The transglutaminase, Factor XIIIA, is present in articular chondrocytes

OBJECTIVES: The transglutaminase (TGase) family includes seven different enzymes that catalyse a protein cross-linking reaction resulting in structural and functional alterations in substrate proteins. TGase activity is easily measureable in mature articular cartilage where it may contribute to CPPD deposition disease through its actions on growth factors, crystal components or extracellular matrix proteins. In contrast, low levels of TGase activity are found in chondrocytes from young animals. We previously demonstrated type II TGase protein in articular chondrocytes. Earlier work also suggested the presence of another form of TGase in chondrocytes. We sought to determine if articular chondrocytes contain the TGase, Factor XIIIA (FXIIIA). METHODS: Western blots with FXIIIA antibody were used to detect FXIIIA in young and old porcine articular chondrocytes and articular cartilage vesicles (ACVs). The presence of FXIIIA mRNA was confirmed by RT-PCR. RESULTS: Old chondrocyte conditioned medium, cytosol, and membrane fractions contained FXIIIA protein on Western blots, while less FXIIIA was detectable in cell fractions or media from young chondrocytes. ACVs also contained FXIIIA. FXIIIA mRNA was demonstrated by PCR in old and young chondrocytes. CONCLUSIONS: FXIIIA is present in articular chondrocytes. FXIIIA levels correlate with TGase activity in chondrocytes. The presence of two forms of TGase in articular chondrocytes suggest an important function for this enzyme family in articular cartilage.     

Growth hormone stimulates insulin-like growth factor I actions on adult articular chondrocytes

We report effects of adding insulin-like growth factor I (IGF-I) and methionyl human growth hormone (GH), alone or in combination, to adult bovine articular chondrocytes plated at high density. Purified human and synthetic IGF-I stimulated chondrocyte DNA and proteoglycan synthesis. GH had no effect on either process. However, GH added in combination with IGF-I increased proteoglycan, cell-associated proteoglycan, and keratan sulfate synthesis over levels observed with IGF-I alone. IGF-I and GH did not alter the hydrodynamic size of proteoglycans or synthesis of collagen. Our results show that GH and IGF-I act together to stimulate adult chondrocyte extracellular matrix synthesis.     

Growth differentiation factor-5 stimulates the growth and anabolic metabolism of articular chondrocytes

Objective： To observe the effect of growth differentiation factor-5 （GDF-5） on the growth and anabolic metabolism of articular chondrocytes. Methods： The articular chondrocytes isolated from rats were treated with various concentrations of rmGDF-5, and the growth of chondrocytes measured by MTT assay, the cellular cartilage matrices formation detected sulfated glycosaminoglycan by Alcian blue staining and type Ⅱ collagen by RT-PCR, the collagen phenotypic expression of chondrocytes detected by immunofluorescence. Results： After 7 days culture, MTT assay showed that GDF-5 enhanced the growth of chondrocytes in a dose-dependent manner, RT-PCR showed that GDF-5 clearly induced the synthesis of type Ⅱcollagen because of the col2al mRNA band more and more strong in a dose-dependent. Chondrocytes were cultured with GDF-5 for 14 days, the intensity of Alcian blue staining was greatly enhanced, especially, at a high concentration of 1000ng/ml, and GDF-5 enhanced the accumulation of the Alcian blue-stainable material in a concentration-dependent manner and in a does-dependent manner. Chondrocytes were cultured with GDF-5 for 21 days, immunofluorescent staining of type Ⅱ collagen was clear, the type Ⅰ and Ⅹ collagen were negative. Conclusion： GDF-5 enhanced the growth of mature articular chondrocytes, and stimulated the cellular cartilage matrices formation, but did not change the collagen phenotypic expression of chondrocytes in mono-layer culture.     

AG-041R stimulates cartilage matrix synthesis without promoting terminal differentiation in rat articular chondrocytes

OBJECTIVE: AG-041R, a novel indolin-2-one derivative, has recently been demonstrated to induce systemic hyaline cartilage hyperplasia in rats. The aim of this study was to characterize its anabolic actions on chondrocytes. DESIGN: Chondrocytes were isolated from knee joints of 5-week-old SD rats. Effects of AG-041R on cartilage matrix synthesis were examined by measuring [(35)S]sulfate incorporation into proteoglycans, Alcian blue staining, and Northern blotting of cartilage matrix genes. ALP activity, mineral deposition and the expression of markers for hypertrophic chondrocytes, were assessed for terminal differentiation of chondrocytes. Roles of endogenous TGF-beta/BMPs and MEK1/Erk signaling in the action of AG-041R were investigated using the neutralizing soluble receptors and the MEK1 inhibitor. RESULTS: AG-041R accelerated proteoglycan synthesis assessed by both [(35)S]sulfate incorporation and Alcian blue stainable extracellular matrix accumulation. It also up-regulated the gene expression of type II collagen and aggrecan, as well as tenascin, a marker for articular cartilage. In contrast, AG-041R suppressed ALP activity, mineralization, and the gene expression of type X collagen and Cbfa1, indicating that AG-041R prevents chondrocyte terminal differentiation. AG-041R increased in BMP-2 mRNA, and the neutralizing soluble receptor for BMPs reversed the stimulatory effects of AG-041R on cartilage matrix synthesis. Moreover, AG-041R activated MEK1/Erk pathway, which was revealed to prevent chondrocyte terminal differentiation. CONCLUSION: AG-041R stimulates cartilage matrix synthesis without promoting terminal differentiation in rat articular chondrocytes, which is mediated at least in part by endogenous BMPs and Erk. The data demonstrates that AG-041R has a potential to be a useful therapeutic agent for articular cartilage disorders.     

关节软骨细胞的凋亡

细胞凋亡(Apoptosis)又称程序性细胞死亡(Programmed Cell Death,PCD),为Kerr于1972年首次描述,是一种不同于坏死的细胞死亡方式.通常认为导致细胞凋亡的程序(即自杀程序)事先就装配在细胞中,如该程序在机体的内、外因素作用下被启动,细胞凋亡即开始发生.     

Arachidonic acid metabolism in articular chondrocytes

In this study rabbit articular chondrocytes were cultured and the cells were labeled with 3H-arachidonic acid and 14C-stearic acid. 3H incorporation reached a plateau at four hours and 14C-incorporation reached a plateau at 24 hours. The 3H was associated mainly with phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) at the 2-position while 14C was found at the 1-position. When the double-labeled cells were incubated with bradykinin or ionophore A23187, a significant release of 3H into the medium was observed, while the 14C release was small. Approximately 90% of the 3H released was arachidonic acid. Small amounts of the released 3H were no longer associated with stearic acid; it was converted mainly into prostaglandin E2 (PGE2). When stimulated by either bradykinin or ionophore, a significant 3H loss was observed in cellular PC while there were no significant 3H changes in other phospholipids, triacylglycerols (TGs), or diacylglycerols (DGs). Although 14C of cellular lysophosphatidylcholine (lyso-PC) was not increased significantly, the 3H seemed to be released from the 2-position of PC by the action of phospholipase A2. There was no significant change in the breakdown of PC between palmitoyl-arachidonyl (16:0/20:4) and stearoyl-arachidonyl (18:0/20:4) species. Both A23187 and bradykinin may activate phospholipase A2, releasing arachidonic acid equally from the 2-position of PCs having either palmitic acid or stearic acid at the 1-position. Some of this material is converted to PGE2, but this conversion is low compared to other cell types.     

关节软骨细胞库

本文介绍了关节软骨细胞的分离、冷冻保存和复苏技术，并在阐明胶原酶的细胞毒性作用的基础上，提出了分阶段消化法，以减少分离细胞的损伤，由于低温保护剂ＤＭＳＯ也具有细胞毒性，因而应尽量缩短ＤＭＳＯ与软骨细胞在４℃以上的接触时间。降温和复温速度是影响细胞存活的关键因素。降温速度从４℃～－８０℃以Ｉ℃／ｍｉｎ为佳，之后迅速投入－１９６℃的液氮中长期保存，细胞复苏应采用３７℃水浴快速复温法，虽然检测冻存细胞的存活率可采用苔盼蓝拒染试验，但最可靠的方法是细胞培养，利用关节软骨细胞库进行细胞培养、电镜观察和激光流式细胞计量分析，结果证明冻存软骨细胞复苏后仍具有正常的结构和形态并保持新陈代谢和自我复制功能。     

Tissue transglutaminase localization and activity regulation in the extracellular matrix of articular cartilage.

Tissue transglutaminase (tTG) catalyzes a Ca2+-dependent transglutaminase (TGase) activity which cross-links proteins and stabilizes many tissues [C.S. Greenberg et al. FASEB J. 5 (1991) 3071]. Because cartilage is subjected to great stress in vivo, an enzyme that strengthens and stabilizes tissue could play an integral role in maintaining cartilage integrity. The purpose of this study was to determine if active tTG is present in the extracellular matrix (ECM) of adult human osteoarthritic articular cartilage. Using a TGase activity assay along with immunolabeling for tTG of cartilage sections, TGase activity and tTG immunoreactivity were localized in the ECM in cartilage sections, predominantly in the superficial layer. Previous in vitro studies have demonstrated that the Mg-GTP complex inhibits the TGase activity of tTG [T.S. Lai et al. J. Biol. Chem. 273 (1998) 1776]. To investigate the in situ regulation of the TGase activity of tTG, a TGase activity assay was done with a dose response of GTP, measuring incorporation of fluorescein cadaverine. TGase activity was inhibited by GTP in a similar manner as in vitro. These results not only confirm tTG presence in the ECM. but also indicate tTG as the major TGase activity of the ECM. Secondly, the study provides a possible mechanism by which extracellular tTG is regulated in vivo.     

Biological freezing of human articular chondrocytes

AIM: To preserve viable, metabolically active chondrocytes cultured in alginate beads at -196 degrees C for further use in in vitro and in vivo studies. METHODS: Human articular chondrocytes were isolated from femoral condyles within 24 h post mortem. To optimize the biological freezing procedure, the chondrocytes were control-rate frozen in different concentrations of dimethyl sulfoxide (DMSO) in Dulbecco's MEM supplemented with 10% FCS before being thawed and the cell viability was determined by Trypan Blue exclusion test. To investigate the effect of control-rate freezing on chondrocyte metabolism, control-rate frozen chondrocytes in 5% DMSO were thawed and cultured in gelled agarose for 2 weeks. Non-frozen chondrocytes cultured in agarose served as controls. Furthermore, human articular chondrocytes were cultured in 2% alginate beads for 2 weeks after which the beads were incubated with 5% DMSO for 0 h, 2.5 h, 5 h and 10 h and frozen at -196 degrees C. Non-frozen alginate beads containing chondrocytes and incubated with 5% DMSO served as a control. After 2 weeks in culture, chondrocytes in agarose or in alginate were sulfated with 10 microCi(35)SO(4)/ml for 48 h. The total production of aggrecans, and the aggrecan subtypes, were subsequently determined. RESULTS: Five percent DMSO in the culture medium was the optimal condition to control-rate freeze and recover viable and functional isolated chondrocytes. Total aggrecan synthesis of control-rate frozen chondrocytes cultured in gelled agarose was not significantly reduced when compared with control cells. The proportion of aggrecan in the aggregate form of control-rate frozen chondrocytes kept in agarose remained unaltered. Chondrocytes, control-rate frozen in the alginate matrix, showed a 0-30% decrease in total aggrecan synthesis rates in culture when compared with the non-frozen chondrocytes. The optimal pre-incubation time of the alginate beads with 5% DMSO was 5 h, without any change in aggrecan synthesis rates when compared with the control situation. Shorter pre-incubation times resulted in an insufficient diffusion of DMSO into the beads and in cell death. There was no difference in the synthesis of the different aggrecan subtypes between frozen and non-frozen chondrocytes in alginate. CONCLUSION: Human articular chondrocytes can be stored at -196 degrees C for 24 h without important decreases in their aggrecan synthesis rates when control-rate frozen as a cell suspension in 5% DMSO. Proportions of the aggrecan subtypes (monomers, aggregates) synthesized by chondrocytes cultured in agarose remained unchanged. The control-rate freezing procedure in the alginate beads pre-incubated with 5% DMSO for 5 h produced no decrease in total aggrecan synthesis rates and no change in the synthesized aggrecan subtypes. Further experiments have to confirm the suitability of this freezing method for long-term storage of chondrocytes allowing us to set up a 'chondrocyte' bank for further use in in vitro and in vivo manipulations.     

Comparison of gene expression patterns in articular cartilage and dedifferentiated articular chondrocytes

During monolayer culture, articular chondrocytes dedifferentiate into fibroblast‐like cells. The mechanisms underlying this process are poorly understood. We sought to further characterize dedifferentiation by identifying an extended panel of genes that distinguish articular cartilage from dedifferentiated chondrocytes. Thirty‐nine candidate marker‐genes were identified from previous studies on articular‐cartilage gene‐expression. Real‐time PCR was used to evaluate the mRNA levels for these candidates in calf articular cartilage and dedifferentiated articular chondrocytes. Twenty‐two of the candidate marker genes exhibited at least a two‐fold difference in gene expression in the two cell types. Twelve of these genes had at least a ten‐fold difference in gene expression. Tenascin C (TNC), type I collagen (COL1A1), and hypoxia‐inducible factor 1 alpha (HIF1α) showed the highest relative expression levels in dedifferentiated chonodrocytes. Type II collagen (COL2A1), type XI collagen (COL11A2), and superficial zone protein (SZP) showed the highest relative expression levels in articular cartilage. In contrast to previous findings, fibromodulin mRNA, and protein levels were higher in dedifferentiated chondrocytes. Compared to smaller subsets of markers, this panel of 12 highly differentially expressed genes may more precisely distinguish articular cartilage from dedifferentiated chondrocytes. Since many of the genes up‐regulated in dedifferentiated chondrocytes are also expressed during cartilage development, dedifferentiated chondrocytes may possess features of cartilage precursor cells. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:234–245, 2012     

玻璃化冻存对兔关节软骨细胞存活率及代谢活性的影响

目的探索理想的关节软骨细胞低温保存方法。方法分别采用玻璃化冻存法、程序性降温法及梯度降温法对兔关节软骨细胞进行低温保存,通过流式细胞仪计数及阿尔新蓝染色等方法了解复苏后软骨细胞的存活率、凋亡率及生物活性。结果采用玻璃化冻存法保存的软骨细胞存活率为(86.57±1.67)%,明显高于传统的程序性降温法及梯度降温法;玻璃化冻存对软骨细胞合成糖胺多糖的能力有一定影响,但与新鲜未冷冻组相比,差异无统计学意义(P>0.05)。结论玻璃化冻存法较传统的保存方法能显著提高冷冻保存后兔关节软骨细胞的存活率,并能维持细胞的生物活性,是一种理想的软骨细胞低温保存方法。     

Xenografts of articular chondrocytes in the nude mouse

Subcutaneous transplantation of articular chondrocytes isolated enzymatically from immature rabbits and dogs into athymic (nu/nu) mice resulted in the formation of hyaline cartilaginous nodules. Graft rejection was seen when the cells were injected into heterozygous (nu/+) mice. Radiosulfate-labeled proteoglycan extracted from the xenografts had a high buoyant density and was digested by chondroitinase ABC. A monomeric preparation of proteoglycan (A1-D1) contained a small quantity of aggregate as assessed by gel chromatography and gel electrophoresis. Almost no incorporation of 3H-thymidine was found by autoradiography. The matrix did not become calcified over the course of 42 days. The nude mouse system lends itself to testing a variety of problems in the biology of cartilage. These include the redifferentiation of chondrocytes following dedifferentiation in vitro. Species differences were found in this regard. The nodules formed by rabbit articular chondrocytes, grown in monolayer culture for up to 14 days, had a hyaline chondroid character. Dog chondrocytes that had "dedifferentiated" during 14 days of culture prior to transplantation, formed a graft that had a sparse fibrous rather than hyaline matrix.     

Xenografts of articular chondrocytes in the nude mouse

Summary Subcutaneous transplantation of articular chondrocytes isolated enzymatically from immature rabbits and dogs into athymic (nu/nu) mice resulted in the formation of hyaline cartilaginous nodules. Graft rejection was seen when the cells were injected into heterozygous (nu/+) mice. Radiosulfate-labeled proteoglycan extracted from the xenografts had a high buoyant density and was digested by chondroitinase ABC. A monomeric preparation of proteoglycan (A1-D1) contained a small quantity of aggregate as assessed by gel chromatography and gel electrophoresis. Almost no incorporation of³H-thymidine was found by autoradiography. The matrix did not become calcified over the course of 42 days. The nude mouse system lends itself to testing a variety of problems in the biology of cartilage. These include the redifferentiation of chondrocytes following dedifferentiation in vitro. Species differences were found in this regard. The nodules formed by rabbit articular chondrocytes, grown in monolayer culture for up to 14 days, had a hyaline chondroid character. Dog chondrocytes that had “dedifferentiated,” during 14 days of culture prior to transplantation, formed a graft that had a sparse fibrous rather than hyaline matrix.     

Studies on cryopreservation of articular cartilage chondrocytes

We used cartilage cells isolated from bovine articular cartilage in experiments to: (1) determine the toxicity of cryopreservatives (glycerol and dimethyl sulphoxide) on chondrocytes, (2) evaluate methods of freezing chondrocytes to maximize viability after freezing, and (3) examine the biosynthetic activity of frozen and thawed chondrocytes in culture. Results showed that the toxicity of cryopreservatives to chondrocytes is dependent on the time and temperature of exposure as well as on the concentration of the cryopreservative. Maximum viability was obtained by a two-stage freezing procedure using a slow cooling period initially, with equilibration of the cells at -40 degrees Celsius before further rapid freezing to -80 degrees Celsius. After seventy-two hours in culture, chondrocytes that had been frozen using this protocol synthesized products that appeared by column chromatography to form proteoglycan aggregates. Clinical Relevance: One of the reasons for failure of frozen osteochondral allografts is the deterioration of joint function after transplantation due to degeneration of the articular cartilage. An important factor in the survival of these cartilage grafts may be preservation of the viability of the chondrocytes during storage and maintenance of the cell's ability to function following storage. In this study we evaluated the ability to store chondrocytes in a frozen state with the aid of cryopreservatives. The results confirmed that chondrocytes will survive freezing and remain capable of functioning in the same manner as fresh chondrocytes. This suggests that chondrocytes in articular cartilage should be able to survive freezing. The pursuit of methods of preserving articular cartilage by freezing appears to be warranted.