limited self-regeneration ability of cartilage often leads to inappropriate biochemistry and structure of healed tissue. Considering Impairments of traditional treatments, cell-based therapies are promising. The rapid ex vivo expansion and chondrogenic differentiation capability make dental pulp stem cells (DPSCs) a favorable cell type for therapeutic application, however strategies in order to efficient cartilage tissue-like production are imperative. In the present study the potential role of hypoxia mimicking agent, cobalt chloride (CoCl2), on chondrogenic differentiation of human DPSCs was surveyed. Cell viability assay used to obtain the optimum dose and exposure time of CoCl2. DPSCs were differentiated in pellet culture system after CoCl2 pretreatment. Chondrogenic differentiation efficiency was evaluated by histological and immunohistological analyses. The results showed that CoCl2 led to increased pellet size, integrity and matrix deposition with organizations more resembled typical cartilage lacuna structure. Furthermore, CoCl2 could improve differentiation by elevated chondrogenic markers, glycosaminoglycans (GAGs) and collagen II expression. CoCl2 pretreatment mitigated hypertrophy, as well, which was reflected in decreased collagen X expression. Alkaline phosphatase (ALP) specific activity did not change significantly by CoCl2 preconditioning. Based on current study hypoxia mimicking agent, CoCl2, could be suggested to promote DPSCs chondrogenic differentiation.     

A neocartilage ideal for extracellular matrix macromolecule immunolocalization

A neocartilage construct readily amenable to microscopy and biomechanical studies is described. Porcine articular cartilage was digested with a mixture of dispase and collagenase for chondrons or pronase and collagenase for chondrocytes. Chondrons or chondrocytes plated in 96-well plates were fixed and immunolabeled in situ for fluorescence microscopy at days 4 and 11. Collagen types I and II, aggrecan, and MMP-13 expression was assayed by semiquantitative RT-PCR. Cell numbers were analyzed by MTT assay. Chondrons and chondrocytes produced neocartilage that could be handled with minimal tearing on day 3 and none on day 11. Some cell division occurred between days 4 and 7. In both cultures, chondrocytes were surrounded by a thin rim of type VI collagen and osteopontin. Type II collagen, keratan sulfate, and tenascin were abundant throughout. At day 3, cells were rounded but by day 11 flattened cells were visible in the substratum. Continued synthesis of aggrecan and type II collagen mRNA indicated maintenance of the chondrocyte phenotype. The neocartilage was easy to immunolabel in situ without the need for sectioning, and individual cells were readily observed by microscopy. The versatility of these constructs makes them ideal for microscopy and for biomechanical studies.     

Adhesion of tissue-engineered cartilate to native cartilage

Reconstruction of cartilaginous defects to correct both craniofacial deformities and joint surface irregularities remains a challenging and controversial clinical problem. It has been shown that tissue-engineered cartilage can be produced in a nude mouse model. Before tissue-engineered cartilage is used clinically to fill in joint defects or to reconstruct auricular or nasal cartilaginous defects, it is important to determine whether it will integrate with or adhere to the adjacent native cartilage at the recipient site. The purpose of this study was to determine whether tissue-engineered cartilage would adhere to adjacent cartilage in vivo. Tissue-engineered cartilage was produced using a fibrin glue polymer (80 mg/cc purified porcine fibrinogen polymerized with 50 U/cc bovine thrombin) mixed with fresh swine articular chondrocytes. The polymer/chondrocyte mixture was sandwiched between two 6-mm-diameter discs of fresh articular cartilage. These constructs were surgically inserted into a subcutaneous pocket on the backs of nude mice (n = 15). The constructs were harvested 6 weeks later and assessed histologically, biomechanically, and by electron microscopy. Control samples consisted of cartilage discs held together by fibrin glue alone (no chondrocytes) (n = 10). Histologic evaluation of the experimental constructs revealed a layer of neocartilage between the two native cartilage discs. The neocartilage appeared to fill all irregularities along the surface of the cartilage discs. Safranin-O and toluidine blue staining indicated the presence of glycosaminoglycans and collagen, respectively. Control samples showed no evidence of neocartilage formation. Electron microscopy of the neocartilage revealed the formation of collagen fibers similar in appearance to the normal cartilage matrix in the adjacent native cartilage discs. The interface between the neocartilage and the native cartilage demonstrated neocartilage matrix directly adjacent to the normal cartilage matrix without any gaps or intervening capsule. The mechanical properties of the experimental constructs, as calculated from stress-strain curves, differed significantly from those of the control samples. The mean modulus for the experimental group was 0.74 +/- 0.22 MPa, which was 3.5 times greater than that of the control group (p < 0.0002). The mean tensile strength of the experimental group was 0.064 +/- 0.024 MPa, which was 62.6 times greater than that of the control group (p < 0.0002). The mean failure strain of the experimental group was 0.16 +/- 0.061 percent, which was 4.3 times greater than that of the control group (p < 0.0002). Finally, the mean fracture energy of the experimental group was 0.00049 +/- 0.00032 J, which was 15.6 times greater than that of the control group. Failure occurred in all cases at the interface between neocartilage and native cartilage. This study demonstrated that tissue-engineered cartilage produced using a fibrin-based polymer does adhere to adjacent native cartilage and can be used to join two separate pieces of cartilage in the nude mouse model. Cartilage pieces joined in this way can withstand forces significantly greater than those tolerated by cartilage samplesjoined only by fibrin glue.     

Stimulation of chondrocytes in vitro by gene transfer with plasmids coding for epidermal growth factor (hEGF) and basic fibroblast growth factor (bFGF)

Human epidermal growth factor (hEGF) and basic fibroblast growth factor (bFGF) influence critical characteristics of chondrocytes. The effects on metabolism and differentiation were evaluated following transfection using specific plasmids coding for both cytokines.Chondrocytes were isolated from femoral head cartilage of patients undergoing a hip arthroplasty for femoral neck fracture. Following collagenase-digestion, cells were cultured in monolayers, and cell proliferation, glucosaminoglycan-production and collagen type II expression were monitored 10 days after isolation.Addition of recombinant hEGF and bFGF resulted in a significant increase in cell proliferation and glucosaminoglycan production. Chondrocytes were transfected with vectors coding for either hEGF or bFGF and the production of these proteins was measured in supernatants by ELISA. Expression kinetics showed different patterns: hEGF was detectable 2.5 days following transfection and peaked at day 5.5, whereas bFGF-production reached its maximum 1.5 days after transfection, declining thereafter. Chondrocytes endogenously produced significant amounts of bFGF within 5 days following isolation. Proliferation of hEGF-transfected cells increased up to 81%; bFGF-transfection caused an increase up to 76%. Similarly, glucosaminoglycan-production was enhanced up to 120% by hEGF-transfection and 37% by bFGF transfection, respectively. Collagen type II production decreased following transfection with both plasmids.Temporary in vitro gene transfer of the growth factors hEGF and bFGF provides a method to stimulate chondrocyte proliferation and induces signs of dedifferentiation, which would limit a reasonable clinical application.