关节软骨修复机制中相关信号分子的研究进展

关节软骨损伤后,在进行性退变过程中,分解代谢水平上调,软骨细胞会分泌多种炎症因子,原有的细胞表型也逐渐改变。因此长期以来,广大研究者针对促软骨细胞合成代谢、维持软骨细胞表型稳定做了大量的研究,并发现在一系列软骨修复过程中有多种分子信号通路参与。本篇综述重点介绍关节软骨修复中关键的信号分子,如:转化生长因子β(TGF-β)、骨形态发生蛋白(BMP)等,并揭示和探讨这些分子在软骨损伤及修复过程中的作用,以便相关领域研究者广泛深入地了解软骨损伤修复过程中的分子机制,并在此基础上寻找软骨修复治疗靶点和更优的生物学治疗方法。     

软骨组织工程中力学因素的影响及应用

力学因素是软骨组织工程中的重要影响因素之一。近年来的研究表明，力学作用可以刺激细胞因子及激素的分泌，改变三维支架上培养的软骨细胞的新陈代谢，从而促进软骨组织的生长与重建。目前已经有诸多关于体外构建软骨组织的报道，但对于其中的力学因素的影响（包括力学因素对软骨细胞增殖的促进及力学刺激的传导机制等）还没有完全认识。就以上几方面做一综述，并简单介绍生物反应器在软骨组织工程中的应用。     

软骨组织工程中力学因素的影响及应用

力学因素是软骨组织工程中的重要影响因素之一。近年来的研究表明,力学作用可以刺激细胞因子及激素的分泌,改变三维支架上培养的软骨细胞的新陈代谢,从而促进软骨组织的生长与重建。目前已经有诸多关于体外构建软骨组织的报道,但对于其中的力学因素的影响(包括力学因素对软骨细胞增殖的促进及力学刺激的传导机制等)还没有完全认识。就以上几方面做一综述,并简单介绍生物反应器在软骨组织工程中的应用。     

关节软骨生物力学与力学生物学 2022 年研究进展

关节软骨是动关节内骨表面具有弹性的负重结缔组织，能提供低磨损润滑、缓冲震荡、传递载荷等支撑作用，具有层级纤维复合结构和优异的力学性能。软骨内没有血管、神经和淋巴，代谢缓慢，损伤后难以实现自我修复。目前，高发的关节炎疾病仍是基础与临床研究的一大热点。关节软骨是力学敏感组织，力学环境影响着组织不同方向的发展。2022年，学者们继续对关节软骨的生物力学与力学生物学开展大量研究；对软骨形态、功能与力学状态，以及不同条件下软骨力学状态的研究报道较多；研究设计了一些软骨相关的动物、组织及细胞水平的加载装置，也开展了体外及在体力学载荷下软骨退变、损伤的修复研究，获得了重要的修复方法及手段。关节软骨的生物力学与力学生物学研究是关节炎、软骨缺损及修复的基础，关节软骨损伤修复定量力学条件的影响还需体内和体外深入研究。     

间歇静水压对ATDC5软骨细胞分化早期的影响

文题释义： 间歇静水压：软骨细胞的新陈代谢及发育,除了有必要的营养成分支持外,力学刺激也是必不可少的,而力学刺激又有许多方式,如剪切力、流水动力、持续压力、间歇压力等,这些力学刺激对软骨细胞的诱导分化结果不尽相同,而最接近生理状态下的力学刺激最有利于软骨细胞的发育与分化,间歇静水压就是模仿关节运动的方式,把细胞放在培养液里间断对细胞进行力学刺激,进而促进细胞分化和发育。 软骨组织工程：软骨组织损伤后很难再生,如何修复受损伤的软骨组织是目前国际关注的焦点,利用工程学原理,重新构建新的软骨组织,是修复软骨组织最有效的方法,但构建新的软骨组织非常复杂,需要能够分化成软骨细胞的干细胞,需要分化所必需的培养液、培养支架、力学环境等因素,还需要稳定的生长发育环境,因此从种子细胞到软骨细胞,最后形成软骨组织是一个复杂的生物工程。 背景：软骨组织修复是组织工程研究的重要领域,如何利用工程学技术有效将种子细胞定向分化成软骨细胞是组织工程的重点和难点。目前,单纯应用各种定向诱导培养试剂很难使其分化为成熟稳定的软骨细胞,正是在这一背景下,作者利用ATDC5软骨细胞的特点,除了应用有效的培养液处理外,还采用间歇净水压的压力刺激方法,对其定向诱导分化进行早期研究。 目的：了解间歇静水压对ATDC5软骨细胞早期软骨方向分化成熟的影响。 方法：将ATDC5软骨细胞株在单层条件下培养,3 d细胞贴壁良好,并形成复层,而后在密封条件下进行间歇静水压(施加强度10 MPa,加压频率1 Hz,4 h/d)培养,设立无间歇静水压且其他条件相同的培养细胞为对照组。在第4,7,11,14,17天,通过显微镜观察细胞形态变化,应用Real-time PCR检测Aggrecan,COL-2,SOX-9的mRNA表达水平。 结果与结论：经间歇静水压作用后,ATDC5细胞表现出较明显的斑块样改变和细胞浓聚现象;Aggrecan、COL-2 mRNA表达水平明显升高,SOX-9 mRNA虽然与对照组变化不大,但也出现了先抑后扬的特点。结果表明,间歇静水压影响ATDC5软骨细胞向软骨方向分化的基因表达,促进软骨特征基质的分泌,利于向软骨细胞分化成熟。 ORCID: 0000-0003-0911-8294(张强) 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     

软骨干/祖细胞修复软骨损伤的研究进展

关节软骨损伤是临床常见的运动损伤,然而关节软骨缺乏血管、神经支配,损伤后难以自身完全再生修复,缺乏有效的治疗会导致骨关节炎(OA)的发生。随着组织工程学的发展,近期研究发现,软骨组织中存在具有多向分化潜能的软骨干/祖细胞(CSPC),可以分化成高度同质化的软骨细胞。这种细胞在软骨损伤后自发迁移并参与软骨损伤组织修复和再生,有望成为治疗软骨损伤的新方法。本文对CSPC的分布、特征及其在软骨损伤和OA中的作用和应用进行综述,旨在为CSPC的研究应用及软骨损伤的治疗提供理论基础,为临床上治疗软骨损伤开拓新方向。     

TRPV4和PIEZOs介导软骨细胞力转导的研究进展EI北大核心

软骨细胞转导机械力信号的力转导过程在关节软骨生长、发育和骨性关节炎(OA)过程中发挥着重要作用。在关节软骨中,细胞外基质代谢活动调控着软骨细胞的基质力学微环境。因此,理解软骨细胞感受基质力学微环境的力转导机制能够通过精准靶向机械力感受器有效建立OA治疗策略。近十几年,大量研究证实力敏感离子通道TRPV4和PIEZOs在关节软骨细胞中大量表达,而且介导机械力相关的软骨重塑和损伤响应。本文综述了软骨细胞感受基质力学微环境过程中TRPV4和PIEZOs介导的力感受和力响应方面的相关研究,同时也介绍了TRPV4和PIEZOs介导炎症信号调控骨性关节炎方面的进展。本文为理解关节软骨发病机制和寻求可能药物靶点,以及设计细胞诱导性生物材料提供了新思路。     

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：     

述评：关节软骨的细胞与分子生物力学

关节软骨是覆盖在动关节骨端表面的一层低摩擦、可承载负荷的水合软组织，其力学性能通过软骨细胞的新陈代谢来维持。物理因素，如关节载荷对软骨细胞的新陈代谢有很大的调节作用。本文对关节软骨和软骨细胞的力学性能和理论模型进行了综述，重点强调物理调节在软骨细胞生物合成和组织维护中的作用，以及在软骨功能性组织工程修复与再生医学中的应用。文中首先描述了关节软骨的分子构成、超微结构及其张力、压力、剪切力等力学性能，着重强调其相互关系。然后，通过介绍已广泛使用的结构模型，即双相和三相混合理论，对组织的力学－电化学行为进行阐述，并着重介绍简化复杂的三相理论的最新研究进展。最后，对软骨细胞和软骨素的机械性能和理论模型进行回顾总结，从而加深对细胞外基质中软骨细胞内及周围力学－电化学信号的认识。根据混合相理论，关节软骨的流动依赖性和非流动依赖性的粘弹性、溶胀行为和电动特性已经在理论上得到了成功阐述。文献中混合相理论用于软骨细胞以及周围细胞外基质的推广也为体内细胞行为物理调节机制的研究提供了新的思路。总之，应用强大的混合相理论、新的实验技术、新的或常规的办法来研究软骨细胞和基质之间的相互作用，可能有助于关节软骨工程学研究的成功。     

软骨细胞力学环境的跨尺度计算

目的通过跨尺度计算,比较表层和深层软骨细胞的周边力学环境。方法建立软骨细胞两相力学模型,将软骨两相力学模型里的结果映射到细胞模型对应边界上作为边界条件。计算细胞模型得到软骨细胞的周边力学环境并进行分析。结果深层软骨细胞及周边应力比表层细胞的小一半,但都远小于细胞外的应力。软骨细胞周围基质(pericellular matrix,PCM)承担了细胞外的高应力,显著降低了细胞内的应力。两处细胞周围的间隙流动方向完全相反。结论软骨承载能力使深层软骨细胞附近应力显著降低,保护了深层软骨细胞及软骨下骨。PCM承担了细胞外围高应力,保证了软骨细胞生存工作所需的低应力环境。两处细胞周围相反的间隙流动支持了由表层关节液渗透及软骨下骨营养泵入构成的软骨双向营养供给学说。     

从基质黏弹性和钙信号深入理解骨性关节炎发病机制

近年来,科学家们普遍认为细胞外基质(extracellular matrix,ECM)力学特性是调控细胞力学生物学行为的一个重要影响因素.几乎所有的活组织和ECM都具有显著的黏弹性特征.大量研究证实,细胞能够感受和响应ECM力学特性.细胞通过基于整合素的黏附、力敏感离子通道激活以及下游信号通路的激活来感受基质力学特性的...     

能量代谢对骨关节炎作用的研究进展

骨关节炎 （Osteoarthritis,OA） 是一种以软骨损伤为主要特征的慢性疾病,目前因其发病机制未详而尚无靶向药物。近些年诸多研究表明,线粒体功能障碍、葡萄糖代谢紊乱以及脂质代谢异常等能量代谢问题所导致的软骨细胞功能衰退加剧了OA的恶化。现有研究表明,从能量代谢途径改善软骨细胞状态,能达到缓解疼痛、减轻炎症、延缓进展与改善功能等OA治疗目的。在本综述中,我们聚焦软骨细胞能量代谢与OA软骨损伤的起因与发展的关联,旨在厘清OA的潜在治疗思路。     

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.     

Articular chondrocytes express connexin 43 hemichannels and P2 receptors – a putative mechanoreceptor complex involving the primary cilium?

Mechanical loading is essential for the health and homeostasis of articular cartilage, although the fundamental mechanotransduction pathways are unclear. Previous studies have demonstrated that cyclic compression up‐regulates proteoglycan synthesis via an intracellular Ca²⁺ signalling pathway, mediated by the release of ATP. However, the mechanism(s) of ATP release has not been elucidated. The present study examines expression of the putative mechanosensitive ATP‐release channel, connexin 43 and whether it is expressed on the chondrocyte primary cilium, which acts as a mechanosensor in a variety of other cell types. In addition the study characterized the expression of a range of purine receptors through which ATP may activate downstream signalling events controlling cell function. Bovine articular chondrocytes were isolated by sequential enzyme digestion and seeded in agarose constructs. To verify the presence of functional hemichannels, Lucifer yellow (LY) uptake into viable cells was quantified following treatment with a hemichannel agonist (EGTA) and antagonist (flufenamic acid). LY uptake was observed in 45% of chondrocytes, increasing to 83% following EGTA treatment (P < 0.001). Treatment with the hemichannel blocker, flufenamic acid, significantly decreased LY uptake to less than 5% with and without EGTA. Immunofluorescence and confocal microscopy confirmed the presence of primary cilia and the expression of connexin 43. Approximately 50% of bovine chondrocyte primary cilia were decorated with connexin 43. Human chondrocytes in situ within cartilage explants also expressed connexin 43 hemichannels. However, expression was confined to the upper 200 µm of the tissue closest to the articular surface. Immunofluorescence revealed the expression of a range of P2X and P2Y receptor subtypes within human articular cartilage. In conclusion, the expression of functional hemichannels by articular chondrocytes may represent the mechanism through which mechanical loading activates ATP release as part of a purinergic mechanotransduction pathway. Furthermore, the expression of connexin 43 on the chondrocyte primary cilium suggests the possible involvement of the cilium in this pathway.     

Relationship among biomechanical, biochemical, and cellular changes associated with osteoarthritis

Articular cartilage that lines the surface of long bones is a multilayered material. The superficial layer consists of collagen fibrils and chondrocytes that run parallel to the joint surface. In the deeper layers, the collagen fibrils are more randomly arranged and support vertical units termed chondrons containing rows of chondrocytes. In the deepest layers, the collagen fibrils run almost vertically and ultimately insert into the underlying subchondral bone. Osteoarthritis (OA) is a disease that affects articular cartilage and is characterized by enzymatic and mechanical breakdown of the extracellular matrix, leading to cartilage degeneration, exposure of subchondral bone, pain, and limited joint motion. Changes in mechanical properties of articular cartilage associated with OA include decreases in modulus and ultimate tensile strength. These changes parallel the changes observed after enzymatic degradation of either collagen or proteoglycans in cartilage. Results of recent viscoelastic studies on articular cartilage suggest that the elastic modulus of collagen and fibril lengths decrease in OA and are associated with a loss of the superficial zone and a decreased ability of articular cartilage to store elastic energy during locomotion. It is suggested that osteoarthritic changes to cartilage involve enzymatic degradation of matrix components and fibril fragmentation that is promoted by subsequent mechanical loading.     

Integrin-Dependent Signal Cascades in Chondrocyte Mechanotransduction

Mechanical forces influence chondrocyte metabolism and are critically important for maintenance of normal cartilage structure and integrity. In cells of the musculoskeletal system and mechanoresponsive cells in other tissues, integrins seem to be involved in the mechanotransduction process. Integrin activity is important in the early cellular responses to mechanical stimulation, regulating activation of a number of intracellularcascades that induce changes in gene expression and tissue remodeling. In normal human articular chondrocytes, integrin activation, consequent to mechanical stimulation in vitro, results in tyrosine phosphorylation of regulatory proteins and subsequent secretion of autocrine and paracrine acting soluble mediators including substance P and interleukin 4. Significant differences in signaling events and cellular responses are seen when normal and osteoarthritic chondrocytes are mechanically stimulated. These differences may relate to differences in integrin expression and function. Improved comprehension of how integrins mediate chondrocyte responses to mechanical stimulation, and how cross talk between integrin signaling, extracellular matrix, and autocrine/paracrine signaling molecules regulate mechanotransduction and cellular reactions are necessary for further understanding of how load influences cartilage structure.     

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。     

Indian Hedgehog,a critical modulator in osteoarthritis,could be a potential therapeutic target for attenuating cartilage degeneration disease

The Hedgehog (Hh) family of proteins consists of Indian hedgehog (Ihh), sonic hedgehog (Shh), and desert hedgehog (Dhh). These proteins serve as essential regulators in a variety of developmental events. Ihh is mainly produced and secreted by prehypertrophic chondrocytes and regulates chondrocyte hypertrophy and endochondral bone formation during growth plate development. Tissue-specific deletion of the Ihh gene (targeted by Col2a1-Cre) causes early lethality in mice. Transgenic mice with induced Ihh expression exhibit increased chondrocyte hypertrophy and cartilage damage resembling human osteoarthritis (OA). During OA development, chondrocytes recapitulate the differentiation process that happens during the fetal status and which does not occur to an appreciable degree in adult articular cartilage. Ihh expression is up-regulated in human OA cartilage, and this upregulation correlates with OA progression and changes in chondrocyte morphology. A genetic study in mice further showed that conditional deletion of Ihh in chondrocytes attenuates OA progression, suggesting the possibility that blocking Ihh signaling can be used as a therapeutic approach to prevent or delay cartilage degeneration. However, Ihh gene deletion is currently not a therapeutic option as it is lethal in animals. RNA interference (RNAi) provides a means to knockdown Ihh without the severe side effects caused by chemical inhibitors. The currently available delivery methods for RNAi are nanoparticles and liposomes. Both have problems that need to be addressed. In the future, it will be necessary to develop a safe and effective RNAi delivery system to target Ihh signaling for preventing and treating OA.     

Regulation of p38 MAPK phosphorylation inhibits chondrocyte apoptosis in response to heat stress or mechanical stress

Activation of p38 MAPK has been associated with a stress response and with apoptotic processes. However, the function of p38 MAPK in chondrocytes is not clearly understood. In this study, we analyzed the expression of p38 MAPK in chondrocytes and investigated the function of p38 MAPK in response to heat stress and mechanical stress. Chondrocytes were isolated from human cartilage and cultured. Expression of p38 and phosphorylated p38 in cartilage of patients with osteoarthritis (OA) was compared to those in normal cartilage by immunohistochemistry and Western blotting. Human knee chondrocytes were exposed to heat stress or mechanical stress. Normal knee chondrocytes were pre-treated with SB203580 or p38 small interfering RNA (siRNA) before induction of heat stress or mechanical stress. Chondrocyte apoptosis was detected by TUNEL staining and Western blotting of cleaved caspases. OA and normal chondrocytes expressed p38; however, OA chondrocytes showed much higher phosphorylated p38 compared to normal chondrocytes. Heat stress or mechanical stress induced apoptosis and increased phosphorylated p38 in normal chondrocytes. The TUNEL positive cells and expression levels of phosphorylated p38 in response to stress decreased when chondrocytes were incubated with SB203580 or transfected with siRNA against p38. In conclusion, we have demonstrated that heat stress or mechanical stress increased chondrocyte apoptosis via phosphorylation of p38. Stress-induced chondrocyte apoptosis decreased due to inhibition of p38 MAPK activation. In contrast, the phosphorylation of p38 MAPK increased in OA chondrocytes. Our results show that down-regulation of p38 MAPK activation inhibits chondrocyte death induced by heat stress or mechanical stress.     

Ultrastructural change of the subchondral bone increases the severity of cartilage damage in osteoporotic osteoarthritis of the knee in rabbits

Osteoporotic osteoarthritis is a phenotype of osteoarthritis (OA) manifested as fragile and osteoporotic subchondral bone. However, the ultrastructural features of subchondral bone in osteoporosis OA have not been determined. The study was aimed to investigate the ultrastructural dynamic changes of subchondral bone in osteoporotic OA model and how the ultrastructural damage in the subchondral bone caused by osteoporosis deteriorated the cartilage damage in OA. Eighteen rabbits were equally randomized to three groups, including the control, the OA and the osteoporotic OA groups. The structural changes of cartilage were evaluated by HE and safranin-O fast green staining, the Mankin’s grading system was used to assess the stage of OA progression. And microstructural or ultrastructural changes in subchondral bone were assessed by micro-computed tomography or by scanning electron microscopy. According to the changes of cartilage histopathology, the OA group was in the early pathological stage of OA while the osteoporotic OA group was in the middle stage of OA based on Mankin’s grading system. In addition, the damage of cartilage surface, reduction in the number of chondrocytes and the matrix staining were more increased in the osteoporotic OA group compared to the OA group. Compared to the OA group, the subchondral bone in the microstructure and ultrastructure in the osteoporotic OA group showed more microfracture changes in trabecular bone with more destructions of the tree-like mesh. Moreover, the collagen fibers were random rough with a fewer amount of bone lacunae in subchondral cortical plate in the osteoporotic OA group compared to the OA group. These findings indicated that the subchondral bone ultrastructure in the osteoporotic OA model was characterized by the destruction of the network structure and collagen fibers. The subchondral bone ultrastructural damage caused by osteoporosis may change mechanical properties of the upper cartilage and aggravate OA cartilage. Therefore, early diagnosis and treatment of osteoporosis is of great significance to prevent early OA from further developing osteoporotic OA.