Cartilage and bone metabolism in rheumatoid arthritis. Differences between rapid and slow progression of disease identified by serum markers of cartilage metabolism.

Serum concentrations of specific cartilage and bone molecules reflecting tissue turnover were measured in two well-defined patient groups with early rheumatoid arthritis with distinctly different disease outcome to see if early differences in their levels are prognostic of the rate of joint destruction. Compared with a matched normal population, increased concentrations of cartilage oligomeric matrix protein (COMP) were found in all patients who developed rapid hip joint destruction. In contrast, levels of a putative marker of cartilage aggrecan synthesis, the chondroitin sulfate epitope 846, were increased only in patients with slow joint destruction. Levels of bone sialoprotein (BSP) were increased in both groups, as were levels of the C-propeptide of type II procollagen (CPII), a marker of collagen II synthesis. The increased concentrations of the 846 epitope in patients with slow joint destruction suggest increased aggrecan synthesis. The low levels of the 846 epitope in patients with rapid joint destruction, concomitant with elevated levels of CPII, suggest a selective increase in collagen synthesis. The elevated BSP levels indicate an increased bone turnover in both groups. Thus elevated serum levels of COMP may indicate an unfavorable prognosis for rapid joint destruction, whereas elevated 846 epitope indicates a more favorable prognosis.     

软骨终板通透性对兔退变腰椎间盘Ⅱ型胶原表达的影响

目的探讨软骨终板通透性对兔退变腰椎间盘Ⅱ型胶原表达的影响。方法 6月龄新西兰大白兔14只,建立退变腰椎间盘模型后饲养8周。8周后处死并手术切取腰段椎间盘每只6个,随机分为A组和B组,其中A组为对照组,B组骨蜡封闭上下软骨终板。两组兔退变椎间盘在体外进行整体器官培养。在培养前以及培养7 d和14 d时分别用Mitotracker Green荧光探针、免疫组化SP法和RT-PCR方法对椎间盘中髓核的细胞活力、Ⅱ型胶原及Ⅱ型胶原mRNA的表达情况进行评估。结果经过7 d的体外培养之后,两组的荧光强度、Ⅱ型胶原的灰度值与培养前比较降低不显著(P0.05),而椎间盘髓核Ⅱ型胶原的mRNA表达与与培养前比较差异显著(P0.05),两组间荧光强度、Ⅱ型胶原的灰度值及椎间盘髓核Ⅱ型胶原的mRNA表达对比差异均无统计学意义(P0.05)。经过14 d的培养,两组的荧光强度分别降低了18.6%与31.3%,与培养之前的荧光强度相比以及两组之间相比差异均具有统计学意义(P0.05);两组的Ⅱ型胶原的灰度值均有提升,与培养之前的Ⅱ型胶原的灰度值相比差异具有统计学意义(P0.01),两组间Ⅱ型胶原的灰度值经比较差异显著,具有统计学意义(P0.01);两组Ⅱ型胶原mRNA的表达比培养前以及培养7 d时明显下降(P0.05),两组之间比较差异显著具有统计学意义(P0.05)。结论降低软骨终板的通透性可以在短时间内(14 d)降低细胞活力和Ⅱ型胶原及其mRNA的表达,加速兔腰椎间盘的退变。     

Interleukin 1 suppresses expression of cartilage-specific types II and IX collagens and increases types I and III collagens in human chondrocytes.

In inflammatory diseases such as rheumatoid arthritis, functions of chondrocytes including synthesis of matrix proteins and proteinases are altered through interactions with cells of the infiltrating pannus. One of the major secreted products of mononuclear inflammatory cells is IL-1. In this study we found that recombinant human IL-1 beta suppressed synthesis of cartilage-specific type II collagen by cultured human costal chondrocytes associated with decreased steady state levels of alpha 1 (II) and alpha 1(IX) procollagen mRNAs. In contrast, IL-1 increased synthesis of types I and III collagens and levels of alpha 1(I), alpha 2(I), and alpha 1(III) procollagen mRNAs, as we described previously using human articular chondrocytes and synovial fibroblasts. This stimulatory effect of IL-1 was observed only when IL-1-stimulated PGE2 synthesis was blocked by the cyclooxygenase inhibitor indomethacin. The suppression of type II collagen mRNA levels by IL-1 alone was not due to IL-1-stimulated PGE2, since addition of indomethacin did not reverse, but actually potentiated, this inhibition. Continuous exposure of freshly isolated chondrocytes from day 2 of culture to approximately half-maximal concentrations of IL-1 (2.5 pM) completely suppressed levels of type II collagen mRNA and increased levels of types I and III collagen mRNAs, thereby reversing the ratio of alpha 1(II)/alpha 1(I) procollagen mRNAs from greater than 6.0 to less than 1.0 by day 7. IL-1, therefore, can modify, at a pretranslational level, the relative amounts of the different types of collagen synthesized in cartilage and thereby could be responsible for the inappropriate repair of cartilage matrix in inflammatory conditions.     

Do the intervertebral disc cells respond to different levels of hydrostatic pressure?

OBJECTIVE: To test the hypothesis that hydrostatic pressure directly affects the synthesis of collagen and proteoglycan by intervertebral disc cells. DESIGN: By the use of pressure vessels, hydrostatic pressure was applied to intervertebral disc cells cultured in alginate. BACKGROUND: The influence of compression (both hydrostatic and axial) on chondrocyte metabolism was examined in a number of earlier studies. However, in most of these studies, articular cartilage, not intervertebral disc was used, and in none of these was hydrostatic pressure applied to intervertebral disc cells cultured in alginate. METHODS: Fresh cells were harvested from the lumbar intervertebral discs of dogs. Before their suspension in an alginate gel system, the cells were plated and expanded until they reached confluence. Then, by use of the alginate gel system, the cells were exposed (for up to 9 days) to specific values of hydrostatic pressure inside two stainless steel pressure vessels. One vessel was kept at 0.35 MPa and the other at atmospheric pressure (approximately 0.1 MPa). The effects of 0.35 MPa were compared against atmospheric pressure by measuring the incorporation of [3H]-proline and [35S]-sulfate into collagen and proteoglycans, respectively, for the anulus cells and nucleus cells separately, and by determining whether this incorporation was reflected by changes in the levels of mRNA for aggrecan and Types I and II collagen. RESULTS: Proteoglycan synthesis was inhibited at 0.35 MPa as compared to atmospheric pressure for both the nucleus and anulus cells, whereas collagen synthesis was stimulated in the nucleus cells, but inhibited in the anulus cells. The mRNA levels of collagen 1A and collagen 2A decreased in the anulus but showed a differential response in the nucleus (collagen 1A increased, while collagen 2A decreased). The mRNA levels for aggrecan core protein decreased in the anulus and increased in the nucleus. CONCLUSIONS: Hydrostatic pressure directly affects the synthesis of collagen and proteoglycan by the intervertebral disc cells. RELEVANCE: This in vitro study reveals the direct effect of hydrostatic pressure on disc cells, in the absence of other factors. However, circumspection must be applied when comparisons between these results, from in vitro experiments on dog disc cells, are extrapolated and applied to the whole discs of humans.     

颈椎终板Modic改变分布特点与退变的关系

背景：关于Modic改变在腰椎中分布及特点的相关性研究比较多,而在颈椎中的相关研究则较少。目的：分析颈椎终板Modic改变的临床分布特点,并探讨其发生与颈椎退变的相关性。方法：随机抽取因颈肩痛行颈椎MRl和常规X射线榆查的患者共200例,年龄20-83岁。记录MRl颈椎Modic改变发生的节段,改变类型,改变的位置等,并分析其发生与性别、年龄、椎间盘退变节段及程度、椎间隙高度、颈椎曲度的相关性。结果与结论：200例共计1200个颈椎椎间盘中23例（11.5％）,29个（2.4％）个椎间盘邻近终板发生Modic改变。I型8例（4％）,10个椎间盘（0.8％）;II型13例（6.5％）,16个椎间盘（113％）;III型2例（1.0％）,3个椎间盘（O.25％）。按照各个椎间盘节段发病数统计,C2/3节段O个,C3/4节段4个,C4／5节段6个,C5/6节段12个,C6/7节段7个,C6/7T1节段0个,发病率分别为O％,0.33％,O.5％,1.0％,O.58％,0％。结果表明颈椎终板也存在Modic改变的现象,但发生率较腰椎低,II型最多见,I型次之,III型最为少见,多发生于C5/6椎间盘,多位于邻近终板的后方。50岁以上为其好发年龄,其发生与年龄、椎间盘退变、椎间盘节段及颈椎曲度之间存在相关性。     

Anatomie, Physiologie und Pathophysiologie der Bandscheiben – eine Literaturübersicht

In order to understand the degeneration of intervertebral discs, it is necessary to have knowledge on the physiology and anatomy of healthy intervertebral discs. In part I the current state of knowledge on the anatomy, biochemistry and physiology of intervertebral discs will be presented and part II will deal with the fundamentals of the degeneration of intervertebral discs. Intervertebral discs age faster than nearly all other tissues because the nutrient supply is impeded due to the lack of vascularisation. The most important structural alterations in the degenerative process are decreases in water content and osmotic pressure, especially in the nucleus pulposus, the inner part of the annulus fibrosus, as well as wear of the matrix. The decreasing osmotic pressure strengthens the opening of existing tears despite a decrease in shearing forces in the annulus. Fibrous alterations of the nucleus, breakdown in structure of the annulus and alterations to the vertebral bodies and the end plates occur most commonly in the first two and in the fifth to seventh decades of life. The alterations are more severe in the lower than in the upper vertebral discs and precede the formation of fissures and tears. The time scale shows a strong correlation with fissures and tears which appear in the first decade of life. In contrast edge lesions arise independently from them and appear much later.     

不同代次免髓核细胞的形态学特征和生物学性状

背景：椎间盘退变是个慢性、复杂的过程,然而椎间盘退变其发生机制尚未完全阐明,很难自行修复。近年来研究细胞移植治疗椎间盘退行性变尚处在实验室阶段。研究髓核细胞的生物学性状可为研究椎间盘退变机制、组织工程构建椎间盘、基因治疗等提供理论依据。目的：研究兔不同代次髓核细胞的生物学特性,旨在找出合适的种子细胞去治疗椎间盘退变性疾病。方法：从新西兰大耳白兔椎间盘髓核组织中,分离并培养髓核细胞同时进行培养传代,对原代及第3,4代髓核细胞进行苏木精一伊红染色观察细胞形态学变化;甲苯胺蓝染色和免疫细胞化学法检测髓核细胞内聚集蛋白聚糖和Ⅱ型胶原的表达;反转录PCR法测定Ⅱ型胶原和聚合蛋白聚糖mRNA的表达水平,观察各代髓核细胞生物学特性的变化。结果与结论：兔椎间盘髓核细胞可以在体外培养并进行传代,原代髓核细胞一般需7d左右贴壁,形状呈类圆形或多角形,原代和第3代髓核细胞都呈圆形或多角形,活力较强,苏木精-伊红染色后细胞核被染成均一蓝黑色,胞浆呈现淡粉色;髓核细胞经过甲苯胺蓝染色后,胞浆内呈现天蓝色,通过Ⅱ型胶原免疫组织化学染色后,胞浆内表现为黄褐色沉淀。到第4代细胞出现退变,Ⅱ型胶原和聚合蛋白聚糖mRNA的表达水平较前几代细胞显著下降。前3代的髓核细胞代谢旺盛,表型一致,聚集蛋白聚糖和Ⅱ型胶原表达正常,传第4代后髓核细胞开始出现衰老、退变。     

Studies of the articular cartilage proteoglycan aggrecan in health and osteoarthritis. Evidence for molecular heterogeneity and extensive molecular changes in disease.

Changes in the structure of the proteoglycan aggrecan (PG) of articular cartilage were determined immunochemically by RIA and gel chromatography and related to cartilage degeneration documented histologically by the Mankin grading system. Monoclonal antibodies to glycosaminoglycan epitopes were used. In all cartilages, three chondroitin sulfate (CS)-rich populations of large size were observed in addition to a smaller keratan sulfate (KS)-rich population. In grades 7-13 OA cartilages (phase II), molecules were significantly larger than the equivalent molecules of grades 2-6 (phase I). CS chain lengths remained unchanged. In most OA cartilages, a CS epitope 846 was elevated in content, this being most marked in phase II (mean: fivefold). Loss of uronic acid, KS, and hyaluronic acid were only pronounced in phase II OA because of variations in normal contents. Aggregation of PG was unchanged (50-60%) or reduced in OA cartilages, but molecules bearing epitope 846 exhibited almost complete aggregation in normal cartilages. This study provides evidence for the capacity of OA cartilage to synthesize new aggrecan molecules to replace those damaged and lost by disease-related changes. It also defines two phases of PG change in OA: an early predominantly degenerate phase I followed by a net reparative phase II accompanied by net loss of these molecules.     

The effects of pulsed low-intensity ultrasound on chondrocyte viability, proliferation, gene expression and matrix production

This study was designed to examine the effects of pulsed low-intensity ultrasound (PLIUS) on chondrocyte viability, proliferation, matrix production and gene expression. Chondrocytes were isolated from the distal part of the sternum of 16-day-old chick embryos and cultured in alginate beads. PLIUS at 2 mW/cm(2) (group PLIUS(2)) and 30 mW/cm(2) (group PLIUS(30)) was applied to chondrocytes for a single 20-min treatment. A control group was treated without PLIUS. The viability of chondrocytes was not affected by exposure to PLIUS. PLIUS influenced chondrocyte proliferation in an intensity-dependent manner. By day 7 after application of PLIUS, the gene expression and synthesis of aggrecan was the same as in the controls. At this same time point, the expression and synthesis of type II collagen was not different between the controls and PLIUS(30), but was increased in PLIUS(2). PLIUS was shown to inhibit the expression of type X collagen. This inhibition of chondrocyte hypertrophy may prove to be significant in the management of cartilage degeneration.     

六味地黄丸含药血清对椎间盘I型和Ⅱ型胶原表达的影响

背景：胶原纤维是维持椎间盘正常结构与功能的重要物质,椎间盘中胶原的类型及含量的变化,影响整个椎间盘的生理和病理状态,I型及Ⅱ型胶原是椎间盘胶原的主要组分。目的：观察六味地黄丸含药血清对肿瘤坏死因子a致伤兔椎间盘I型、Ⅱ型胶原蛋白和基因表达的影响。方法：将15只新西兰白兔带终板的45个L2-L5椎间盘随机分为空白第1天组、空白第14天组、肿瘤坏死因子a组、中药血清组、肿瘤坏死因子a＋中药血清组。后两种培养液中分别含5ug/L肿瘤坏死因子a和体积分数10％六味地黄丸血清,培养14d后收集髓核和纤维环观察。结果与结论：RT—PCR和WesternBlot检测结果表明,随着培养时间的延长,纤维环中I型胶原及髓核中Ⅱ型胶原蛋白和基因表达明显降低;加入肿瘤坏死因子a可加速这种变化,而含药血清可延缓I,Ⅱ型胶原蛋白和基因表达变化。提示六味地黄丸可通过稳定I,Ⅱ型胶原蛋白和基因表达,对椎间盘细胞外基质具有保护作用。     

Regulation of procollagen metabolism in the pressure-overloaded rat heart.

To determine the molecular events responsible for the disproportionate accumulation of myocardial fibrillar collagens during sustained hypertension, we examined the in vivo rate of procollagen synthesis, collagen accumulation, and intracellular procollagen degradation 1-16 wk after abdominal aortic banding in young rats. These measurements were correlated with tissue mRNA levels for type I and type III procollagen polypeptides. Banded animals developed moderate, sustained hypertension and mild left ventricular hypertrophy. Increased type III procollagen mRNA levels were detected early after banding and persisted for the entire observation period. Disproportionate collagen accumulation without histological evidence of fibrosis was noted within 1 wk after hypertension induction. Fibrillar collagen accumulation at this time point resulted not from a major increase in procollagen synthesis, but rather a marked decrease in the rate of intracellular procollagen degradation. Interstitial fibrosis, however, was observed 16 wk after banding. Type I procollagen mRNA levels were increased six-fold, but only after 16 wk of hypertension. These results correlated well with the results of in vivo procollagen synthesis experiments at 16 wk, which demonstrated a threefold increase in left ventricular procollagen biosynthesis. We conclude that pretranslational as well as posttranslational mechanisms regulate fibrillar collagen deposition in the myocardial extracellular matrix during sustained hypertension.     

髓核细胞移植抑制椎间盘退变

背景：近年来,椎间盘细胞移植和椎间盘移植修复椎间盘退变研究取得较大的进展。目的：探讨髓核细胞的体外培养方法以及髓核细胞移植在抑制椎间盘退变研究中的作用。方法：通过数据库检索的方式探讨髓核细胞移植抑制椎间盘退变的研究。髓核细胞的主要功能是产生胶原和蛋白聚糖,改进髓核细胞的培养方法,使培养后的髓核细胞数量增多,合成和分泌细胞外基质增加,通过髓核细胞移植来修复退变的椎间盘。结果与结论：通过三维小球聚集培养、微载体旋转立体培养等方法可以使髓核细胞数量增多,肝细胞生长因子对髓核细胞增殖产生促进作用。髓核细胞移植可以恢复退变椎间盘高度,促进退变椎间盘内蛋白多糖和Ⅱ型胶原的生物合成和分泌。随着对椎间盘退变研究的不断深入,髓核细胞移植对退变椎间盘可能是一种重要的治疗手段。     

Damage to type II collagen in aging and osteoarthritis starts at the articular surface, originates around chondrocytes, and extends into the cartilage with progressive degeneration.

Enhanced denaturation of type II collagen fibrils in femoral condylar cartilage in osteoarthritis (OA) has recently been quantitated immunochemically (Hollander, A.P., T.F. Heathfield, C. Webber, Y. Iwata, R. Bourne, C. Rorabeck, and A.R. Poole. 1994. J. Clin. Invest. 93:1722-1732). Using the same antibody that only reacts with denatured type II collagen, we investigated with immunoperoxidase histochemistry (results were graded for analysis) the sites of the denaturation (loss of triple helix) of this molecule in human aging (at autopsy, n= 11) and progressively degenerate (by Mankin grade [MG]) OA (at arthroplasty, n= 51) knee condylar cartilages. Up to 41 yr, most aging cartilages (3 of 4) (MG 0-4) showed very little denaturation. In most older cartilages, (4 of 7) (MG 2-4), staining was observed in the superficial and mid zones. This pattern of collagen II denaturation was also seen in all OA specimens with increased staining extending to the deep zone with increasing MG. Collagen II staining correlated directly both with MG and collagen II denaturation measured by immunoassay. Cartilage fibrillation occurred in OA cartilages with increased penetration of the staining for collagen II denaturation into the mid and deep zones and where denaturation was more pronounced by immunoassay. Thus in both aging and OA the first damage to type II collagen occurs in the superficial and upper mid zone (low MG) extending to the lower mid and deep zones with increasing degeneration (increasing MG). Initial damage is always seen around chondrocytes implicating them in the denaturation of type II collagen.     

胰岛素样生长因子1及血小板源性生长因子对人退变髓核细胞生物学活性的影响

背景：有研究表明胰岛素样生长因子1和血小板源性生长因子可抑制人椎间盘细胞凋亡。目的：观察在体外培养条件下胰岛素样生长因子1、血小板源性生长因子对人退变髓核细胞生物学活性的影响。方法：体外单层培养人退变髓核细胞,通过免疫组织化学鉴定细胞。对传3代人退变髓核细胞采用分别不同生长因子干预,实验分4组：胰岛素样生长因子1组,血小板源性生长因子组,胰岛素样生长因子1＋血小板源性生长因子组及对照组。结果与结论：胰岛素样生长因子1、血小板源性生长因子均可促进细胞增殖,促进细胞合成Ⅱ型胶原和聚集蛋白聚糖,其中胰岛素样生长因子1促Ⅱ型胶原合成作用强于血小板源性生长因子（P〈0.05）,血小板源性生长因子促蛋白聚糖合成作用强于胰岛素样生长因子1（P〈0.05）。胰岛素样生长因子1促进细胞合成Ⅰ型胶原,血小板源性生长因子抑制细胞合成Ⅰ型胶原。结果证实,胰岛素样生长因子1、血小板源性生长因子均可通过促进细胞增殖、促进细胞合成Ⅱ型胶原和聚集蛋白聚糖,从而提高人退变髓核细胞的生物学活性。     

过氧化物酶Ⅱ对体外培养人退变腰椎间盘髓核细胞的抑制作用

背景:椎间盘退变可导致髓核细胞数量的减少,过氧化物酶Ⅱ可参与细胞的抗氧化损伤、细胞分裂、分化、信号转导和凋亡等过程的调控.过氧化物酶Ⅱ对椎间盘退变有促进作用,但其机制仍不清楚.目的:观察过氧化物酶Ⅱ对体外培养的人退变腰椎间盘髓核细胞的活性和Ⅱ型胶原合成的影响.方法:体外培养人退变腰椎间盘髓核细胞,设置对照组及过氧化物酶Ⅱ10,100和1 000 ng/L组.对照组不含氧化物酶Ⅱ,其他3组加入相应剂量的氧化物酶Ⅱ.应用免疫组化法鉴定髓核细胞,并采用cck-8试剂盒检测细胞增殖情况,于加过氧化物酶Ⅱ后第3和7天分别取对照组及各组细胞上清液,采用双抗体夹心酶联免疫吸附实验测定Ⅱ型胶原表达情况.结果与结论:在体外培养的人退变腰椎间盘髓核细胞,随加入的过氧化物酶Ⅱ质量浓度的增加,椎间盘髓核细胞数量和Ⅱ型胶原合成逐渐减少(P＜0 01).提示过氧化物酶Ⅱ对椎间盘髓核细胞的数量和Ⅱ型胶原合成有明显的抑制作用,并呈剂量依赖关系.以此推测过氧化物酶Ⅱ对髓核细胞的抑制作用可能是导致椎间盘退变的一种促发因素.     

体外阻断基质细胞衍生因子1/CXC趋化因子受体4信号通路后人关节软骨组织Ⅱ型胶原和聚集蛋白聚糖的表达

背景：基质细胞衍生因子1/CXC趋化因子受体4信号通路在骨关节炎的发病中起关键作用。目的：探讨AMD3100体外阻断基质细胞衍生因子1/CXC趋化因子受体4信号通路后对培养的关节软骨组织Ⅱ型胶原、聚集蛋白聚糖mRNA表达及Ⅱ型胶原蛋白表达的影响。方法：将骨关节炎软骨和创伤性截肢患者正常软骨分别分为3个小组即实验组,实验对照组,空白对照组。将其置于含基质细胞衍生因子1和AMD3100,含基质细胞衍生因子1和MAB310,只含基质细胞衍生因子1的培养液培养。结果与结论：实验组软骨组织内的Ⅱ型胶原和聚集蛋白聚糖mRNA表达量、Ⅱ型胶原蛋白含量高于实验对照组和空白对照组（P〈0.05）。可见基质细胞衍生因子1通过基质细胞衍生因子1/CXC趋化因子受体4信号通路诱导人关节软骨中Ⅱ型胶原和聚集蛋白聚糖的降解;AMD3100可阻断该信号通路及减少软骨中Ⅱ型胶原和聚集蛋白聚糖的降解,延缓关节软骨组织的退变;AMD3100不能使已退变的骨关节炎软骨内的Ⅱ型胶原和聚集蛋白聚糖含量恢复到正常水平。     

胰岛素样生长因子1及血小板源性生长因子对人退变髓核细胞生物学活性的影响

背景:有研究表明胰岛素样生长因子1和血小板源性生长因子可抑制人椎间盘细胞凋亡.目的:观察在体外培养条件下胰岛素样生长因子1、血小板源性生长因子对人退变髓核细胞生物学活性的影响.方法:体外单层培养人退变髓核细胞,通过免疫组织化学鉴定细胞.对传3代人退变髓核细胞采用分别不同生长因子干预,实验分4组:胰岛素样生长因子1组,血小板源性生长因子组,胰岛素样生长因子1+血小板源性生长因子组及对照组.结果与结论:胰岛素样生长因子1、血小板源性生长因子均可促进细胞增殖,促进细胞合成Ⅱ型胶原和聚集蛋白聚糖,其中胰岛素样生长因子1促Ⅱ型胶原合成作用强于血小板源性生长因子(P < 0.05),血小板源性生长因子促蛋白聚糖合成作用强于胰岛素样生长因子1(P < 0.05).胰岛素样生长因子1促进细胞合成Ⅰ型胶原,血小板源性生长因子抑制细胞合成Ⅰ型胶原.结果证实,胰岛素样生长因子1、血小板源性生长因子均可通过促进细胞增殖、促进细胞合成Ⅱ型胶原和聚集蛋白聚糖,从而提高人退变髓核细胞的生物学活性.     

Increased damage to type II collagen in osteoarthritic articular cartilage detected by a new immunoassay.

A new immunoassay was developed to detect denaturation of type II collagen in osteoarthritis (OA). A peptide, alpha 1 (II)-CB11B, located in the CB11 peptide of type II collagen, was synthesized and used to produce a monoclonal antibody (COL2-3/4m) of the IgG1 (kappa) isotype. This reacts with a defined epitope in denatured but not native type II collagen and the alpha 3 chain of type XI collagen. The latter is present in very small amounts (about 1% wt/wt) in cartilage relative to the alpha 1 (II) chain. By using an enzyme-linked immunosorbent assay, type II collagen denaturation and total type II collagen content were determined. The epitope recognized by the antibody was resistant to cleavage by alpha-chymotrypsin and proteinase K which were used to extract alpha 1 (II)-CB11B from the denatured (alpha-chymotrypsin soluble) and residual native (proteinase K soluble) collagen alpha-chains, respectively, present in human femoral articular cartilage. Type II collagen content was significantly reduced from a mean (range) of 14% (9.2-20.8%) of wet weight in 8 normal cartilages to 10.3% (7.4-15.0%) in 16 OA cartilages. This decrease, which may result in part from an increased hydration, was accompanied by an increase in the percent denaturation of type II collagen in OA to 6.0% of total type II collagen compared with 1.1% in normal tissue. The percent denaturation was ordinarily greater in the more superficial zone than in the deep zone of OA cartilage.     

Performance of new gellan gum hydrogels combined with human articular chondrocytes for cartilage regeneration when subcutaneously implanted in nude mice

Gellan gum is a polysaccharide that has been recently proposed by our group for cartilage tissue‐engineering applications. It is commonly used in the food and pharmaceutical industry and has the ability to form stable gels without the use of harsh reagents. Gellan gum can function as a minimally invasive injectable system, gelling inside the body in situ under physiological conditions and efficiently adapting to the defect site. In this work, gellan gum hydrogels were combined with human articular chondrocytes (hACs) and were subcutaneously implanted in nude mice for 4 weeks. The implants were collected for histological (haematoxylin and eosin and Alcian blue staining), biochemical [dimethylmethylene blue (GAG) assay], molecular (real‐time PCR analyses for collagen types I, II and X, aggrecan) and immunological analyses (immunolocalization of collagen types I and II). The results showed a homogeneous cell distribution and the typical round‐shaped morphology of the chondrocytes within the matrix upon implantation. Proteoglycans synthesis was detected by Alcian blue staining and a statistically significant increase of proteoglycans content was measured with the GAG assay quantified from 1 to 4 weeks of implantation. Real‐time PCR analyses showed a statistically significant upregulation of collagen type II and aggrecan levels in the same periods. The immunological assays suggest deposition of collagen type II along with some collagen type I. The overall data shows that gellan gum hydrogels adequately support the growth and ECM deposition of human articular chondrocytes when implanted subcutaneously in nude mice. Copyright © 2009 John Wiley & Sons, Ltd.