Scaffold-dependent differentiation of human articular chondrocytes

Matrix-associated autologous chondrocyte transplantation (MACT) is a tissue-engineered approach for the treatment of cartilage defects and combines autologous chondrocytes seeded on biomaterials. The objective of the study is the analysis of growth and differentiation behaviour of human articular chondrocytes grown on three different matrices used for MACT. Human articular chondrocytes were kept in monolayer culture for 42 days and then seeded on matrices consisting of either collagen type I/III, hyaluronan, or gelatine. During the culture time of 4 weeks the constructs were analyzed weekly. Morphological criteria were studied by scanning and transmission electron microscopy. The expression of the main type collagens was analyzed by real-time PCR. The collagen type I/III matrix supported a differentiation that closely resembled the tissue organisation of native cartilage, but cell number and type II collagen synthesis were low and differentiation occurred rather late in the cultivation period. The hyaluronan matrix and the gelatine-based matrix supported a rather rapid differentiation, with a high number of cells and a relatively high amount of type II collagen, but there was no spatial assembly that mimicked native cartilage. These facts indicate that the nature of the matrix is of great influence in the differentiation behaviour of dedifferentiated chondrocytes.     

成人关节软骨细胞增殖与人参皂甙Rg1的促进作用

背景：人参总皂甙的主要有效成分为人参皂甙Rg1,有促进细胞增殖、抗衰老、抗细胞凋亡、抗炎及抗自由基等作用,但以往实验对象多为鼠、兔等动物的关节软骨细胞。 目的：体外培养成人软骨细胞,观察人参皂甙Rg1对体外培养的成人关节软骨细胞增殖的促进作用。 方法：实验分2组,Rg1组使用含质量浓度为40 mg/L人参皂甙Rg1的DMEM培养液培养,对照组使用未加人参皂甙的DMEM培养液培养,每日应用倒置相差显微镜观察细胞生长情况至第7天,并进行细胞计数。同时培养至第6天检测软骨细胞中Ⅱ型胶原的表达。 结果与结论：对照组自培养第2天起细胞进入对数增殖期,到第7天细胞数约为接种时的8倍。Rg1组自培养第2天起细胞增殖能力较对照组增强(P < 0.05),到第7天细胞数约为接种时的18倍(P < 0.05)。免疫组织化学SABC法检测结果显示,Rg1组与对照组细胞胞浆均可见Ⅱ型胶原表达,两组差异无显著性意义(P > 0.05)。说明人参皂甙Rg1可促进成人软骨细胞的增殖。     

The role of apoptotic resistance in irradiated adult articular chondrocytes

There have so far been no studies on the apoptosis of adult articular chondrocytes after X-ray irradiation. The purpose of this study was to assess the apoptotic resistance of articular chondrocytes in X-ray radiation, in order to examine the possibility of irradiated allogenic chondrocyte implantation. Adult human chondrocytes of the non-degenerated cartilage group without X-ray irradiation did not show positive cells of Annexin V and PI staining in a 48 h culture. The Annexin V positive chondrocytes did not increase in a radiation dose dependent manner, and the PI positive cells were slightly increased at 30 Gy irradiation. In the degenerated cartilage group, the PI positive chondrocytes without irradiation were present, and both the Annexin V and PI positive chondrocytes increased in a radiation dose dependent manner. The Annexin V and PI positive staining of chondrocytes in the non-degenerated cartilage group was less than that of the degenerated cartilage group in the same dose of X-ray irradiation exposure. Loss of the mitochondrial membrane potential, revealed in an early stage of apoptosis, did not show in the irradiated chondrocytes of the non-degenerated cartilage, but were demonstrated in those of the degenerated cartilage. These results demonstrated that the non-degenerated chondrocytes of X-ray irradiation were highly resistant for apoptosis, and this knowledge could be applied to allogenic chondrocytes implantation.     

Effect of oxygen tension on adult articular chondrocytes in microcarrier bioreactor culture

Tissue-engineering approaches for cartilage repair hold promise for the treatment of cartilage defects. Various methods to prevent or reduce dedifferentiation during chondrocyte expansion are currently under investigation. In the present study we evaluated the effect of oxygen on chondrocyte proliferation, as oxygen has received increased attention as a possible regulator of chondrocyte differentiation and its effect during expansion is uncertain. Therefore, the effect of three oxygen tensions (4, 10.5, and 21%) was investigated in a bioreactor microcarrier culture, which allows precise control of the oxygen tension in the liquid phase. During culture cells acquired a round shape on microcarriers. No differences in proliferation rate of chondrocytes were observed within the range of oxygen tensions evaluated. Cells exhibited predominantly anaerobic metabolism and, per mole of glucose, approximately 2 mol of lactate was produced independent of oxygen tension. Cellular oxygen consumption was comparable for all bioreactor cultures. Nevertheless, specific consumption rates were relatively high (2-4 x 10(-17) mol. cell(-1). s(-1)), in comparison with chondrocytes in cartilage (0.8-2.2 x 10(-18) mol. cell(-1)). Subsequent cartilaginous tissue formation in pellets was not affected as qualitatively assessed by safranin-O staining. At the oxygen concentrations evaluated, no effect of oxygen tension was observed on proliferation, oxygen consumption, and yield of lactate on glucose administration. For future investigations of chondrocytes and oxygen, the bioreactor system, which allows precise control and monitoring of oxygen tension, holds promise.     

Biodegradable polymers in chondrogenesis of human articular chondrocytes

The aim of this study was to evaluate the potential role of polyglycolic acid (PGA), poly(glycolic acid-ε-caprolactone) (PGCL), poly(l-lactic acid-glycolic acid) (PLGA), poly(l-lactic acid-ε-caprolactone, 75:25 (w/w)) [P(LA-CL)25], poly-ε-caprolactone (tetrabutoxy titanium) [PCL(Ti)], and fullerene C-60 dimalonic acid (DMA) in cartilage transplants. After 4 weeks of culture of human articular cartilage, the levels of cell proliferation and differentiation and the expression of cartilage-specific matrix genes were estimated. The relationship between cell differentiation and gap junction protein connexin 43 (Cx43) was also evaluated. All materials except PCL(Ti) retained cell proliferation activities similar to the controls. Cell differentiation levels from the highest to the lowest were in the following order: PGA >> PLGA > PGCL > Control = DMSO > P(LA-CL)25 = PCL(Ti) >> fullerene C-60 DMA. Expression of the collagen type II gene was selectively upregulated for PGA, PGCL, and PLGA and slightly increased for P(LA-CL)25 polymers but was downregulated for fullerene C-60 DMA. Aggrecan gene expression was strongest with PGA and was consistently expressed with other matrices, especially with PGCL and PLGA. However, the expression patterns of the connexin 43 gene were different from the former two genes. Multiple regression analysis revealed a high correlation between cartilage proteoglycans production and expression levels of these three genes. The first two authors contributed equally to this work     

Rapid effects of hypoxia on H+ homeostasis in articular chondrocytes

Articular chondrocytes experience low oxygen (O₂) levels compared with many other tissues, and values fall further in disease states. Chondrocyte intracellular pH (pH_i) is a powerful modulator of matrix synthesis and is principally regulated by Na⁺-H⁺ exchange (NHE). In equine chondrocytes, NHE is inhibited when cells are incubated for 3 h at low O₂, leading to intracellular acidosis. O₂-dependent changes in reactive oxygen species (ROS) levels appear to underlie this effect. The present study examines whether hypoxia can influence chondrocyte NHE activity and pH_i over shorter timescales using the pH-sensitive fluoroprobe BCECF in cells isolated not only from equine cartilage but also from bovine tissue. O₂ levels in initially oxygenated solutions gassed with N₂ fell to approximately 1% within 2 h. A progressive fall in pH_i and acid extrusion capacity was observed, with statistically significant effects (P?<?0.05) apparent within 3 h. For equine and bovine cell populations subjected to step change in O₂ by resuspension in hypoxic (1%) solutions, a decline in acid extrusion and pH_i was observed within 10 min and continued throughout the recording period. This effect represented inhibition of the NHE-mediated fraction of acid extrusion. Cells subjected to hypoxic solutions supplemented with CoCl₂ (100 μM) or antimycin A (100 µM) to raise levels of ROS did not acidify. The conserved nature and rapidity of the response to hypoxia has considerable implications for chondrocyte homeostasis and potentially for the maintenance of cartilage integrity.     

人脱细胞软骨细胞外基质对异种软骨种子细胞的影响

目的观察人脱细胞软骨细胞外基质(hACAM)对异种兔软骨种子细胞增殖和表型的影响。方法将差速梯度离心法制作的人脱细胞软骨细胞外基质,配制成0.5%的浆料,分别铺于6孔细胞培养板和96孔细胞培养板,形成1 mm厚的薄膜,以此培养板为实验组。空白对照组培养板仅使用单纯培养液培养。在培养板上培养兔关节软骨细胞。通过hochest33258染色验证人软骨细胞外基质脱细胞完全与否;分别在第5、15天两个时间点,通过倒置显微镜、甲苯胺蓝染色观察两种培养方法的兔软骨细胞的生长形态、细胞表型和增殖情况,用CCK-8细胞增殖实验比较两种培养方法在第1、3、7、10天的增殖情况。结果通过hochest33258染色发现差速梯度离心的人软骨细胞外基质脱细胞完全。通过倒置显微镜观察第5和15天的实验组细胞增殖情况优于空白对照组,甲苯胺蓝染色的结果也证明这个观点;CCK-8细胞增殖试验显示第7天hACAM组在细胞增殖方面明显地优于单纯培养液的对照组(P=0.0298),hACAM组的软骨细胞与空白组的软骨细胞在第10天的增殖情况相比没有统计学差异。结论 hACAM免疫原性低,无细胞毒性,能够很好地促进异种软骨细胞的增殖。     

Explant culture of human and rabbit articular chondrocytes.

Copious outgrowth of chondrocytes was obtained by explantation from each of three rabbit and one surgically-resected human articular cartilages pretreated briefly with trypsin. In lapine explants, ascorbate (40 micrograms/ml) increased DNA three-fold over control values and resulted in deposition of a chondroid matrix. It doubled radiosulfate incorporation by the outgrowths. Up to 56% of the sulfated glycosaminoglycan synthesized was located in the trypsin-digestible pericellular coat compared with about 15% in previous monolayer cultures. The collagens synthesized were characterized partially. In rabbit cell cultures, the alpha 1:alpha 2 ratio varied from 2.9 to 3.8. In human cultures, an unusual post-alpha 2 peak was observed. The findings suggest an uncoupling of the phenotypic expression of the major cartilaginous macromolecules in the cultures. There were no distinctive differences between chondrocytes derived from normal and fibrillated human cartilage of the same individual.     

单层培养的人关节软骨细胞生物学行为研究

目的：研究单层培养的人关节软骨细胞生物学行为的改变。方法：对来自8例手术切除负重关节非负重区的软骨细胞在10％DMEM中进行单层传代培养，观察细胞形态学、增殖细胞倍增时间（DT）、细胞Ⅱ型胶原免疫组织化学染色以及流式细胞分析细胞增殖指数（P1）和细胞凋亡。结果：随着传代次数的增加，（1）细胞形态由原代的多角形转变为第3、4代的短梭形、第8、9代的成纤维细胞形态；（2）DT在逐渐延长，P1在逐渐减小；（3）细胞分泌的Ⅱ型胶原逐渐减少；（4）细胞凋亡指数未见明显的改变（P〉0．05）。结论：体外单层培养的关节软骨细胞存在着失分化和老化，但第3、4代能较好地保持软骨细胞的生物学行为，可作为种子细胞的来源。     

Low-intensity pulsed ultrasound affects human articular chondrocytes in vitro

We investigated whether low-intensity pulsed ultrasound (LIPUS) stimulates chondrocyte proliferation and matrix production in explants of human articular cartilage obtained from donors suffering from unicompartimental osteoarthritis of the knee, as well as in isolated human chondrocytes in vitro. Chondrocytes and explants were exposed to LIPUS (30 mW/cm²; 20 min/day, 6 days). Stimulation of [³⁵S]-sulphate incorporation into proteoglycans by LIPUS was 1.3-fold higher in degenerative than in collateral monolayers as assessed biochemically and 1.9-fold higher in explants as assessed by autoradiography. LIPUS decreased the number of cell nests containing 1–3 chondrocytes by 1.5 fold in collateral and by 1.6 fold in degenerative explants. LIPUS increased the number of nests containing 4–6 chondrocytes by 4.8 fold in collateral and by 3.9 fold in degenerative explants. This suggests that LIPUS stimulates chondrocyte proliferation and matrix production in chondrocytes of human articular cartilage in vitro. LIPUS might provide a feasible tool for cartilage tissue repair in osteoarthritic patients, since it stimulates chondrocyte proliferation and matrix production. C. M. Korstjens and R. H. H. van der Rijt are first authors.     

In-vitro culture of human chondrocytes from adult subjects

An agarose gel matrix was utilized to grow chondrocytes from human donors of various ages in cell culture. The chondrocytes produced the pericellular matrix characteristic for such cells and synthesized collagen type II as well as glyco-saminoglycans. The latter exhibit the typical distribution pattern of the respective articular cartilage matrix. The electron-microscopic appearance of the cultured chondrocytes closely resembles that of chondrocytes in sections of the original cartilage.     

Three-dimensional tissue engineering of hyaline cartilage: comparison of adult nasal and articular chondrocytes

Adult chondrocytes are less chondrogenic than immature cells, yet it is likely that autologous cells from adult patients will be used clinically for cartilage engineering. The aim of this study was to compare the postexpansion chondrogenic potential of adult nasal and articular chondrocytes. Bovine or human chondrocytes were expanded in monolayer culture, seeded onto polyglycolic acid (PGA) scaffolds, and cultured for 40 days. Engineered cartilage constructs were processed for histological and quantitative analysis of the extracellular matrix and mRNA. Some engineered constructs were implanted in athymic mice for up to six additional weeks before analysis. Using adult bovine tissues as a cell source, nasal chondrocytes generated a matrix with significantly higher fractions of collagen type II and glycosaminoglycans as compared with articular chondrocytes. Human adult nasal chondrocytes proliferated approximately four times faster than human articular chondrocytes in monolayer culture, and had a markedly higher chondrogenic capacity, as assessed by the mRNA and protein analysis of in vitro-engineered constructs. Cartilage engineered from human nasal cells survived and grew during 6 weeks of implantation in vivo whereas articular cartilage constructs failed to survive. In conclusion, for adult patients nasal septum chondrocytes are a better cell source than articular chondrocytes for the in vitro engineering of autologous cartilage grafts. It remains to be established whether cartilage engineered from nasal cells can function effectively when implanted at an articular site.     

胰岛素样生长因子- I与透明质酸促人关节软骨细胞增殖的作用

为了探讨胰岛素样生长因子-I(IGF-I)和透明质酸(HA)联合培养对人关节软骨细胞增殖的影响,在人关节软骨细胞培养液中分别加入不同浓度的IGF-I、HA、IGF-I和 HA,用四氮甲基唑蓝(MTT)法及流式细胞术测定软骨细胞增殖活性的变化.结果显示,10μg/L浓度的IGF-I即可明显促进软骨细胞的增殖,IGF-I浓度为50μg/L时,其促增殖作用达最大值;单独应用HA对软骨细胞的增殖无影响; IGF-I+HA组吸光度值为0.377,较IGF-I组增加10.7%;IGF-I组促增殖的高峰时间为72h,IGF-I+HA组的高峰时间为96h,培养96h后,IGF-I+HA组吸光度值0.389,细胞的增殖指数为28.79±1.24%,较对照组升高更明显.说明IGF-I具有促软骨细胞增殖作用,与HA联合培养促增殖能力更强,具有协同效应,并能延长促丝裂作用的时间.