Regaining chondrocyte phenotype in thermosensitive gel culture

Chondrocyte tissue engineering continues to be a challenging problem. When chondrocytes are duplicated in vitro, it is imperative to obtain an adequate number of cells of optimal phenotype. A temperature-sensitive polymer gel, a copolymer of poly(N-isopropylacrylamide) and acrylic acid (PNiPAAm-co-Aac), has the ability of gelling at 37 degrees C (the lower critical solution temperature, LCST) or above and liquefying below that temperature (Vernon and Gutowska, Macromol. Symp. 1996;109:155-167). The hypothesis of this study was that chondrocytes could (1) duplicate in the copolymer gel; (2) regain their chondrocyte phenotype; and (3) be easily recovered from the gel by simply lowering the temperature below 37 degrees C. Chondrocytes from adult rabbit scapular cartilage were harvested and cultured in a monolayer culture until confluency (approximately 2 weeks). Next, the cells were harvested and seeded into the copolymer gel and cultured for 2-4 weeks. The phenotype of the cultured cells was then characterized. Two groups of control cultures, monolayer and agarose gel, were used to compare their ability to maintain chondrocyte phenotype. The results showed that chondrocytes isolated from rabbit scapula can re-express chondrocyte phenotype in agarose culture and polymer gel culture but not in monolayer culture. Also, cultured chondrocytes can be easily recovered from polymer gel culture by simply lowering the temperature. This new in vitro method of chondrocyte culture is recommended for chondrocyte propagation and regaining chondrocyte phenotype before cell seeding or transplantation.     

Self-assembled octapeptide scaffolds for in vitro chondrocyte culture

Nature has evolved a variety of creative approaches to many aspects of materials synthesis and microstructural control. Molecular self-assembly is a simple and efficient way to fabricate complex nanostructures such as hydrogels. We have recently investigated the gelation properties of a series of ionic-complementary peptides based on the alternation of non-polar hydrophobic and polar hydrophilic residues. In this work we focus on one specific octapeptide, FEFEFKFK (F, phenylalanine; E, glutamic acid; K, lysine). This peptide was shown to self-assemble in solution and form β-sheet-rich nanofibres which, above a critical gelation concentration, entangle to form a self-supporting hydrogel. The fibre morphology of the hydrogel was analysed using transmission electron microscopy and cryo-scanning electron microscopy illustrating a dense fibrillar network of nanometer size fibres. Oscillatory rheology results show that the hydrogel possesses visco-elastic properties. Bovine chondrocytes were used to assess the biocompatibility of the scaffolds over 21 days under two-dimensional (2-D) and three-dimensional (3-D) cell culture conditions, particularly looking at cell morphology, proliferation and matrix deposition. 2-D culture resulted in cell viability and collagen type I deposition. In 3-D culture the mechanically stable gel was shown to support the viability of cells, the retention of cell morphology and collagen type II deposition. Subsequently the scaffold may serve as a template for cartilage tissue engineering.     

Behaviour of epiphyseal mouse chondrocyte populations in monolayer culture

Summary Growth and dedifferentiation of a heterogeneous mouse chondrocyte population, prepared from epiphyses of mouse embryos (day 17 of gestation), were studied in primary monolayer culture. At different times of culture, light and electron microscopic investigations were carried out and the change of collagen types was shown by immunofluorescence microscopy. During the first four days in culture, chondrocytes express their typical phenotype. Round or polygonal cells are embedded in a metachromatically staining matrix and produce type II collagen. After four to eight days in vitro most of the chondrocytes lose their matrix capsule and alter to fibroblast-like cells. Simultaneously, a switch of collagen synthesis to type III and type I collagen occurs, whereas the type II collagen synthesis is stopped. Altered cells and transitional stages have intracellular glycogen like typical chondrocytes, but show phagocytosis and indications of cell migration like fibroblasts. It is proposed that these cells, originating from a subpopulation of epiphyseal cartilage, are able to differentiate and dedifferentiate in vitro.     

The lonely death: chondrocyte apoptosis in TNF-induced arthritis

Inflammatory joint disease typically provokes progressive cartilage damage. The proliferative synovial inflammatory tissue directly invades the cartilage and induces the expression and activation of degrading enzymes such as matrix metalloproteases (MMPs) and aggrecanases. However, also chondrocyte apoptosis has been observed in cartilage samples of inflamed joints. It remains unclear whether this is a secondary phenomenon due to cartilage damage or a primary event initiated by the synovial inflammation. To determine the presence or absence of chondrocyte death in experimental arthritis, we longitudinally assessed proteoglycan depletion and chondrocyte apoptosis in paw sections from human tumor necrosis factor transgenic (hTNFtg) mice and wild-type littermates. Whereas, wild-type mice showed no signs of cartilage damage, hTNFtg mice exhibited progressive proteoglycan loss starting at clinical onset of arthritis. However, we already found the first apoptotic chondrocytes well before cartilage matrix breakdown occurred indicating that chondrocyte death can be induced before matrix resorption. Chondrocyte death could constantly be observed until late stages of arthritis causing a continuous increase in the number of empty cartilage lacunae. As apoptotic cells in cartilage cannot be cleared by phagocytes due to their spatial isolation in the avascular lacunae of cartilage, having no contact to professional or amateur phagocytes. The dying cells are compelled to undergo a "lonely death" inevitable ending up in secondary necrosis giving rise to perpetuation of a pro-inflammatory cascade. These data indicate that chondrocyte death may play a primary role in inflammatory arthritis fueling cartilage inflammation and damage due to secondary necrosis.     

Short-term exposure of cartilage to blood results in chondrocyte apoptosis

Studies have shown that joint bleeding leads to cartilage degradation independent of concurrent synovitis. We hypothesized that the blood-induced cartilage damage is because of increased chondrocyte apoptosis after short-term exposure of whole blood or isolated mononuclear cells plus red blood cells to cartilage. Human cartilage tissue samples were co-cultured for 4 days with whole blood (50% v/v) or with mononuclear cells plus red blood cells (50% v/v equivalents). Cartilage matrix proteoglycan synthesis ((35)SO(4)(2-) incorporation) was determined after 4 days as well as at day 16 (after a 12-day recovery period in the absence of any additions). To test the involvement of apoptosis a specific caspase-3 inhibitor (acDEVDcho, 0 to 500 micro mol/L) as well as a pan-caspase inhibitor (zVADfmk, 0 to 500 micro mol/L) were added. Chondrocyte apoptosis was evaluated by immunohistochemical staining of single-strand DNA and by terminal dUTP nick-end labeling. Cartilage co-cultured with whole blood as well as mononuclear cells plus red blood cells induced a long-term inhibition of proteoglycan synthesis (74% and 78% inhibition on day 16, respectively). Immunohistochemistry showed a threefold increase in apoptotic chondrocytes in cultures with 50% whole blood as well as with mononuclear cells plus red blood cells. Both the specific caspase-3 inhibitor and the pan-caspase inhibitor partially restored proteoglycan synthesis in the cartilage after blood exposure. This effect was accompanied by a decrease in the number of apoptotic chondrocytes. These data suggest that a single joint hemorrhage (a 4-day exposure of cartilage to 50% v/v blood) results in induction of chondrocyte apoptosis, responsible for the observed inability of the chondrocytes to restore the proteoglycan synthesis during recovery from a short-term exposure to blood. This reduced restoration could eventually lead to cartilage degeneration and ultimately joint destruction.     

改良分步消化法培养原代软骨细胞

背景：胰蛋白酶和细菌胶原酶结合使用消化关节软骨基质获得大量纯度高的软骨细胞的方法步骤繁琐、过程复杂,容易污染,但更好的简单易行、安全可靠的方法至今少有报道。 目的：采用改良分步消化法进行软骨细胞培养,以获取大量纯净的软骨细胞。 方法：将新西兰白兔6只随机分2组,酶消化法组运用酶消化法分两步获取原代软骨细胞,对照组用传统法进行原代软骨细胞培养。培养1周后观察两组培养的软骨细胞的生长状态,并进行细胞鉴定、计数,评估改良后的方法对细胞的影响。 结果与结论：酶消化法组采用0.2%Ⅱ型胶原酶消化软骨细胞,与对照组相比,可将6 h以上的消化时间缩短至3 h。两组原代软骨细胞培养24 h均多呈圆形,悬浮状态,48 h后贴壁,培养1周后,两组软骨细胞可铺满培养瓶底。结果证实,采用改良分步消化法进行软骨细胞培养,在缩短了消化时间的同时其细胞生长及形态变化均无改变,可以顺利获取大量纯净的软骨细胞。     

Three-dimensional microenvironments retain chondrocyte phenotypes during proliferation culture

Although autologous chondrocyte implantation has already been in clinical use, chondrocyte dedifferentiation is problematic during proliferation culture. We attempted a three-dimensional (3D) collagen gel culture under chondrocyte proliferation with repeated passaging to prevent the chondrocytes dedifferentiation. Human auricular chondrocytes were cultured in 3D or conventional monolayer conditions, which reached a 1000-fold increase in cell numbers at passages 3 and 4, respectively. During multiplication, the chondrocytes in 3D culture showed greater suppression of collagen type I (COL1) and preservation of collagen type II (COL2) than those in monolayer. Tissue-engineered cartilage made of 3D cells also abundantly accumulated COL2 or proteoglycan and possessed favorable mechanical properties. The advantage of 3D cells may result from the similarity of microenvironments in cell-to-matrix adhesion or cell-to-cell contacts with that of native cartilage. The up-regulation of integrins and down-regulation of cadherins in the 3D cells mimicked the expression pattern of native cartilage, rather than that of monolayer cells. The silencing of integrin beta1 and Ob-cadherin expression by small interfering ribonucleic acid in the cultured chondrocytes led to the promotion of dedifferentiation and redifferentiation, respectively, indicating that the 3D collagen gel culture provided sufficient cell preparation and reduced chondrocyte dedifferentiation, which is regarded as a feasible strategy in autologous chondrocyte implantation.     

Replication of alcelaphine herpesviruses in various cell culture systems and subsequent purification of virus

Summary Cultures of fetal aoudad sheep kidney (FAK), bovine embryonic lung (BEL), and African green monkey kidney (Vero) cells were compared for differential replication of alcelaphine herpesviruses. Cell-free virus appears more rapidly when infected cells are incubated at 33° C rather than at 37° C. Events in the replication and morphologic development of several alcelaphine herpesvirus isolates have been documented using light and electron microscopy. Techniques for indirect immunofluorescence and serum virus neutralization are described. When virus free of host-cell contaminants is desired for biochemical analysis, virus isolates are initially purified on sucrose gradients and then further purified by density gradient centrifugation in Percoll.     

Matrix-mixed culture: new methodology for chondrocyte culture and preparation of cartilage transplants

For cartilage engineering a variety of biomaterials were applied for 3-dimensional chondrocyte embedding and transplantation. In order to find a suitable carrier for the in vitro culture of chondrocytes and the subsequent preparation of cartilage transplants we investigated the feasibility of a combination of the well-established matrices fibrin and alginate. In this work human articular chondrocytes were embedded and cultured either in alginate, a mixture of alginate and fibrin, or in a fibrin gel after the extraction of the alginate component (porous fibrin gel) over a period of 30 days. Histomorphological analysis, electron microscopy, and immunohistochemistry were performed to evaluate the phenotypic changes of the chondrocytes, as well as the quality of the newly formed cartilaginous matrix. Our experiments showed that a mixture of 0.6% alginate with 4.5% fibrin promoted sufficient chondrocyte proliferation and differentiation, resulting in the formation of a specific cartilage matrix. Alginate served as a temporary supportive matrix component during in vitro culture and can be easily removed prior to transplantation. The presented tissue engineering method on the basis of a mixed alginate-fibrin carrier offers the opportunity to create stable cartilage transplants for reconstructive surgery.     

Characteristics of a scaffold-free articular chondrocyte plate grown in rotational culture

We investigated whether articular chondrocytes could form three-dimensional tissue-engineered cartilage in a rotational culture system without a scaffold. A suspension of chondrocytes derived from Japanese white rabbits was inoculated into a mold. Eight hours later, the cell suspension in the mold showed cell aggregation, forming a chondrocyte plate. The mold was removed, and the plate was cultured under static conditions. After 7 days of primary static culture, the plate was cultured under dynamic conditions, using rotational culture. After 2-3 weeks of rotational culture, the chondrocyte plate maintained a constant form and was considered stable enough to be handled with surgical pincers. Conversely, after 3 weeks of static culture, the plate gradually changed into an arch over that time. Histological and immunohistochemical evaluations indicated that the plate had cartilaginous qualities in terms of cell distribution and organization and the production of glycosaminoglycans and type II collagen in rotational cultures. Chondron units were detected with scanning electron microscopy. In contrast, a plate cultivated in static culture for 3 weeks was irregular in shape, and histological analysis indicated irregularly accumulated glycosaminoglycans. TUNEL-positive cells had increased significantly in the central region in 3-week static cultures, compared with those in 3-week rotational cultures. In this study, cartilaginous tissue in a scaffold-free environment has been produced. Significantly rotational cultures produce a construct, which is stable enough to be handled with surgical forceps after only 2 weeks of rotational culture. This system should be useful for implantation in the future.     

A modified cell culture method for autologous chondrocyte transplantation

Autologous chondrocyte transplantation (ACT) is a promising method to treat chondral and osteochondral defects. This study introduced a modified method for cell culture in ACT. Porcine chondrocytes were cultured for 3 weeks under low hydrostatic pressure at 250 Pa. The results showed that the dry weight of the cartilage-like membrane in the loading group was 3.0 times more than the control group (no loading) (p < 0.01), and cell numbers were significantly increased by 3.1 times (p < 0.01) after a 3-week culture. Compared with the fresh tissue sample, the mRNA expression of collagen II was not statistically different and the mRNA of aggrecan was only slightly decreased by 19%. These data suggest that the hydrostatic pressure at this level significantly increased the cell numbers and biosynthesis of cultured chondrocytes.     

Role of mitochondria in apoptosis

Apoptosis is characterized by biochemical processes that are largely conserved throughout evolution. The basic elements of the system comprise caspases, their activators and inhibitors, and regulators of mitochondrial integrity. New evidence reveals the role of mitochondria as the central coordinators of apoptosis. Accordingly, some caspases are sequestered within the mitochondria, and mitochondria contain additional proapoptotic factors. Bcl-2 and Bax homologs regulate the integrity of the mitochondrial outer membrane, which may also serve as a scaffold for the apoptotic machinery.     

Role of mitochondria in apoptosis

Apoptosis is an evolutionary-conserved physiological mechanism to remove cells from an organism. Cellular apoptosis is mediated via an intracellular signalling programme that involves a variety of signalling molecules and cellular organelles including caspases, sphingomyelinases, Bcl-2-like proteins and proteins to cleave the DNA and mitochondria. Mitochondria contain several pro-apoptotic molecules that activate cytosolic proteins to execute apoptosis, block anti-apoptotic proteins in the cytosol and directly cleave nuclear DNA. Mitochondria trap these pro-apoptotic proteins and physically separate pro-apoptotic proteins from their cytoplasmic targets. Apoptosis is then initiated by the release of mitochondrial pro-apoptotic proteins into the cytosol. This process seems to be regulated by Bcl-2-like proteins and several ion channels, in particular the permeability transition pore (PTP) that is activated by almost all pro-apoptotic stimuli.     

Development of PDMS microbioreactor with well-defined and homogenous culture environment for chondrocyte 3-D culture

Perfusion cell culture is believed to provide a stable culture environment due to the continuous supply of nutrients and removal of waste. However, the culture scales used in most cases were large, where the culture conditions can not be regarded as homogenous because of chemical gradients. To improve this, the concept of miniaturization is applied to 3-D cell culture. In this study, a simple perfusion microbioreactor was developed based on mass transport simulation to find out the reasonable culture scales with relatively lower chemical gradients. Besides, PDMS surface was treated with surfactant solution to reduce non-specific serum protein adsorption, which keeps the culture conditions steady. Chondrocyte 3-D culture using the proposed microbioreactors was compared with similar perfusion culture with a larger culture scale. Results showed that surfactant-treated PDMS surface could reduce serum protein adsorption by 85% over the native one. Also, microbioreactors were proved to provide a stable culture environment (e.g. pH) over the culture period. Cell culture scale of 200 μm thick culture construct was justified to have relatively lower chemical gradients than the larger scale perfusion culture. As a whole, the proposed culture system is capable of providing a well-defined and homogenous culture environment.     

12/15-脂氧合酶抑制剂黄芩素对软骨细胞凋亡的抑制作用研究

目的探讨12/15-脂氧合酶抑制剂黄芩素(Baicalein)对硝普钠(SNP)诱导的软骨细胞凋亡的抑制作用及可能机制。方法取8周雄性SD大鼠膝关节软骨,采用Ⅱ型胶原酶消化法提取软骨细胞并体外培养。设置对照组、SNP凋亡组和Baicalein给药组,以0.5 m M SNP作用24小时诱导软骨细胞凋亡,以5 M,25 M,50 M,100 M不同浓度的Baicalein作用细胞,确定最适浓度,对照组仅加入同体积溶剂(蒸馏水)。CCK8法检测药物对细胞毒性;Annexin V/PI双染法检测细胞凋亡;免疫荧光观测凋亡诱导因子(AIF)在软骨细胞中的定位。结果 Baicalein在各浓度下对软骨细胞无明显毒性,与对照组相比,凋亡组软骨细胞活性明显下降(0.01),与凋亡组相比,给药组细胞活性浓度依赖性地升高(0.01);流式检测显示对照组软骨细胞早期凋亡率3.16%,凋亡组27.8%,100 M Baicalein给药组14.1%;免疫荧光检测显示,凋亡组AIF出现核内移位,100 M Baicalein给药组AIF核移位受到抑制。结论 Baicalein通过抑制12/15-脂氧合酶活性,进而抑制AIF从线粒体中的释放及核转位,发挥抗软骨细胞凋亡作用。     

自体血清培养新西兰大白兔关节软骨细胞

目的：改进与探讨新西兰大白兔软骨细胞体外培养的方法。方法：取3月龄新西兰大白兔自体血,制备自体血清备用,无菌条件下取4周龄新西兰大白兔双侧膝关节软骨,采用Ⅱ型胶原酶消化并机械吹打的方法,分离关节软骨细胞并应用新西兰大白兔自体血清进行原代、传代培养;采用形态学观察,甲苯胺蓝染色以及Ⅱ型胶原免疫组织化学方法对膝关节软骨细胞进行鉴定,MTT法检测软骨细胞的增值能力;结果：倒置显微镜下见原代软骨细胞2 h后开始贴壁,8 h可形成单层,24 h即可传代。第1~5代细胞表型稳定,增殖力良好。甲苯胺蓝染色显示培养的软骨细胞核呈深染色,细胞质呈淡蓝色;免疫组织化学显示软骨细胞Ⅱ型胶原呈黄褐色表达,MTT检测显示前5代软骨细胞增值能力强,无明显差异;结论：结果表明自体血培养的前5代新西兰大白兔节软骨细胞均可用于膝骨关节炎的研究。     

血管内皮生长因子和缺氧诱导因子对软骨细胞凋亡的作用

背景：骨关节炎是一种关节疾病,主要影响软骨,随着软骨细胞胞外基质的变化,软骨细胞发生凋亡,血管内皮生长因子在促进血管内皮细胞分裂与增殖、诱导血管生成中起重要作用。缺氧诱导因子是一种在细胞环境中的转录因子,因氧含量而产生不同反应,血管内皮生长因子和缺氧诱导因子在抑制软骨细胞凋亡中的作用受到研究者的重视。 目的：阐述血管内皮生长因子和缺氧诱导因子及其他可能因素对软骨细胞凋亡的影响。 方法：分析、总结近年来软骨细胞凋亡的影响因素的相关文献,从骨关节炎进程中软骨细胞血管内皮生长因子表达变化,血管内皮生长因子和缺氧诱导因子对软骨细胞凋亡的调控等方面进行阐述。 结果与结论：血管内皮生长因子通过上调抑制细胞凋亡因子的表达促进软骨细胞存活,缺氧诱导因子能增加软骨细胞活性和细胞外基质合成,成为抑制骨细胞凋亡的重要靶点,血管内皮生长因子与缺氧诱导因子的相关性还有待进一步研究。 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     

自噬基因在骨关节炎软骨细胞凋亡过程中的保护和平衡效应

背景：软骨细胞在低营养供给下可发生细胞自噬,细胞自噬和细胞坏死、凋亡不同,可以使软骨细胞在营养供给不足时,能够存活下来,可能是软骨细胞自身的重要保护机制之一。 目的：综合阐述自噬基因保护关节软骨及抑制骨关节炎等方面的机制和作用。 方法：应用计算机检索中国知网、万方数据库及PubMed数据库2000年1月至2015年1月关于自噬基因与骨关节炎的相关研究的文章,中文检索词为“自噬基因、骨关节炎、关节软骨、软骨细胞”,英文检索词为“autophagy、osteoarthritis、beclin1、LC3”。选择自噬基因与骨关节炎的相关文章,初检得到269余篇文献,根据纳入标准选择其中的38篇进行综述。  结果与结论：软骨细胞的损伤凋亡是软骨退变的主要机制,不予控制,就可能会进一步发展为骨性关节炎。细胞的损伤凋亡是软骨退变的重要机制之一,因此防止软骨细胞的损伤凋亡,可能有利于损伤软骨的修复,以此来缓解骨关节炎的病情发展。自噬现象能够抑制受损软骨细胞凋亡,发现其可将改变传统治疗骨关节炎的局限,但目前自噬基因与骨关节炎相关的研究还处于初级阶段,特别是对自噬途径在软骨中如何被诱导,如何进行信号转导,如何对软骨细胞生存产生影响等方面的认识还不够全面,有待进一步的研究。