异常高应力导致关节软骨退变机理的形态学研究

通过透射电镜、扫描电镜和光镜，对８７只跟腱切除的ＳＤ大鼠的对侧膝关节软骨进行了观察。发现术后２周，关节软骨表面出现凹陷与裂隙；术后１个月，软骨出现软骨细胞簇，软骨细胞显示旺盛的合成与分泌功能。继而，关节软骨进行性地退变。高应力首先导致软骨基质的破坏，使胶原纤维网架断裂，蛋白多糖丧失。基质破坏使正常的微环境发生变化，导致软骨细胞的退变。软骨细胞退变又可加重基质的损害，形成恶性循环。高应力造成软骨基质损害的早期可引起部分软骨细胞增生，并具有活跃的合成、分泌功能，但随后大部分细胞退变。这是最初的基质损害激发了软骨细胞的修复功能，但随着基质损害的加重，正常微环境遭到破坏，又使这些细胞也迅速退变。     

应力降低导致关节软骨退变机理的形态学研究

制动在限制关节活动的同时形成低应力环境。本研究切除大鼠一侧跟腱，造成该侧后肢膝关节负重降低而保持关节活动的次数及幅度。通过透射电镜、扫描电镜和光镜，对８７只左侧跟腿切除后的ＳＤ大鼠的左膝关节软骨进行观察，实验发现低应力侧软骨发生退变，退变首先出现于软骨细胞，并以细胞功能活动衰退和细胞退变、崩解为主要特点。继而软骨基质出现断裂和破坏，软骨细胞和软骨基质的退变呈进行性发展。作者发现：正常应力环境对维持     

高应力导致兔腰椎小关节骨性关节炎的实验研究

目的:观察兔腰椎小关节加载应力后小关节在应力作用下的病理变化,并探讨其病理机制。方法:将42只新西兰白兔随机分为正常对照组(A组)、椎间盘退变组(B组)、小关节加力组(C组)及椎间盘退变及小关节加力组(D组)。对B组和D组动物采用针刺抽吸髓核法建立椎间盘退变模型,建模6周后分别对C组和D组的L3/4、L5/6小关节加载拉簧造成高应力模型,对A组和B组仅行小关节暴露,分别于术后4个月、8个月取腰椎小关节,采用番红O-快绿染色,进行Mankin评分判断软骨退变程度并分为轻度、中度及重度;免疫组化染色观察细胞因子IL-1β、IL-6、TNF-α在小关节软骨中的表达。结果:不同组间小关节退变程度有显著性差异(F=92.77,P=0.000),组内比较,各组8个月时小关节退变程度较4个月时严重(P<0.05);同一时间段不同组间比较,小关节加力组(C组和D组)的小关节退变程度较小关节未加力组(A组和B组)严重,差异均有显著性(P<0.05),A、B组间及C、D组间比较,小关节退变程度无显著性差异(P>0.05)。三种细胞因子随着软骨退变程度的增加而表达增强,而严重退变的软骨由于软骨细胞数减少表达反而有所下降。结论:高应力可导致腰椎小关节退变,应力作用时间越长,小关节退变越严重。腰椎小关节软骨细胞的退变伴随着炎性细胞因子合成、分泌的改变。     

创伤性关节炎发生机制相关性研究

创伤性关节炎发生机制研究显示,关节软骨凋亡或坏死引起关节软骨数量减少,导致关节软骨退变;关节生物力学及其关节周围环境应力改变引起关节软骨细胞及基质之间的动态平衡破坏,进一步引发关节软骨退变;关节损伤后关节液中细胞炎症介质对关节软骨的作用及介质之间相互作用在关节软骨退变中起着重要作用;关节损伤疾病发展过程及关节软骨代谢均与患者个体基因学关系密切。研究明确关节损伤后各种因素在创伤性关节炎中的作用及各种因素之间的联系,有助于更深入地了解创伤性关节炎的发生机制,更好地制定相关治疗方案,以治愈或延缓创伤性关节炎的发生发展。     

骨水泥阻塞髓腔后骨内压升高对远侧关节影响的实验研究

[目的]探讨骨水泥阻塞股骨近中段骨干髓腔后骨内压升高对远侧关节组织结构的影响。[方法]将26只实验兔分成4组,随机取出8只不做模型而作为正常对照组,其余18只采取左侧股骨髓腔内灌注聚甲基丙烯酸甲酯(PMMA)骨水泥而右侧不灌注,制作骨水泥阻塞股骨近中段骨干髓腔的动物模型,根据不同观察时间段随机分成术后4(T4)、8(T8)和16周(T16)3个观察组。对正常对照和模型动物实验侧股骨远端软骨、软骨下骨和滑膜组织标本,采用HE染色、甲苯胺蓝染色、免疫组织化学检测及透射电镜方法进行组织结构观察。[结果](1)HE染色:关节软骨、软骨下骨和关节滑膜组织损害随时间呈进行性加重,造模16周关节软骨破坏,骨组织结构损害,滑膜组织增生、肿胀。(2)甲苯胺蓝染色:造模16周关节软骨全层失染。(3)免疫组化:①Ⅱ型胶原:造模16周软骨细胞染色阳性。②TGF-β1:造模16周软骨细胞染色阳性,关节滑膜细胞染色阳性。(4)透射电镜:关节软骨和骨细胞损害随时间呈进行性加重,造模16周部分软骨细胞和骨细胞坏死、崩解。[结论]骨水泥阻塞股骨近中段骨干髓腔后导致股骨远端血循环障碍,骨内压升高,持续骨内高压加重了股骨远端的缺血缺氧状态,使股骨远端骨代谢发生紊乱,最终造成股骨远端骨、软骨和滑膜组织发生了退变或坏死。     

失应力及恢复应力作用下关节软骨的变化特点

目的通过对伸膝制动及解除制动兔模型的关节软骨进行组织学及免疫组织化学研究，探讨软骨在失应力及恢复应力作用下的变化特点，从而明确应力对关节软骨正常生长代谢以及修复过程的影响。方法选取成年新西兰雄性大白兔90只，平均体重（2．5±0．2）kg。按照实验要求分为9组，每组10只。制备兔伸膝制动及解除制动模型，对制动2、4、6周及解除制动2、4周的兔膝关节全层软骨进行HE染色及Ⅱ型胶原免疫组织化学染色，比较解除制动前后软骨细胞分布情况及Ⅱ型胶原变化。结果HE染色结果表明制动2、4、6周兔膝关节软骨的退变程度逐渐加重；Ⅱ型胶原免疫组织化学染色结果提示制动早期（2、4周）软骨细胞分泌增强，后期逐渐减弱。随着制动时间增加兔关节软骨病理评分和免疫组化灰度值的变化具有统计学意义（F=234．45，13．03；P〈0．01），制动后自由活动时间增加导致的病理评分和免疫组化灰度值的变化无统计学意义（F=0．14，3．10；P〉0．05）。结论（1）失应力时间越长，关节软骨细胞正常生理活动及其分泌Ⅱ型胶原的能力降低越明显。（2）应力恢复后可引发关节软骨的自行修复，制动少于4周者关节软骨能完全修复，制动大于6周者关节软骨几乎不能修复。失应力时间的长短是决定能否修复的关键。     

脊柱内固定与邻近关节突关节退变程度的关系

目的：研究不同脊柱内固定节段对邻近运动节段关节突关节的影响。方法：设计山羊长节段及短节段脊柱内固定的动物模型,对固定６ 个月后近头侧两个邻近关节突关节进行光镜、扫描电镜及透射电镜的观察。结果：短节段组出现了软骨基质的破坏,部分软骨细胞呈现出活跃的分泌功能,符合软骨早期退变的表现;长节段组软骨基质的破坏加剧,软骨细胞也出现了较严重的退变表现,符合软骨晚期退变的表现。两组近头侧两个邻近关节突关节都出现了退变现象。结论：脊柱内固定导致了邻近运动节段关节突关节的退变。长节段固定后邻近关节突关节早期就出现了较为严重的退变,相比之下短节段固定后邻近关节突关节的退变程度较轻,出现的时间较晚     

软骨细胞体外培养负性相关因素研究进展

软骨细胞体外培养受到众多复杂因素的影响.白介素-1(IL-1)和肿瘤坏死因子-α(TNF-α),可通过核因子-κB等信号途径干扰软骨细胞的正常代谢,两者具有协同作用;IL-6、IL-17、IL-18经相应途径上调炎症和软骨破坏相关基因,基质金属蛋白酶及其抑制剂的平衡失调是引起软骨退变的重要原因;含钙结晶体能通过两个独立的途径引起关节组织退变;过度的应力刺激、低氧、高渗溶液可不同程度地影响软骨细胞生长;各种因素引起的内质网过度紧张可导致软骨细胞调亡,软骨外植和切碎过程能引起IL-1表达升高.     

细胞因子与骨性关节炎软骨退变的研究现状

骨性关节炎是最常见关节疾患之一,是导致中老年残疾的最常见原因,严重危害着中老年人的生活质量。骨性关节炎的主要病理是软骨的改变,软骨细胞分解和合成活动的平衡有助于维持软骨细胞外基质结构和功能的完整性,而软骨细胞的这种平衡受到细胞因子制约,本文就近年来细胞因子与OA软骨退变之间的研究现状综述。     

细胞因子与骨性关节炎软骨退变的研究现状

骨性关节炎是最常见关节疾患之一,是导致中老年残疾的最常见原因,严重危害着中老年人的生活质量。骨性关节炎的主要病理是软骨的改变,软骨细胞分解和合成活动的平衡有助于维持软骨细胞外基质结构和功能的完整性,而软骨细胞的这种平衡受到细胞因子制约,本文就近年来细胞因子与OA软骨退变之间的研究现状综述。     

椎间盘源性腰痛的病理形态学观察

目的观察椎间盘源性腰痛的病理形态学特征,探讨其病理机制。方法选取20例椎间盘源性腰痛的椎间盘手术标本,常规HE染色,光学显微镜观察分析。结果镜下见椎间盘髓核面积减少,髓核中活性软骨细胞少,退变细胞多;纤维环增厚,从内层至外层纤维环可见不同程度的破裂,纤维环软骨细胞减少,软骨基质增多。结论椎间盘源性腰痛的主要致病机制可能是椎间盘形态结构的改变作用。     

Expression of parathyroid hormone-related peptide in human osteoarthritis

To evaluate the involvement of the expression of parathyroid hormone-related peptide gene in human articular cartilage pathology, we performed immunohistochemical staining and in situ hybridization on specimens of femoral head cartilage obtained from 15 patients with osteoarthritis, 11 with rheumatoid arthritis, and 12 control subjects. Parathyroid hormone-related peptide-positive chondrocytes were observed predominantly in degenerated lesions of osteoarthritic tissue and were less evident in rheumatoid arthritic samples, while the normal cartilage expressed little parathyroid hormone-related peptide. In addition, the level of parathyroid hormone-related peptide expression was clearly dependent on the degree of cartilage degeneration; cartilage tissues with moderate degenerative changes contained more positive chondrocytes compared with mildly or severely degenerated cartilage. In situ hybridization confirmed the localization of parathyroid hormone-related peptide protein and demonstrated intense expression of mRNA of the peptide in osteoarthritic samples. This is the first demonstration of parathyroid hormone-related peptide expression in chondrocytes from pathologic articular cartilage of humans. Our results, suggest that parathyroid hormone-related peptide may be involved in the pathophysiology of osteoarthritis.     

BIO alleviated compressive mechanical force‐mediated mandibular cartilage pathological changes through Wnt/β‐catenin signaling activation

Osteoarthritis induced by compressive mechanical force is characterized by decreased chondrocyte proliferation and degradation of the ECM. To examine underlying mechanisms of the pathological changes of mandibular cartilage induced by compressive mechanical force, an established animal model was used to examine Wnt signaling activation by glycogen synthase kinase‐3 beta (GSK3β) inhibitor 6‐Bromoindirubin‐3′‐oxime (BIO) injection in vivo. Histological changes in mandibular cartilage were assessed via hematoxylin & eosin (HE), masson, and alcian blue staining. Immunohistochemistry and real‐time PCR were performed to evaluate activation of the Wnt signaling pathway and chondrocytes proliferation markers. Chondrocytes apoptosis was examined by TUNEL staining. During the compressive mechanical force loading‐mediated process, Wnt signaling was largely inhibited, which showed the inhibited expression of β‐catenin and the increased expression of GSK‐3β. The expression of chondrocytes proliferation markers Ki67, and proliferating cell nuclear antigen (PCNA) also decreased. With BIO injection, the Wnt signaling was restored and the proliferation of mandibular chondrocytes was also increased in the late stage (7 days) of compressive mechanical force loading. Finally, the decreasing mandibular cartilage thickness, the degradation of extracellular matrix, and the erosion of bone trabecula were subsequently restored. Also, the changes of extracellular matrix markers such as collagen II and collagen X, matrix metalloproteases, and inflammatory cytokines were reversed followed by the injection of BIO. In summary, compressive mechanical force decreased endogenously Wnt signaling, leading to impaired proliferation in chondrocytes and degradation in cartilage matrix. Restoration of Wnt signaling largely recovered the proliferation defects and alleviated the pathological changes of mandibular cartilage. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1228–1237, 2018.

     

Matrix vesicle formation in cartilage of scorbutic guinea pigs electron microscope study of serial sections

The cartilage of the costo-chondral junctions of guinea pigs kept on a scorbutigenic diet for 26 days has been examined under the electron microscope and the relationship between matrix vesicles and chondrocytes has been studied in consecutive serial sections.Vitamin C deficiency induces severe changes in cartilage. Matrix calcification is not impaired whereas hardly any matrix synthesis occurs, so that the inorganic substance gradually reaches the zone of proliferating chondrocytes and the cell columns become shortened and distorted. The chondrocytes show various degrees of degeneration and fragmentation and are sometimes calcified.More matrix vesicles are found in the cartilage of scorbutic animals than in that of controls. They are often found in close contact with and seem to be budding from the tips of cell processes. Some of these break away from the cells and their fragments can have homogeneous, amorphous matrix, as if they were undergoing “transformation” into matrix vesicles. Other vesicles are found within cellular lacunae, sometimes together with remnants of degenerated cells.These results show that matrix vesicle formation in the cartilage of scorbutic guinea pigs occurs through three main mechanisms, as in the normal state budding from cell processes, fragmentation of cell processes, and the disruption of degenerated chondrocytes.     

不同幅度周期性拉伸形变对软骨细胞凋亡细胞因子基因水平的影响

目的 探讨兔软骨细胞受到拉伸机械形变后金属蛋白酶(MMP)及转化生长因子-β1(TGF-β1)的影响.方法 取兔原代关节软骨细胞培养方法,利用FLEXERCELL-5000T细胞拉伸系统对单层培养细胞施加拉伸形变,其中分为对照组、低幅度(5%)拉伸组及高幅度(15%)拉伸组,每天作用8 h,拉伸5 d,分别取3、5 d时细胞行实时定量PCR(qRT-PCR)检测MMP3、MMP13、ADAMTS-5、TGF-β1的基因表达.结果 拉伸组MMP3、ADAMTS-5基因水平3 d时拉伸形变导致MMP3、ADAMTS-5均显著升高(P=0.002),5 d时各组差异有统计学意义.3 d时各拉伸组中MMP13均高于对照组,5 d时高幅度拉伸组高于低幅度拉伸组.5 d时拉伸组中TGF-β1均高于对照组,低幅度拉伸组高于高幅度拉伸组.结论 本实验结果明确了软骨细胞中凋亡细胞因子及TGF的基因水平受不同拉伸幅度而旱现不同趋势.TGF-β1在软骨细胞拉伸形变的修复中起到重要作用.

Abstract：

Objective To explore the effects of cyclic tensile stresses of different magnitudes on expressions of apoptosis cytokines and transforming growth factors (TGF) in chondrocytes. Methods Classical methods were used to culture primary chondrocytes in New Zealand rabbits. Mechanical stresses were exerted onto the mono-layer cells cultured by the FLEXERCELL-5000T system. The experiment was conducted in 3 groups: low magnitude stress group (5% ), high magnitude stress group (15% ) and control group. The stresses were applied for 5 days and 8 hours per day. The cultured cells were harvested on days 3 and S to measure gene copies of MMP3 (matrix metalloproteinases), MMP13, ADMATS-5 (a disintegrin and metalloproteinase with thrombospondin motifs), and TGF-β1 by quantitative RT-PCR. Results Expressions of ADMATS-5 and MMP3 were significantly enhanced in each stress group on day 3 but weakened on day 5 ( P = 0. 002). Expressions of MMP13 were significantly higher in the stress groups than in the control group on day 3, and significantly higher in the high magnitude stress group than in the low one on day 5 ( P = 0. 002) .Levels of TGF-β1 on day 5 were significantly higher in the stress groups than in the control group, and higher in the low magnitude stress group than in the high one (P = 0. 002). Conclusions Gene expressions of apoptosis cytokines and TGF in chondrocytes are differently influenced by cyclic tensile stresses of different magnitudes. TGF -β1 may play an important role in pathological repair of the deformed chondrocytes following tensile stress.     

Activation of annexin II and V expression, terminal differentiation, mineralization and apoptosis in human osteoarthritic cartilage

OBJECTIVE: To test the hypothesis that terminal differentiation of chondrocytes in human osteoarthritic cartilage might lead to the failure of repair mechanisms and might cause progressive loss of structure and function of articular cartilage. DESIGN: Markers for terminally differentiated chondrocytes, such as alkaline phosphatase, annexin II, annexin V and type X collagen, were detected by immunohistochemical analysis of human normal and osteoarthritic knee cartilage from medial and lateral femoral condyles. Apoptosis in these specimens was detected using the TUNEL labeling. Mineralization and matrix vesicles were detected by alizarin red S staining and electron microscopic analysis. RESULTS: Alkaline phosphatase, annexin II, annexin V and type X collagen were expressed by chondrocytes in the upper zone of early stage and late stage human osteoarthritic cartilage. However, these proteins, which are typically expressed in hypertrophic and calcifying growth plate cartilage, were not detectable in the upper, middle and deep zones of healthy human articular cartilage. TUNEL labeling of normal and osteoarthritic human cartilage sections provided evidence that chondrocytes in the upper zone of late stage osteoarthritic cartilage undergo apoptotic changes. In addition, mineral deposits were detected in the upper zone of late stage osteoarthritic cartilage. Needle-like mineral crystals were often associated with matrix vesicles in these areas, as seen in calcifying growth plate cartilage. CONCLUSION: Human osteoarthritic chondrocytes adjacent to the joint space undergo terminal differentiation, release alkaline phosphatase-, annexin II- and annexin V-containing matrix vesicles, which initiate mineral formation, and eventually die by apoptosis. Thus, these cells resume phenotypic changes similar to terminal differentiation of chondrocytes in growth plate cartilage culminating in the destruction of articular cartilage in osteoarthritis.     

Type II collagen synthesis in the articular cartilage of a rabbit model of osteoarthritis: expression of type II collagen C-propeptide and mRNA especially during early-stage osteoarthritis

Background The aim of this study was to observe time course changes in type II collagen synthesis in various regions of articular cartilage affected with osteoarthritis (OA) by examining the expression of type II collagen C-propeptide (pCOL II-C) and mRNA in a rabbit OA model. Methods Osteoarthritis was experimentally induced by partial lateral meniscectomy in the knees of Japanese white rabbits. The cartilage of the animals was then examined histologically over time. The degenerative area of articular cartilage was divided into three areas, according to the degree of degeneration. The ability to synthesize type II collagen was estimated by the immunohistological staining of pCOL II-C and the in situ hybridization of mRNA in type II collagen. Results The positive rate of pCOL II-C immunostaining in chondrocytes was highest in the central-degenerative region 1 week after surgery, and the highest rate in the para-degenerative region was observed 2 and 4 weeks after surgery. The percentage of pCOL II-C positive cells increased as the histological degeneration score increased to moderate degeneration and then decreased with further progression of the severity of cartilage degeneration. Examination by in situ hybridization revealed that the regions marked by strong pCOL II-C mRNA expression were similar to those indicated by the immunohistology results. Conclusions These results suggest that the type II collagen-synthesizing potential of chondrocytes is highest in moderately degenerated areas of OA articular cartilage. Cartilage repair continues to be seen even as OA advances, although the reaction varies depending on the stage of OA.     

Chondrocyte transplantation

The transplantation of chondrocytes as a treatment to repair defects and degeneration in hyaline articular cartilage is being tested in numerous laboratory and clinical settings. This has included transplanting chondrocytes grown in tissue culture that were procured from non-weight-bearing areas of the affected joint to transplanting allografts with living chondrocytes in their intact cartilaginous matrix. Reported success with transplanting host and donor chondrocytes has varied and widespread application of these techniques still awaits more definitive studies. The clinician needs more evidence that the transplanted chondrocytes maintain their viability and that they synthesize the appropriate extracellular matrix. This new matrix needs to reproduce the functional, mechanical, and long-term wear properties of the native articular cartilage. Chondrocyte transplantation also merits further monitoring for possible delayed immunogenicity or for any signs of neoplastic potential. This exciting technology and its potential application to damaged and degenerated articular cartilage remains a stimulus to encourage further scientific work. Duplicating the unique and complex interrelations of the chondrocytes, matrix, and various bioactive factors is still some years away from general patient care.     

Effects of high molecular weight hyaluronan on the distribution and movement of proteoglycan around chondrocytes cultured in alginate beads

OBJECTIVE: To evaluate the effects of high molecular weight hyaluronan (HA) on the distribution and movement of proteoglycan (PG) formed around rabbit chondrocytes cultured in alginate beads. DESIGN: Rooster comb-derived HA (MW 8x10(5) Da) was co-polymerized in alginate gel to study the direct effects of extrinsic HA on chondrocytes. PG metabolism of rabbit chondrocytes cultured in alginate beads was examined by measuring the incorporation of [(35)S]sulfate into glycosaminoglycan in two distinct regions, the cells with their cell-associated matrix (CM) and the further-removed matrix (FRM). Immunohistochemical analysis was performed using monoclonal antibodies against chondroitin sulfate and keratan sulfate. Autoradiography using degenerated cartilage tissue from the rabbit osteoarthritis (OA) model was performed to discover the effect of HA on the distribution of newly-synthesized PG in the cartilage tissue. RESULTS: The incorporation of [(35)S]sulfate into newly-synthesized PG in the cells with CM decreased with the addition of 0.125-1.0 mg/ml HA, while the incorporation in the FRM increased. These effects of HA on the distribution of newly-synthesized PG were the same either in chondrocytes with CM or chondrocytes without CM. Immunohistochemical analysis showed that staining of PG in the CM was decreased and staining in the FRM was increased in the HA treated group compared to the control group. Autoradiography using degenerated cartilage tissue from the rabbit OA model indicated that [(35)S]-labeled macromolecules showed a more diffuse distribution in the HA treated group compared with the control group. CONCLUSION: These results indicate that extrinsic HA could affect the movement of newly-synthesized PG from the CM to the FRM in both alginate beads and cartilage tissue.     

力学刺激对软骨细胞整合素亚单位的调控

骨关节炎（osteoarthritis,OA）是以关节软骨退变、关节缘骨质增生为主要改变的疾病。机械应力可以调节细胞的多种功能,而整合素作为细胞表面应力受体之一,主要介导细胞与细胞外基质（extracellularmatrix,ECM）间的黏附,在传导力学信号从而调节细胞的生理功能方面起着重要作用。因此,在软骨病变早中期选择适当的良性刺激（如推拿手法）作用于软骨,调控整合素的表达影响软骨细胞的功能,修复损伤的软骨细胞,延缓关节软骨退变,这对于骨关节炎的治疗有重要意义。