bFGF对人关节软骨细胞增殖和凋亡的影响

目的：观察碱性成纤维细胞生长因子(bFGF)对体外单层培养的人负重关节软骨细胞的增殖和凋亡的影响。方法：对8例来自人负重关节软骨细胞系进行体外单层传代培养，从第2代起试验组培养液中加入bFGF，观察细胞的倍增时间(DT)、流式细胞分析细胞增殖周期和凋亡指数，并与无bFGF组相比较。结果：与未加bFGF相比较，加用10μg／ml bFGF后，DT缩短，S期细胞数升高，细胞凋亡无变化。结论：bFGF能促进培养的人关节软骨细胞生长，不引起细胞凋亡。     

线粒体DNA突变与骨关节病

线粒体DNA(m tDNA)是唯一的核外遗传物质,由于其独特的结构特征,m tDNA比nDNA更容易损伤。活性氧(reactive oxygen spec ies,ROS)是有氧代谢的产物,可通过多种机制损伤m tDNA,m tDNA突变又可导致ROS过度产生,二者形成恶性循环。骨关节病与m tDNA突变有关,m tDNA突变可导致ROS过度产生、关节软骨细胞过度凋亡,以及关节软骨细胞老化、氧化及炎症应激和滑膜的氧化损伤。     

关节软骨损伤的治疗（上）

关节软骨属于透明软骨，自身没有血供，无淋巴引流，无神经分布，软骨细胞是高度分化的组织细胞，成熟的关节软骨中软骨细胞的分裂能力非常有限，因此软骨损伤后修复能力差。早在1743年Hunter就提出“骨软骨一旦破坏，就不能得到修复”的观点，而关节软骨损伤及其修复问题，长期以来也一直是困扰骨科基础研究与临床治疗的棘手问题。随着社会的现代化进程和人口的老龄化，由于外伤和退变引起的关节软骨损伤患者显著增多，为此如何解决关节软骨修复的问题日益显得重要。     

硒和富里酸对软骨细胞的生物效应

本研究应用人胚软骨细胞培养技术,实验观察了硒和富里酸(FA)对软骨细胞生长、谷胱甘肽过氧化酶(GSH-Px)活力和粘多糖合成功能的影响,并探讨了富里酸作用与过氧化损伤的可能联系,以期为某些软骨性疾病的病因学和发病学研究提供新的途径和线索。     

结缔组织生长因子与关节软骨的修复

背景：结缔组织生长因子有刺激间充质细胞向软骨细胞分化的潜能,能促进软骨细胞的增殖和分化,可促进关节软骨细胞Ⅱ型胶原及蛋白多糖的表达,与其他生长因子一起在关节软骨修复的过程中发挥着重要作用。目的：重点就结缔组织生长因子的结构,在关节软骨修复中的功能,与其他物质的相互作用方面作一综述。方法：以“connective tissue growth factor,connective tissue growth factor and articular cartilage,articular cartilage damage,articular cartilage repairment”为英文检索词,以“关节软骨损伤”为中文检索词,检索PubMed数据库、中国知网-cnki数据库1980年1月至2014年7月有关关节软骨损伤修复的文献,排除与软骨损伤的修复重建相关性不强、以及内容重复、陈旧的文献。共保留32篇文献进行综述。结果与结论：结缔组织生长因子有刺激间充质细胞向软骨细胞分化的潜能,能促进软骨细胞的增殖、分化和成熟,可维持胞外基质合成以及平衡,可促进关节软骨细胞Ⅱ型胶原及蛋白多糖的表达,与其他生长因子一起在关节软骨修复的过程中发挥着重要作用。结缔组织生长因子是软骨细胞生长、增殖、分化的关键生长因子之一,贯穿软骨修复整个过程。研究发现,骨性关节炎患者的关节软骨细胞对成纤维细胞生长因子1和结缔组织生长因子表达呈现明显相关性增加。结缔组织生长因子通过对关节软骨组织的信号通路及与其他组织内的细胞因子彼此作用对关节软骨细胞及基质发挥作用。 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;组织工程全文链接：     

关节软骨损伤后体外培养软骨细胞的功能变化

背景：关节软骨损伤可以影响软骨细胞功能,诱发创伤性骨关节炎。 目的：观察关节软骨损伤后体外培养的软骨细胞功能的变化。 方法：通过酶消化法分离培养高能量、低能量撞击后和正常兔膝关节透明软骨细胞,观察创伤能量对软骨细胞生存能力的影响;检测软骨细胞合成蛋白多糖和Ⅱ型胶原能力,检测细胞中白细胞介素1β和核转录因子κB mRNA表达水平,检测细胞合成白细胞介素1β和基质金属蛋白酶1的表达。 结果与结论：高能量和低能量关节软骨损伤后,软骨细胞的存活率下降,原代细胞的贴壁细胞数量减少,贴壁时间延长,生长曲线下移,细胞甲苯胺蓝染色异染反应减弱,Ⅱ型胶原免疫组化染色强度减弱,软骨细胞中白细胞介素1β和核转录因子κB mRNA表达水平上升,细胞培养液中白细胞介素1β和基质金属蛋白酶1的质量浓度升高,其中高能量组效果更为显著(P < 0.05)。说明关节软骨损伤后软骨细胞的功能受到影响,受损程度与创伤强度及炎性细胞因子的表达相关。     

英科学家从成人骨骼中找到软骨干细胞

英国卡迪夫大学科学家宣布,他们在成人关节的软骨组织中找到一种特定的干细胞,通过实验室培育能够产生足够多的软骨细胞,这一发现为关节炎患者和软骨损伤患者带来了新的治疗希望。目前,对于年轻的软骨损伤病人,通常的治疗方法是,用受损软骨周围的健康软骨培育新的软骨细胞,再将     

胎儿关节和兔关节软骨细胞的体外培养

本文报告了人胎儿关节软骨细胞和兔关节软骨细胞的体外培养,结果表明：胎儿关节软骨细胞和兔关节软骨细胞在胰蛋白酶和胶原酶作用下,在无ＣＯ２培养条件下,获得了大量的软骨细胞,且能生长分裂,复制。为将此法用于生物材料的人工软骨研究奠定了基础。     

双极射频能量处理时间可影响软骨细胞活性和软骨表面轮廓

背景：射频能量已用于处理关节软骨损伤,但其安全性受到质疑。 目的：在体外模拟关节镜条件下使用双极射频能量处理软骨损伤,分别通过葡萄胺聚糖检测和扫描电镜观察评估软骨细胞的活力和表面轮廓。 方法：无菌条件下,应用双极射频能量分别处理新鲜牛膝关节软骨10,20,30,40和50 s。处理后取下全层关节软骨,每个标本分为2部分,一部分通过检测葡萄胺聚糖释出率评估软骨细胞的活性,另一部分通过扫描电镜评估软骨的表面轮廓。 结果与结论：双极射频能量对软骨细胞造成时间相关的有害效应,软骨细胞的活性与处理时间呈负相关。表面轮廓的光滑度与处理时间呈正相关,处理时间大于20 s才可充分光滑软骨表面,达到扫描电镜评分的2分。提示应用双极射频能量处理关节软骨损伤时,软骨细胞的活性和表面轮廓的光滑度与处理时间相关,应谨慎使用射频能量处理关节软骨损伤直至其长期效应得到评估。     

钼对培养心肌细胞的双向性影响

黄嘌呤-黄嘌呤氧化酶系统(X-XOD)可诱发培养心肌细胞的自由基损伤。鉴于钼是黄嘌呤氧化酶(XOD)的重要组分,而且钼对于心肌细胞有保护作用还是有损伤作用的报道不尽一致,本实验拟以心肌细胞动作电位与自发性搏动为指标,探讨钼对心肌细胞机能影响的规律。 实验方法取新生昆明种小鼠与Wistar大鼠的乳鼠;胎龄4～6个月,水囊引产的人胚心尖,分别经0.1％胰蛋白酶分离心肌细胞,然后将其置入聚苯乙烯培养瓶,在36.5℃、5％CO_2和95％空气的孵箱内培养。常规培养基由80％199及20％小牛血清组成。小鼠心肌细     

Inflammatory synovial fluid microenvironment drives primary human chondrocytes to actively take part in inflammatory joint diseases

The role of human chondrocytes in the pathogenesis of cartilage degradation in rheumatic joint diseases has presently gained increasing interest. An active chondrocyte participation in local inflammation may play a role in the initiation and progression of inflammatory joint diseases and in a disruption of cartilage repair mechanisms resulting in cartilage degradation. In the present study, we hypothesized that inflammatory synovial fluid triggers human chondrocytes to actively take part in inflammatory processes in rheumatic joint diseases. Primary human chondrocytes were incubated in synovial fluids gained from patients with rheumatoid arthritis, psoriasis arthritis and reactive arthritis. The detection of vital cell numbers was determined by using Casy Cell Counter System. Apoptosis was measured by Annexin-V and 7AAD staining. Cytokine and chemokine secretion was determined by a multiplex suspension array. Detection of vital cells showed a highly significant decrease in chondrocyte numbers. Flow cytometry demonstrated a significant increase in apoptotic chondrocytes after the incubation. An active secretion of cytokines such as MCP-1 and MIF by chondrocytes was observed. The inflammatory synovial fluid microenvironment mediates apoptosis and cell death of chondrocytes. Moreover, in terms of cytokine secretion, it also induces an active participation of chondrocytes in ongoing inflammation.     

Oxygen-derived free radicals in postischemic tissue injury

It is now clear that oxygen-derived free radicals play an important part in several models of experimentally induced reperfusion injury. Although there are certainly multiple components to clinical ischemic and reperfusion injury, it appears likely that free-radical production may make a major contribution at certain stages in the progression of the injury. The primary source of superoxide in reperfused reoxygenated tissues appears to be the enzyme xanthine oxidase, released during ischemia by a calcium-triggered proteolytic attack on xanthine dehydrogenase. Reperfused tissues are protected in a variety of laboratory models by scavengers of superoxide radicals or hydroxyl radicals or by allopurinol or other inhibitors of xanthine oxidase. Dysfunction induced by free radicals may thus be a major component of ischemic diseases of the heart, bowel, liver, kidney, and brain.     

Pannocytes: distinctive cells found in rheumatoid arthritis articular cartilage erosions.

A distinctive cell was identified from sites of rheumatoid arthritis cartilage injury. Similar cells are not found in lesions of osteoarthritis cartilage. We have designated them as pannocytes (PCs). Their rhomboid morphology differs from the bipolar shape of fibroblast-like synoviocytes or the spherical configuration of primary human articular chondrocytes. Chondrocytes are short-lived, whereas the original PC line grew for 25 passages before becoming senescent. Features in common with cultured primary chondrocytes include maximal proliferation in response to transforming growth factor-beta a catabolic response to interleukin-1 beta, collagenase production, and mRNA for the induced lymphocyte antigen and inducible nitric oxide synthase. Despite the presence of the inducible nitric oxide synthase message, PCs do not produce NO either constitutively or when cytokine stimulated. Each of the mesenchymal cells, fibroblast-like synoviocytes, primary chondrocytes, and PCs have the gene for type I collagen, but the type II collagen gene is detected only in primary chondrocytes. PCs can be distinguished from fibroblast-like synoviocytes and primary chondrocytes by their morphology, bright VCAM-1 staining, and growth response to cytokines and growth factors. Their prolonged life span in vitro suggests that PCs might represent an earlier stage of mesenchymal cell differentiation, and they could have a heretofore unrecognized role in rheumatoid arthritis joint destruction.     

Reaction of antibody to mycobacterial 65 kDa heat-shock protein with human chondrocytes.

We report on the reactivities of four monoclonal antibodies generated against mycobacterial proteins to human chondrocytes, cells in cartilage which may be subject to immune attack in rheumatoid arthritis. Only one of the monoclonal antibodies, ML30, which had been shown previously to react with a human homologue to heat-shock protein (hsp), reacted strongly to chondrocytes. By immunocytochemical methods using fixed chondrocytes, ML30 reacted to cytoplasmic constituents in a granular pattern. There were no marked qualitative differences in the staining intensities and patterns of chondrocytes kept at ambient temperatures and those subjected to 42 degrees C heat treatment. No significant staining was observed with normal peripheral blood mononuclear cells. By indirect immunofluorescence, the distribution of ML30 reactive constituents was very low on the cell surface. Reactivities of each of the monoclonal antibodies were tested on frozen sections of cartilage. Significant reactivity was found only with ML30, and the staining was only associated with chondrocytes, not with the cartilage matrix surrounding the cells. These findings may have significance in view of the possibility that an hsp homologue may be a target for inciting or perpetuating the autoimmune process in rheumatoid arthritis.     

Ischemia-induced vascular changes: role of xanthine oxidase and hydroxyl radicals

The results of previous studies indicate that oxygen-derived free radicals are responsible for the increased vascular permeability produced by 1 h of intestinal ischemia. The aims of this study were 1) to test the hypothesis that the enzyme xanthine oxidase is the source of oxygen radicals in the ischemic bowel and 2) to assess the role of the hydroxyl radical in the ischemia-induced vascular injury. The capillary osmotic reflection coefficient was estimated from lymphatic protein flux data in the cat ileum for the following conditions: ischemia, ischemia plus pretreatment with allopurinol (a xanthine oxidase inhibitor), and ischemia plus pretreatment with dimethyl sulfoxide (a hydroxyl radical scavenger). The increased vascular permeability produced by ischemia was largely prevented by pretreatment with either allopurinol or dimethyl sulfoxide. These findings support the hypothesis that xanthine oxidase is the source of oxygen radicals produced during ischemia. The results also indicate that hydroxyl radicals, derived from the superoxide anion, are primarily responsible for the vascular injury associated with intestinal ischemia.     

Distribution of chondrocytes containing alpha-smooth muscle actin in human normal, osteoarthrotic, and transplanted articular cartilage

The aim of our study was to evaluate the occurrence of chondrocytes containing alpha-smooth muscle actin in human normal and diseased cartilage. Immunohistochemistry using monoclonal antibodies for alpha-smooth actin, muscle-specific actin, S-100 protein, CD 34, and desmin was performed on samples of human articular cartilage obtained at autopsy following sudden death, during total hip and knee replacement for osteoarthritis, or after femoral neck fracture in patients without symptoms of osteoarthritis. Moreover, the layers of residual cartilage from chondral posttraumatic defects obtained during preoperative arthroscopy and of newly formed cartilage after autologous-chondrocyte transplantation (Hyalograft C) obtained during second-look arthroscopy were also examined by immunohistochemistry and RT PCR. Our study showed that a significant percentage of articular chondrocytes express alpha-smooth muscle actin in healthy, diseased, and regenerated articular cartilage. Alpha-actin positive chondrocytes (18%) were observed predominantly in the upper zone of normal articular cartilage. By contrast, only approximately 10% of cartilage cells in the deep region stained for this contractile actin isoform. Actin-positive chondrocytes (myochondrocytes) are formed predominantly in response to injury to the osteoarthrotic cartilage, at sites of defective healing, and in newly formed cartilage after autologous chondrocyte transplantation. Fibrocartilage is present in some of these conditions, and it is known that this tissue contains chondrocytes with actin. The presence of myochondrocytes in the surface layer of normal articular cartilage indicates that this region probably plays an important role in maintaining cartilage integrity. Myochondrocytes may utilize the contractile actin isoform in manipulating the extracellular matrix of articular cartilage. It is also possible that actin-containing chondrocytes have a higher potential for regeneration in contrast to chondrocytes that do not contain this contractile material in their cytoplasm.     

p38MAPK mediates IL-1-induced down-regulation of aggrecan gene expression in human chondrocytes

The pleiotropic cytokine interleukin 1 (IL-1) is considered to be the principal inducer of mediators of cartilage degradation in both, osteoarthritis (OA) and rheumatoid arthritis (RA). IL-1 activates numerous signaling pathways involved in cartilage destruction and dedifferentiation of chondrocytes. In this study, we analyzed expression and functional effects of IL-1 in human chondrocytes. We found an IL-1-induced reduction in the expression of the cartilage specific proteoglycan aggrecan as an indicator for the IL-1-mediated dedifferentiation of chondrocytes. To block the IL-1-induced signaling pathways specifically, we incubated human chondrocytes and cartilage explants with IL-1 in the presence of different signal transduction inhibitors and analyzed their effect on aggrecan mRNA expression and IL-6 secretion. IL-6 has been found to act synergistically in the IL-1-induced suppression of the proteoglycan synthesis in chondrocytes. Our results led to the identification of p38MAPK and/or PI3K/JNK as being crucial for IL-1-induced IL-6 secretion by chondrocytes. IL-1-induced down-regulation of aggrecan expression was found to be mediated by p38MAPK and/or ERK1/2. The identification and characterization of these signaling pathways will enable us to develop new modulation strategies for therapeutic use in inflammatory joint diseases.     

Hedgehog信号通路阻断剂环巴胺体外对佐剂性关节炎大鼠的关节软骨细胞增殖的影响

目的探讨Hedgehog(Hh)信号通路抑制剂环巴胺体外对佐剂性关节炎大鼠(AA)模型的关节软骨细胞增殖的影响和部分机制。方法弗氏完全佐剂诱导AA大鼠,测量关节炎指数和继发性足肿胀度,HE染色观察两组软骨组织生长情况;取AA大鼠踝关节软骨组织,采用胰蛋白酶-胶原酶法分离、培养、鉴定,环巴胺(0、0.05、0.5、5、20μmol/L)体外给药,MTT法检测AA大鼠踝关节软骨细胞增殖,Annexin V-FITC/PI双染检测AA大鼠踝关节软骨细胞凋亡,Western blotting检测AA大鼠踝关节软骨细胞Shh、Ptch1、Gli1的蛋白表达。结果弗氏完全佐剂诱导后,与正常大鼠相比,AA大鼠关节炎指数和继发性足肿胀度明显升高,HE染色显示,AA大鼠踝关节软骨组织有破坏;甲苯胺蓝和Ⅱ型胶原鉴定体外成功培养AA大鼠踝关节软骨细胞;体外给药环巴胺(0.05、0.5、5、20μmol/L)可升高AA模型关节软骨细胞增殖,流式细胞检测结果显示,环巴胺能降低AA模型软骨细胞凋亡率;与未用环巴胺组相比,环巴胺(0.5、5、20μmol/L)给药对AA软骨细胞中Hh信号通路相关蛋白(Shh、Ptch1、Gli1)表达显著下降。结论弗氏完全佐剂诱导建立AA大鼠模型成立,体外给药环巴胺可抑制AA大鼠软骨细胞的增殖,抑制软骨细胞的凋亡,该作用与抑制AA大鼠软骨细胞Hh信号有关。     

Tissue-specific gene expression in chondrocytes grown on three-dimensional hyaluronic acid scaffolds

The re-differentiation capacities of human articular and chick embryo sternal chondrocytes were evaluated by culture on HYAFF-11 and its sulphate derivative, HYAFF-11-S, polymers derived from the benzyl esterification of hyaluronate. Initial results showed that the HYAFF-11-S material promoted the highest rate of chondrocyte proliferation. RNA isolated from human and chick embryo chondrocytes cultured in Petri dishes, HYAFF-11 or HYAFF-11-S were subjected to semi-quantitative RT-PCR analyses. Human collagen types I, II, X, human Sox9 and aggrecan, chick collagen types I, II, IX and X were analysed. Results showed that human collagen type II mRNA expression was upregulated on HYAFF-11 biomaterials. In particular, a high level of collagen type IIB expression was associated with three-dimensional culture conditions, and the HYAFF-11 material was the most supportive for human collagen type X mRNA expression. Human Sox9 mRNA levels were constantly maintained in monolayer cell culture conditions over a period of 21 days, while these were upregulated when chondrocytes were cultured on HYAFF-11 and HYAFF-11S. Furthermore, chick collagen type IIA and IIB mRNA expression was detected after only 7 days of HYAFF-11 culture. Chick collagen type IX mRNA expression decreased in scaffold cultures over time. Histochemical staining performed in engineered cartilage revealed the presence of a de novo synthesized glycosaminoglycan-rich extracellular matrix; immunohistochemistry confirmed the deposition of collagen type II. This study showed that the three-dimensional HYAFF-11 culture system is both an effective chondrocyte delivery system for the treatment of articular cartilage defects, and an excellent in vitro model for studying cartilage differentiation.