Demonstration of increased proteoglycan turnover in cartilage explants from dogs with experimental osteoarthritis

The turnover of proteoglycans (assessed by the release into the medium of newly synthesised [35S]-proteoglycan) in explant cultures of articular cartilage from various anatomical sites of the knee joints (stifle) of mature beagles with experimental osteoarthritis has been studied with the following findings: (a) The proportion of newly synthesised proteoglycans released from cartilage explants maintained in vitro was generally increased for cartilage from operated compared with nonoperated control joints. (b) At 3 weeks after surgery there was a significant increase in the release of [35S]-proteoglycans from explants of the lateral and medial tibial plateaux of operated joints compared with sham-operated joints but not from other sites. On the other hand, when this comparison was made at 3 to 6 months after surgery, significant increases in the release of [35S]-proteoglycans were observed from cartilage of all anatomical areas except the patellar groove. (c) The release of [35S]-proteoglycan from cartilage explant cultures was dependent on live chondrocytes, since freeze-thawing the tissue immediately after labelling markedly reduced the release from both normal and osteoarthritic cartilage.     

Analysis of changes in proteoglycan content in murine articular cartilage using image analysis

The extracellular matrix of articular cartilage consists mainly of type II collagen and large aggregating proteoglycan (aggrecan). During arthritis and other joint diseases, the proteoglycan (PG) level of cartilage matrix is diminished, leading to impairment of normal joint function. A new method is described for measuring the changes in PG content of murine articular cartilage. The method is based on the automated densitometric analysis of patellar cartilage of standard, safranin O-stained sections of whole murine knee joints. It appeared to be possible to measure optical density in parallel layers of articular cartilage with high reproducibility. Approximately 25 sections can be evaluated within 1 h. Measuring a single section 10 times resulted in a coefficient of variation (CV) of 0.1-1.4%. A mean CV of 5-14% was calculated when a group of 18 sections was analyzed in quintuplicate. To validate the procedure, changes in PG content induced by arthritis or by intra-articular injection of TGFbeta-1 were analyzed by the image analysis method, the dimethylmethylene blue (DMB) assay and by visual grading. Although not a quantitive method, the newly developed image analysis method appeared to be more sensitive in detecting significant change in PG content of murine articular cartilage than the DMB method or visual grading. The image analysis method makes it possible to measure changes in PG content of specific areas of articular cartilage with higher sensitivity than the DMB method and eliminating the bias inherent to visual grading by human observers.     

Analysis of experimental immune synovitis cartilage using monoclonal antibodies reactive to rabbit proteoglycan

This study details the macromolecular changes in cartilage involving proteoglycan molecules in an animal model of rheumatoid arthritis. In experimental chronic immune synovitis, fluorescein-conjugated mouse IgG and three monoclonal antibodies (MAbs 2G2, 2E9, and 6C9) portraying differing fine antigenic specificity for rabbit cartilage proteoglycan monomer were utilized to detail alterations in cartilage proteoglycan. In normal and IgG-immune animals, fluorescein isothiocynate (FITC)-conjugated MAbs 2G2 and 2E9 stained cellular/pericellular (C/PC) region intensely. FITC-MAb 2G2 stained cartilage interterritorial matrix as well. FITC-MAb 6C9 stained only C/PC area lightly but did not stain matrix. A marked decrease in staining intensity with FITC-MAb 2G2 was noted in cartilage sections derived from animals with immune synovitis. A corresponding increase in staining of cartilage was noted with FITC-MAb 6C9. The augmented staining of articular cartilage with FITC-MAb 6C9 was most prominent in femoral condyle tissue sections, which corresponded to the cartilaginous area, with the greatest severity in gross pathology. There was a slight augmentation of staining with FITC-MAb 2E9, especially in the C/PC area of medial/femoral cartilage. In addition, the animals with immune synovitis showed abortive cartilage repair exemplified by the presence of chondrocyte cloning (up to 20 cells) which correlated with increased FITC-MAb 2G2 staining. The differential MAb staining patterns of cartilaginous tissues obtained utilizing FITC-conjugated monoclonal antibodies with known fine antigenic specificity indicates a modulation of proteoglycans involving predominantly core protein epitopes in the articular cartilage of animals with chronic immune synovitis.     

Nitric oxide and proteoglycan turnover in rabbit articular cartilage

Articular chondrocytes are known to synthesize large amounts of nitric oxide in response to exposure to interleukin-1, but the role of this radical in proteoglycan turnover remains controversial. In this study, we used two different inhibitors of nitric oxide synthase, N^G-methyl-L -arginine and thiocitrulline, to study the effects of nitric oxide on the synthesis and breakdown of proteoglycan in rabbit articular cartilage. Synthes of nitric oxide by cartilage slices in response to treatment with interleukin-1 and a partially purified mixture of synovial cytokines known as chondrocyte-activating factors peaked during the first 2 days of culture and then fell to low levels, despite daily replenishment with fresh medium and cytokines to the cultures. The production of nitric oxide was completely inhibited by N^G-methyl-L -arginine and thiocitrulline. Interleukin-1 and the chondrocyte-activating factors inhibited proteoglycan synthesis and accelerated proteoglycan breat down in the slices of cartilage. Both nitric oxide synthase inhibitors substantially counteracted the suppression of prdteoglycan synthesis but exacerbated proteoglycan catabolism occurring in response to interleukin-1 and the chondrocyte-activating factors. The accelerated eatabolism was associated with increased levels of matrix metalloproteinases in the conditioned medium. This dual effect of nitric oxide complicates decision making with regard to the possible clinical applications of nitric oxide agonists or antagonists in diseases of cartilage.     

Intra-articular sodium hyaluronate in osteoarthritis of the knee: a multicenter, double-blind study

The efficacy and the safety of intra-articular injections of sodium hyaluronate were studied in patients with osteoarthritis of the knee in a randomized multicenter double-blind study. Two hundred and nine patients received five injections of either 25 mg hyaluronate/2.5 ml (verum, N = 102) or 0.25 mg hyaluronate/2.5 ml (control, N = 107) at weekly intervals. Seven patients in each group were excluded from the protocol-correct efficacy analysis. The Lequesne Index, the first main criterion, showed a significant superiority of the verum-treated patients after the third injection up to the final follow-up examination 9 weeks after the last injection (MANOVA, P < 0.025). The consumption of paracetamol was defined as a complementary main criterion that did not reveal significant differences between the treatment groups. Most of the individual secondary endpoints demonstrated a much better response to the active treatment without reaching the significance level in the intergroup comparisons for the single time-points. Side-effects were confined to local reactions of minor severity and short duration in four patients (six events) of the verum group and in five patients of the control group. Clinical chemistry and hematology remained essentially unchanged.     

Chondro-osseous metaplasia, bone density and patellar cartilage proteoglycan content in the osteoarthritis of STR/ORT mice

The evolution of osteoarthritis (OA) was compared in male and female STR/ORT mice and in male CBA mice with particular emphasis on the changes in para-articular structures which occur before the classical degenerative phenomena in the articular cartilage of the knee. In male STR but not female STR mice or male CBA mice, chondro-osseous metaplasia was found in the tendinous structures which surround the joint and in the major ligamentous entheses such as the patellar ligament. This change was detectable from the age of three months. By contrast, changes in the articular surfaces of the knee were never detected before four months while the total patellar proteoglycan content in male STR animals increased from three months. Line density profile measurements on radiographs of the knees revealed that changes in the bone density of the sub-chondral zone of the articular surfaces only increased in affected animals after the development of severe OA changes in the joint. It is suggested that the model of OA which develops in male STR/ORT mice is secondary to soft tissue changes, particularly chondro-osseous metaplasia in para-articular structures.     

关节内注射富含血小板血浆在兔膝骨关节炎动物模型中的作用

目的评估关节内注射富含血小板血浆(PRP)对胶原酶诱导的兔膝骨关节炎(OA)动物模型的影响。方法 20只新西兰大白兔在超声引导下行右侧膝关节内胶原酶注射,建立兔OA动物模型。实验的第4周随机分为PRP组和生理盐水组,每组10只,每周给药1次,共4次。实验第9周取关节软骨观察软骨总体形态及软骨微形态的变化。结果 PRP组软骨总体形态评分为(1.7±0.3)分,明显低于生理盐水组的(3.5±0.6),分,差异有统计学意义(P0.05),提示PRP组软骨总体形态损伤较小;PRP组软骨微形态评分为(3.2±1.0)分,明显低于生理盐水组的(7.4±1.2)分,差异有统计学意义(P0.05),提示PRP组软骨微形态损伤较小。结论关节内注射PRP有保护软骨作用。     

同种软骨移植后胶原含量变化的实验研究

采用生化手段测定了豚鼠异体软骨移植后1～20周软骨胶原含量的变化,结果显示:术后第3周起软骨胶原含量开始下降,至第4周达最低点(P<0.05),此后逐渐回升,至第8周时已接近于正常组并维持在接近于对照组的水平(P>0.05)。提示可采用生化测定胶原在同种软骨移植后不同时期的含量变化来评价异体软骨移植后的结构和弹性变化。     

同种软骨移植后胶原含量变化的实验研究

采用生化手段测定了豚鼠异体软骨移植后１～２０周软骨胶原含量的变化，结果显示：术后第３周起软骨胶原含量开始下降，至第４周达最低点（Ｐ＜０．０５），此后逐渐回升，至第８周时已接近于正常组并维持在接近于对照组的水平（Ｐ＞０．０５）。提示可采用生化测定胶原在同种软骨移植后不同时期的含量变化来评价异体软骨移植后的结构和弹性变化。     

Mechanical and biochemical changes in the superficial zone of articular cartilage in canine experimental osteoarthritis

The changes in the tensile mechanical properties and biochemical composition of the superficial zone of articular cartilage were examined in a canine model of early osteoarthritis generated by transection of the anterior cruciate ligament. Sixteen weeks following ligament transection, the tensile stiffness of the articular cartilage was decreased by 44% and the ion-induced stress relaxation of the tissue was increased by 57% compared with the contralateral control. Biochemical analyses indicated that the water content of the experimental tissue was increased by 13%, which was reflected as an apparent 37% decrease in the proteoglycan content and a 36% decrease in the collagen content (expressed per wet weight). The hydroxypyridinium crosslink density was decreased in the experimental tissue by 11%. A significant negative correlation was found between the ion-induced stress relaxation and the hydroxypyridinium crosslink density in both control tissue (R = ?0.56) and experimental tissue (R = ?0.70). No correlation was noted between the tensile stiffness and the biochemical composition of the tissue. These results suggest that, in the superficial zone of articular cartilage, the structure of the tissue may play a more important role than the composition in the determination of its mechanical properties. A major event observed in the model of early osteoarthritis appears to be the disruption and remodeling of the collagen network in the superficial zone of the articular cartilage.     

Immunological analysis of proteoglycan structural changes in the early stage of experimental osteoarthritic canine cartilage lesions.

Specific modifications of the proteoglycan (PG) structure of osteoarthritic (OA) dog cartilage in relation to endogenous metalloprotease activity were examined using murine anti-proteoglycan monoclonal antibodies (MoAbs). OA lesions were induced over a period of 8 weeks in crossbred dogs (Pond-Nuki model). The articular cartilage was removed and homogenized in a Tris buffer, pH 7.5, and then divided into four groups: direct PG extraction, no addition, presence of 1 mM p-aminophenyl mercuric acetate (APMA), and presence of 1 mM APMA and 10 mM o-phenanthroline, incubated for 42 h at 37 degrees C followed by PG extraction. MoAbs reactive with PG protein and carbohydrate epitopes included 1C6, 3B3, 5D4, D1B2, and m4D6. The results showed marked alterations induced by APMA activation of the endogenous metalloproteases. PG changes were apparent at at least three sites: one was either in the hyaluronic acid-binding region or between the hyaluronic-binding region and the G2 globular domain, another was between the keratan-sulfate-rich domain and the chondroitin sulfate-attachment domain, and a third was in the chondroitin sulfate-attachment domain. Constitutive metalloprotease activity resulted in less marked PG alterations with preservation of functional PG aggregation to hyaluronan.     

软骨代谢标志物对骨关节炎软骨改变的反映

目的研究血清中蛋白聚糖和Ⅱ型胶原水平与骨关节炎严重程度间的关系,及对手术后软骨代谢改变的反映。方法研究对象包括65例膝骨关节炎患者及22名正常人。45例患者分别行不同方式手术治疗,术后半年复查。正常人及患者术前和复查时抽取静脉血,摄下肢负重位X线片。应用酶联免疫吸附方法检测血清样本中蛋白聚糖及Ⅱ型胶原的水平。结果骨关节炎患者血清中蛋白聚糖及Ⅱ型胶原水平均显著高于正常对照组,差异有显著性意义(P<0.05)。血清中蛋白聚糖和Ⅱ型胶原水平在轻型骨关节炎组升高,在关节明显狭窄组水平最高,而在严重狭窄组明显降低。术后半年,全膝关节置换术组血清中蛋白聚糖水平较术前明显下降(P<0.05);截骨术组血清中Ⅱ型胶原水平较术前明显下降(P<0.05)。结论血清中蛋白聚糖和Ⅱ型胶原的水平与骨关节炎软骨的破坏程度、软骨细胞的合成反应及软骨的总量有关。蛋白聚糖和Ⅱ型胶原水平是反映软骨代谢改变的较敏感的指标。     

An experimental study on the pathogenesis of osteoarthritis--histological and biochemical changes of proteoglycan in the osteoarthritic cartilage of rabbit in the early stage-- (author's transl)

Osteoarthritis is generally a slowly progressive destructive disease of joints. As the disease is asymptomatic in its initial phases, it was detectable only at the later stage when radiological changes and clinical symptom had developed. The biochemical and cellular changes that had been recorded were those of the later clinically recognizable stage of the disease. Because the time of onset in not known, the initial events which might be the most important to investigate the pathogenesis of osteoarthritis, are virtually impossible to investigate except in experimentally induced models of osteoarthritis. For the purpose of investigating the pathogenesis of degenerative osteoarthritis, experimental osteoarthritic model was prepared. Section of the medial collateral and both cruciate ligaments combined with resection of the medial meniscus in adult rabbit knees showed progressive histological changes similar to those in human osteoarthritis. Therefore, qualitative and progressive alteration of the cartilage matrix proteoglycan in experimental osteoarthritis of the early stages were investigated with time. Biochemical analysis of the cartilage from experimental osteoarthritic model demonstrated a decrease in the content of glycosaminoglycan and increase in the rate of the synthesis of glycosaminoglycan as compared to the normals. The analysis of disaccharide chain revealed a decrease in the concentration of kelatan sulfate and chondroitin-6 sulfate, along with an increase in the concentration of chondroitin-4 sulfate. These findings, which were quite consistent with those in human osteoarthritis and were contrary to those in aging, indicate the attempts of cells to compensate for the proteoglycan deficiency in osteoarthritic cartilage by synthesizing large quantities of glycosaminoglycans of immature pattern. On the other hand, synthesized aggregated proteoglycans in arthritic cartilage had shown a polydispersive pattern in CsCl density gradient compared to that of normal one, which had shown a single curved pattern. Further, newly synthesized proteoglycan in osteoarthritic cartilage seemed to be polydispersive compared with control proteoglycan in molecular sizes on Sepharose 2B gel chromatography under non-aggregated dissociative condition. Large molecular size proteoglycan composed of small molecular size glycosaminoglycan on its polysaccharide chain and small molecular size proteoglycan composed of large molecular size glycosaminoglycan. This might represent the expression of repair mechanism in the damaged cartilage, or phenotypic transformation of extracellular macromolecule in osteoarthritis.     

Intra-articular injections and articular cartilage metabolism. An experimental study in rabbits.

We studied the effects of repeated intra-articular injections of sterile 140 mM NaCl solution on articular cartilage in adult rabbits. After 20 injections into the knee joints over a period of 4 weeks, chondrocyte glucosaminoglycan synthesis was evenly reduced in all cartilage layers, accompanied by a significant proteoglycan depletion of the matrix which was most marked in the superficial half of the cartilage. These and other changes only partially reversed during a further 4-week period after the injections had been stopped. Our data underline the need for a clear-cut indication for intra-articular injections. The microtrauma caused by injection, in conjunction with the introduction of a carrier solution into the joint, may, at least when repeated at short intervals, lead to measurable damage to the articular cartilage.     

Effects of SKI 306X,a new herbal agent,on proteoglycan degradation in cartilage explant culture and collagenase-induced rabbit osteoarthritis model

OBJECTIVE: Protective effects of SKI 306X, a natural herbal product extracted from three herbs Clematis mandshurica, Trichosanthes kirilowii, and Prunella vulgaris, on articular cartilage was examined and compared with other osteoarthritis (OA) drugs using in vitro and in vivo models. METHODS: In vitro culture of rabbit articular cartilage explants was used as a model to measure the effects of drugs on the matrix degradation. The recombinant human interleukin-1alpha (rhIL-1alpha, 5 ng/ml) was added to induce proteoglycan (PG) degradation and the degree of PG degradation was assessed by measuring the amount of glycosaminoglycan (GAG) released into the culture medium. In in vivo experiment, collagenase was intraarticularly injected twice into the right knee joint of rabbits to induce OA-like change, and test agents were orally administered once a day for 28 days. The degrees of OA-like changes were evaluated through a histological examination. RESULTS: In vitro study revealed SKI 306X inhibited the degradation of PG in a concentration-dependent manner. Trichosanthes kirilowii, which is one of the major components of SKI 306X, also significantly inhibited the GAG release in cartilage explant culture at 0.3 and 0.1 mg/ml. Dexamethasone and NSAIDs, such as diclofenac and rofecoxib, had no significant effects on the suppression of PG degradation. In in vivo studies, OA-like degeneration of the articular cartilage and synovial tissue was induced by injecting collagenase into the right knee joint of mature rabbits. At a dose of 200 mg/kg, SKI 306X reduced the OA-like histological changes, whereas diclofenac had no effect at 10 mg/kg. CONCLUSION: These results indicate that SKI 306X inhibited PG degradation in cartilage explant culture, and its prophylactic administration significantly protected the knee joint of rabbit from OA-like change in collagenase-induced experimental OA model. This strongly suggests that SKI 306X can be a good OA agent with some cartilage protection activity.     

兔骨关节炎软骨缺损动物模型的建立

目的探讨建立一种便捷实用的兔骨关节炎软骨缺损动物模型的方法，以适应软骨组织工程技术修复骨关节炎软骨缺损研究的要求。方法5-7月龄新西兰大白兔22只，雌雄不限，体重2．5～3．0kg。根据侧别，分为改良侧（左侧膝）及对照侧（右侧膝）。改良侧分别切除兔内侧半月板、前十字韧带并在股骨髌沟部制造一直径4mm，深3mm的软骨缺损，对照侧仅切除内侧半月板和前十字韧带。分别在术后第3周和第6周在双侧股骨髁部和髌沟部取材，比较两种骨关节炎动物模型的大体形态及病理变化，进行Mankin评分及统计学分析。结果术后6周，改良侧股骨髌沟软骨缺损仍明显存在，但缺损面直径减小，股骨髌沟墨汁染色均达Ⅳ级，光学显微镜下示股骨髌沟软骨缺损达钙化层以下；而对照侧股骨髌沟墨汁染色均未达Ⅳ级。术后3周，改良侧股骨髌沟部Mankin法OA评分（11．82±1．07）分，对照侧（2．37±0．62）分；术后6周，改良侧股骨髌沟部Mankin法OA评分（13．29±1．15）分，对照侧（5．65±1．03）分；改良侧与对照侧股骨髌沟部Mankin评分比较差异有统计学意义（P〈0．01），但股骨髁部Mankin评分两组比较，差异无统计学意义（P〉0．05）。改良侧关节软骨退变进行性加重。结论改良侧和对照侧均能获得满意的骨关节炎动物模型。改良侧在股骨髌沟处形成一个明显的陈旧性软骨缺损，为应用软骨组织工程技术研究骨关节炎软骨缺损修复提供了一种便捷实用的动物模型。     

吡咯喹啉醌对兔骨关节炎模型关节软骨的保护作用

目的 观察吡咯喹啉醌(PQQ)对兔骨关节炎模型关节软骨的保护作用.方法 Hulth法建立日本大耳白兔右侧膝关节骨关节炎模型;实验组兔术后给予关节腔内注射1×104 μmol/LPQQ,0.2 ml/kg,3d1次,连续6周;对照组兔行关节腔内等量生理盐水注射;伪手术组兔仅暴露关节,不进行药物处理.术后6周行膝关节前后位X线检查,并行Kellgren-Lawrance评分;观察股骨髁关节面大体形态并行软骨Pelletier JP评分;制备切片行苏木素-伊红(HE)染色,光镜观察、Mankin评分;行Ⅱ型胶原免疫组织化学染色;原位末端转移酶标记(TUNEL)法检测软骨细胞凋亡;透射电镜下观察软骨细胞线粒体形态和数目的改变.结果 实验组、对照组和伪手术组Kellgren-Lawrance 评分为1.49±0.45、5.69±1.08、0.28±0.06;JP评分为1.46±0.42、2.75±0.60、0.08 ±0.04;膝关节组织学Mankin评分为2.68±0.86、6.95±1.46、0.25 ±0.06;凋亡软骨细胞所占总细胞数目的比例为(5.60±1.20)％、(35.30±4.90)％、(0.70±0.30)％;实验组、对照组和伪手术组电镜下软骨细胞线粒体数为3.40±0.68、1.24±0.45、6.40±1.20;上述各指标组间比较差异有统计学意义(P＜0.05).实验组免疫组织化学染色可见Ⅱ型胶原纤维组织结构良好,而对照组软骨Ⅱ型胶原缺失.结论 关节腔内注射PQQ可保护软骨细胞,减轻关节软骨结构及功能的破坏,减缓关节软骨退变.PQQ可能是通过保护软骨细胞线粒体而发挥作用.     

Cyclodextrin polysulphate protects articular cartilage in experimental lapine knee osteoarthritis

OBJECTIVE: To evaluate the in vivo chondroprotective effect of cyclodextrin polysulphate (CDPS) in a rabbit model of experimental osteoarthritis (OA). DESIGN: Experimental OA was induced in rabbits by anterior cruciate ligament transection (ACLT). Forty-eight hours post-surgery, the rabbits were randomised into three treatment groups (n=15 in each group) and a sham-operated control group. The rabbits were either injected subcutaneously with saline, 0.25mg/kg CDPS or 1mg/kg CDPS once a week for a period of 12 weeks, and their weight was monitored as a parameter for their general status. The animals were then sacrificed for macroscopic and histological assessment of the knee joints. RESULTS: At the lowest dose, CDPS treatment was unable to induce a significant improvement of cartilage degradation vs the saline control in the experimentally induced knee OA. However, subcutaneous injections of 1mg/kg CDPS induced a marked inhibition (P<0.05) of osteophyte formation. Additionally, a significant reduction of cartilage degradation revealed an overall chondroprotective effect of CDPS at a concentration of 1mg/kg. No significant effects on weight gain were noted. CONCLUSIONS: Systemic administration of CDPS is able to protect cartilage in vivo and can therefore be considered as a chondroprotective agent with structure modifying capacities.