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。     

自噬与软骨细胞生存及软骨损伤

背景：自噬是细胞通过溶酶体途径处理内源性底物的过程,它普遍存在于机体细胞中,又被看作是细胞Ⅱ型程序性死亡,自噬可能是正常软骨细胞的一种保护或平衡机制。 目的：就自噬与软骨及其损伤相关性的最新研究进展进行讨论,旨在对自噬在软骨及其损伤修复中的作用有一个更好的理解。 方法：应用计算机检索中国知网、万方数据库及PubMed数据库最近20年有关自噬与软骨损伤方面的文献,中文检索词为“自噬、软骨、软骨细胞”,英文检索词为“autophagy、cartilage、chondrocytes、beclin1、LC3”。 结果与结论：软骨细胞能感受关节内微环境变化而做出应答,以调整细胞基质代谢,维持关节软骨生物学功能,而软骨细胞所处低氧环境是引起细胞自噬的重要因素。自噬是正常软骨细胞的一种平衡或者保护机制,虽然自噬与软骨及其损伤相关性在近些年的研究中取得了长足的进步,但不得不承认其仍处于初级阶段,在分子水平上一些Atg的发现加深了对自噬的认识,但在软骨中如何诱导自噬途径,自噬信号是如何传导的,对软骨细胞生存会产生怎样的影响等方面的了解还不够丰富,有待广大学者进一步研究。     

Chondrogenic Differentiation of Amniotic Fluid Stem Cells and Their Potential for Regenerative Therapy

Chronic articular cartilage defects are the most common disabling conditions of humans in the western world. The incidence for cartilage defects is increasing with age and the most prominent risk factors are overweight and sports associated overloading. Damage of articular cartilage frequently leads to osteoarthritis due to the aneural and avascular nature of articular cartilage, which impairs regeneration and repair. Hence, patients affected by cartilage defects will benefit from a cell-based transplantation strategy. Autologous chondrocytes, mesenchymal stem cells and embryonic stem cells are suitable donor cells for regeneration approaches and most recently the discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options. It is the aim of this review to summarize recent advances in the use of amniotic fluid stem cells as novel cell sources for the treatment of articular cartilage defects. Molecular aspects of articular cartilage formation as well as degeneration are summarized and the role of growth factor triggered signaling pathways, scaffolds, hypoxia and autophagy during the process of chondrogenic differentiation are discussed.     

Morphological Study: Ultrastructural Aspects of Articular Cartilage and Subchondral Bone in Patients Affected by Post‐Traumatic Shoulder Instability

Post‐traumatic shoulder instability is a frequent condition in active population, representing one of most disabling pathologies, due to altered balance involving joints. No data are so far available on early ultrastructural osteo‐chondral damages, associated with the onset of invalidating pathologies, like osteoarthritis‐OA. Biopsies of glenoid articular cartilage and sub‐chondral bone were taken from 10 adult patients underwent arthroscopic stabilization. Observations were performed under Transmission Electron Microscopy‐TEM in tangential, arcuate and radial layers of the articular cartilage and in the sub‐chondral bone. In tangential and arcuate layers chondrocytes display normal and very well preserved ultrastructure, probably due to the synovial liquid supply; otherwise, throughout the radial layer (un‐calcified and calcified) chondrocytes show various degrees of degeneration; occasionally, in the radial layer evidences of apoptosis/autophagy were also observed. Concerning sub‐chondral bone, osteocytes next to the calcified cartilage also show signs of degeneration, while osteocytes farther from the osteo‐chondral border display normal ultrastructure, probably due to the bone vascular supply. The ultrastructural features of the osteo‐chondral complex are not age‐dependent. This study represents the first complete ultrastructural investigation of the articular osteo‐chondral complex in shoulder instability, evaluating the state of preservation/viability of both chondrocytes and osteocytes throughout the successive layers of articular cartilage and sub‐chondral bone. Preliminary observations here collected represent the morphological basis for further deepening of pathogenesis related to shoulder instability, enhancing the relationship between cell shape and microenvironment; in particular, they could be useful in understanding if the early surgical treatment in shoulder instability could avoid the onset of OA. Anat Rec, 300:1208–1218, 2017. © 2016 Wiley Periodicals, Inc.     

骨关节炎：关节软骨退变的相关研究与进展

背景：骨关节炎关节软骨退变被认为是不可逆的病理改变,但其病因及发病机制不清。 目的：总结并探讨骨关节炎的病因、发病机制及相关的生物标志物的最新基础研究进展。 方法：由第一作者用计算机检索中国期刊全文数据库(CNKI：2000/2010)和Medline数据库(2000/2010),检索词分别为“骨关节炎,关节软骨,退行性变,基础研究,生物标志物”和“Osteoarthritis,Articular cartilage,degradation,basic research,Biological markers”,语言分别设定为中文和英文。从引起骨关节炎发病的相关因素的作用及相关的生物标志物的变化2方面进行总结,对病理、生物化学和免疫等相关因素及生物标志物的研究现状进行介绍。 结果与结论：共检索到60篇文章,按纳入和排除标准对文献进行筛选,共纳入20篇文章。结果发现,关节软骨退行性变过程中可出现Ⅱ型原胶原羧基端前肽及Ⅱ型胶原羧基端肽等细胞因子的升高,基质金属蛋白酶、生物自由基、一氧化氮及细胞和体液免疫等参与了骨关节炎退行性变的过程,但其具体发病机制目前仍不明确。     

正常关节软骨和骨关节炎关节软骨细胞中血管活性肠肽表达的比较

背景：神经肽的发现给骨关节炎的治疗带来了新的希望,但神经肽的表达与骨关节炎发病以及软骨退变程度的关系尚不清楚。 目的：观察血管活性肠肽在正常关节软骨和不同退变程度骨关节炎软骨中的表达,以及血管活性肠肽表达与骨关节炎发病及软骨退变程度的关系。 方法：选取2007-03/11中南大学湘雅医院骨科进行关节置换的骨关节炎患者的关节软骨标本26个,选取因外伤行截肢的膝关节软骨或股骨颈暴力骨折的股骨头正常关节软骨标本10个为对照,根据大体观察凿取正常和骨关节炎不同退变程度软骨块50个,再根据关节软骨改良Mankin病理评分法进行分组,采用免疫组织化学染色检测软骨组织中血管活性肠肽的表达和分布。 结果与结论：各关节软骨中均可见到血管活性肠肽阳性神经纤维,正常关节软骨中血管活性肠肽的表达明显高于骨关节炎关节软骨(P < 0.05)。且血管活性肠肽表达与软骨改良Mankin病理评分呈负相关(r=-0.896,P < 0.05)。说明血管活性肠肽低表达与关节软骨退变程度、骨关节炎病程进展有关,可能是关节软骨退变、骨关节炎发病的机制之一。     

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.     

骨性关节炎患者退变软骨及滑膜组织中细胞因子的表达

背景：骨性关节炎的炎症反应是由软骨细胞、滑膜组织分泌的细胞因子所介导的。关节软骨和滑膜组织内含有多种细胞因子,在关节软骨的损伤修复中起着重要的调节作用。 目的：分析软骨细胞、滑膜组织分泌的细胞因子与骨性关节炎发病的关系及影响。 方法：由第一作者应用计算机检索万方数据库(www.wanfangdata.com.cn),PubMed数据库(www.ncbi.nlm.nih.guv/pubmed)检索时间：2005至2010年。检索词为“骨性关节炎,退变,软骨组织,细胞因子”。计算机初检得到146篇文献,阅读标题和摘要进行初筛,排除因研究目的与此文无关的86篇,内容重复性的研究40篇,保留21篇骨性关节炎患者退变软骨及滑膜组织中各种细胞因子作用及影响的相关文献作进一步分析。 结果与结论：细胞因子主要是指活化的免疫细胞和某些基质细胞分泌的一类非特异调节免疫应答和介导炎症反应的小分子蛋白质,包括由淋巴细胞产生的淋巴因子,单核巨噬细胞产生的单核因子及其他细胞因子等。关节滑膜细胞分泌的细胞因子可部分解释骨性关节炎的病理过程,在炎症关节中起着重要的作用。虽然越来越多的学者重视到滑膜细胞、软骨细胞分泌细胞因子的作用,但主要是研究外源性细胞因子对软骨细胞或滑膜细胞的影响,而其内源性细胞因子在骨性关节炎发病中的作用却未广泛开展研究。     

Cell sources for the regeneration of articular cartilage: the past,the horizon and the future

Avascular, aneural articular cartilage has a low capacity for self‐repair and as a consequence is highly susceptible to degradative diseases such as osteoarthritis. Thus the development of cell‐based therapies that repair focal defects in otherwise healthy articular cartilage is an important research target, aiming both to delay the onset of degradative diseases and to decrease the need for joint replacement surgery. This review will discuss the cell sources which are currently being investigated for the generation of chondrogenic cells. Autologous chondrocyte implantation using chondrocytes expanded ex vivo was the first chondrogenic cellular therapy to be used clinically. However, limitations in expansion potential have led to the investigation of adult mesenchymal stem cells as an alternative cell source and these therapies are beginning to enter clinical trials. The chondrogenic potential of human embryonic stem cells will also be discussed as a developmentally relevant cell source, which has the potential to generate chondrocytes with phenotype closer to that of articular cartilage. The clinical application of these chondrogenic cells is much further away as protocols and tissue engineering strategies require additional optimization. The efficacy of these cell types in the regeneration of articular cartilage tissue that is capable of withstanding biomechanical loading will be evaluated according to the developing regulatory framework to determine the most appropriate cellular therapy for adoption across an expanding patient population.     

Osteoarthritis in the context of ageing and evolution. Loss of chondrocyte differentiation block during ageing

Ageing is the main risk factor of primary osteoarthritis (OA) and OA is the disease most strongly correlated with ageing. Both in humans and other animals OA development appears to be not strictly time-dependent but to hold pace with ageing processes. A characteristic of OA is deviant behaviour of chondrocytes in articular cartilage. These chondrocytes resemble terminal differentiated chondrocytes in growth plates and actively produce matrix degrading enzymes. The latter results in cartilage degeneration and eventually OA. We postulate that at a young age progression of chondrocyte differentiation is actively blocked in articular cartilage. This block declines when the evolutionary pressure to maintain this block, after reproductive life, is minimized. The loss of this differentiation block, maybe as a result of changes in chondrocyte TGF beta signalling, results in combination with normal joint loading in cartilage degeneration and OA.     

Immunohistochemical detection of estrogen receptor α in articular chondrocytes from cows, pigs and humans: in situ and in vitro results

Clinical observations suggest that estrogens are involved in the pathogenesis of postmenopausal osteoarthritis, but only little is known about the influence of these hormones on articular cartilage cells. The effect of estradiol is mediated by estrogen receptors α and β. The goal of the present study was to search for estrogen receptor α in articular tissue from cows, pigs and humans by immunohistochemistry to form a basis for in vitro studies. In addition, we also tried to detect estrogen receptor α in cultivated articular chondrocytes from cows and bulls under certain culture conditions. Estrogen receptor α is detected by the use of antibody 13H2 in articular chondrocytes from cows, bulls, pigs and humans. Chondrocytes are physiologically exposed to reduced oxygen tension. In isolated articular chondrocytes from cows and bulls incubated either with 21% O₂ or with 5% O₂ positive cells were also found. These positive results therefore encourage testing the influence of estradiol on cultivated articular cartilage cells in these species under different culture conditions.     

Passive role of articular chondrocytes in quinolone-induced arthropathy in guinea pigs

The role of articular chondrocytes and matrix degrading enzymes such as collagenase and neutral protease in the pathogenesis of quinolone-induced cartilage degeneration was investigated in immature guinea pigs. Articular cartilage from nalidixic acid (NA) treated guinea pigs was examined for the presence of protease activity or the ex vivo synthesis of collagenase at various times post-treatment. Histologic evaluation of knee joints confirmed the presence of degenerative changes in the matrix, but increased collagenase synthesis or protease activity were not detected. A separate group of animals was used to determine the importance of articular chondrocytes in the lesion generation. These cells were killed by intra-articular injection of the glycolysis inhibitor iodoacetic acid (IA) prior to treatment of the animals with NA. Typical "blister-like" their presence was not required for lesion development. Cartilage exposed to IA only did not exhibit "blister-like" lesions indicating that chondrocyte death and proteoglycan loss in conjunction with frictional forces associated with load-bearing were not sufficient to induce major matrical disruptive changes during the period of this study. These results indicate that articular chondrocytes are not actively involved in inducing the degenerative changes and provide no evidence for involvement of collagenase or neutral protease in the pathogenesis of the lesion.     

丝裂原活化蛋白激酶信号通路对骨关节炎软骨的作用

背景：骨关节炎的主要病理过程是软骨损伤,而软骨细胞间信号转导的异常是软骨损伤的重要因素。 目的：综合分析丝裂原活化蛋白激酶信号通路的最新进展,进一步分析丝裂原活化蛋白激酶信号转导通路在骨关节炎软骨中的作用。 方法：由第一作者用计算机检索中国期刊全文数据库(CNKI：1990/2011)和Pubmed数据库(1990/2011),检索词分别为骨性关节炎,关节软骨,ERK,JNK,P38,MAPK signaling palhway,osteoarthritis, chondrocytes,语言分别设定为中文和英文。纳入有关MAPKs信号通路及其相关蛋白激酶对骨关节炎软骨作用的研究,排除重复性研究。 结果与结论：保留32篇文献进一步分析。结果发现,丝裂原活化蛋白激酶信号转导通路在细胞内具有生物进化的高度保守性,通过保守的三级酶促级联反应激活转录因子,调节特定的基因表达。目前已证实,丝裂原活化蛋白激酶信号参与并调控关节软骨中软骨细胞的增殖、分化、凋亡,而丝裂原活化蛋白激酶信号的失调在骨关节炎的发生、发展中扮演着十分重要的角色。明确丝裂原活化蛋白激酶信号通路在骨关节炎发病机制中的确切作用将有助于骨性关节炎的靶向治疗。     

Coumestrol Counteracts Interleukin-1β-Induced Catabolic Effects by Suppressing Inflammation in Primary Rat Chondrocytes

In the present study, we investigated the anti-catabolic effects of coumestrol, a phytoestrogen derived from herbal plants, against interleukin-1β-induced cartilage degeneration in primary rat chondrocytes and articular cartilage. Coumestrol did not affect the viability of human normal oral keratinocytes and primary rat chondrocytes treated for 24 h and 21 days, respectively. Although coumestrol did not significantly increase the proteoglycan contents in long-term culture, it abolished the interleukin-1β-induced loss of proteoglycans in primary rat chondrocytes and knee articular cartilage. Furthermore, coumestrol suppressed the expression of matrix-degrading enzymes such as matrix metalloproteinase-13, ?3, and ?1 in primary rat chondrocytes stimulated with interleukin-1β. Moreover, the expression of catabolic factors such as nitric oxide synthase, cyclooxygenase-2, prostaglandin E₂, and inflammatory cytokines in interleukin-1β-stimulated primary rat chondrocytes was suppressed by coumestrol. In summary, these results indicate that coumestrol counteracts the catabolic effects induced by interleukin-1β through the suppression of inflammation. Therefore, based on its biological activity and safety profile, coumestrol could be used as a potential anti-catabolic biomaterial for osteoarthritis.     

Age-related changes in the articular cartilage of human sacroiliac joint

 Iliac and sacral articular cartilage of 25 human sacroiliac joints (1–93 years) are examined by light microscopy and immunohistochemistry in order to gain further insight into the nature and progress of degenerative changes appearing during aging. These changes can already be seen in younger adults as compared to cartilage degeneration known in other diarthrodial joints. Structural differences between sacral and iliac cartilage can already be observed in the infant: the sacral auricular facet is covered with a hyaline articular cartilage, reaching 4 mm in thickness in the adult and staining intensely blue with alcian blue at pH1. Iliac cartilage of the newborn is composed of a dense fibrillar network of thick collagen bundles, crossing each other at approximately right angles. A faint staining with alcian blue suggests a low content of acidic glycosaminoglycans. In the adult, iliac cartilage becomes hyaline and its maximal thickness reaches 1–2 mm. Both articular facets exhibit morphological changes during aging that are more pronounced in the iliac cartilage and resemble osteoarthritic degeneration; the staining pattern of the extracellular matrix becomes inhomogenous, chondrocytes are arranged in clusters and the articular surface develops superficial irregularities and fissures. Sometimes fibrous tissue fills up these defects. Nevertheless, large areas of iliac cartilage remain hyaline in nature. Sacral articular cartilage often remains largely unaltered until old age. The sacral subchondral bone plate is usually thin and shows spongiosa trabeculae inserted at right angles, suggesting a perpendicular load on the articular facet. Iliac subchondral spongiosa shows no definite alignment and joins the thickened subchondral bone plate in an oblique direction. The iliac cartilage therefore seems to be stressed predominantly by shearing forces, arising from the changing monopodal support of the pelvis during locomotion. The subchondral bone plate on both the iliac and sacral auricular facet is penetrated by blood vessels that come into close contact with the overlying articular cartilage. These vessels may contribute to the high incidence of rheumatoid and inflammatory diseases in the human sacroiliac joint. Immunolabelling with an antibody against type II collagen reveals a diminished immunoreactivity in the upper half of adult sacral cartilage and only a faint and irregular labelling in the iliac cartilage. Type I collagen can be detected in a superficial layer on the sacral articular surface and around chondrocyte clusters in iliac cartilage, as in dedifferentiating chondrocytes during the development of osteoarthritis. Accepted: 22 April 1998     

Immunohistochemical detection of estrogen receptor alpha in articular chondrocytes from cows, pigs and humans: in situ and in vitro results.

Clinical observations suggest that estrogens are involved in the pathogenesis of postmenopausal osteoarthritis, but only little is known about the influence of these hormones on articular cartilage cells. The effect of estradiol is mediated by estrogen receptors alpha and beta. The goal of the present study was to search for estrogen receptor alpha in articular tissue from cows, pigs and humans by immunohistochemistry to form a basis for in vitro studies. In addition, we also tried to detect estrogen receptor alpha in cultivated articular chondrocytes from cows and bulls under certain culture conditions. Estrogen receptor alpha is detected by the use of antibody 13H2 in articular chondrocytes from cows, bulls, pigs and humans. Chondrocytes are physiologically exposed to reduced oxygen tension. In isolated articular chondrocytes from cows and bulls incubated either with 21% O2 or with 5% O2 positive cells were also found. These positive results therefore encourage testing the influence of estradiol on cultivated articular cartilage cells in these species under different culture conditions.     

Autophagy Prior to Chondrocyte Cell Death During the Degeneration of Meckel's Cartilage

The central portion of Meckel's cartilage degenerates almost immediately after birth. Whether autophagy is involved in this process remains unclear. Thus, to explore the role of autophagy during this process, we have detected the expression of autophagy and apoptosis‐related markers in embryonic mice. In E15, Beclin1 and LC3 expressions were weak and negative in Meckel's cartilage, respectively. In E16, chondrocytes of the central portion became hypertrophic. Moderate immunoreactivities of Beclin1 and LC3 were observed in prehypertrophic and hypertrophic chondrocytes of the central portion. In E17, the degradation occurred in the central portion and expanded anteriorly and posteriorly. Beclin1 expression was observed in Meckel's cartilage with an increase in the hypertrophic chondrocytes of the central portion. The expression of LC3 was detected specifically in terminally differentiated hypertrophic chondrocytes. The mRNA expressions of LC3 and Beclin1 from E15 to E17 significantly increased. This result is in accord with the histologic findings. Terminal deoxynucleotidyltransferase‐mediated dUTP‐biotin nick‐end labeling assay and Caspase 3 expression demonstrated that apoptosis was detected in the lateral part of terminal hypertrophic chondrocytes along the degeneration area of Meckel's cartilage. In addition, Bcl2 expression increased significantly from E15 to E17. These results indicate that autophagy is involved in hypertrophic chondrocytes during the degradation of Meckel's cartilage and occurs prior to chondrocyte cell death during this process. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Observations on the senescence of cells derived from articular cartilage

In the experiments described here, we have sought to determine whether primary cultures of cells derived from articular cartilage will, upon subsequent subculture, undergo in vitro senescence in a manner analogous to that described for several other types of diploid cell. Using cells from the articular cartilage of rabbits, dogs and man, we have established that the population doubling capacity of cultures of these cells is directly related to the specific lifespan of the donor organism. Furthermore, the doubling capacity of the initial cultures of lapine articular chondrocytes is inversely related to the age of the donor rabbit. By these criteria, serially passaged primary cultures of cells derived from articular cartilage appear, a priori, to be a valid system for studies of cellular ageing. Monolayer cultures of lapine chondrocytes appear to "dedifferentiate" after several passages. However, the same cells can be grown as clones, under which conditions they appear to retain better their differentiated properties. Even under these circumstances, lapine articular chondrocytes have a limited capacity for growth, which can be calculated to approximate to the same average number of cell divisions as undergone by monolayer cultures. Lapine chondrocytes frequently transform into established lines of fibroblastic cells. Transformation of canine chondrocytes was more rare, while human chondrocytes have not been observed to transform. This suggests that resistance to transformation is somehow related to lifespan. In addition to furthering our understanding of cellular ageing, studies of the senescence of articular chondrocytes could provide new insights into the aetiology of primary osteoarthritis.