Intra-articular injections and articular cartilage metabolism

Summary 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.Recipient of grant no. Ne 308/1-1 from the Deutsche Forschungsgemeinschaft, Bonn, Germany     

Effect of human interleukin-1 on cartilage metabolism

This study was undertaken to elucidate the effect of human interleukin-1 (IL-1) on the cartilage metabolism, with H235SO4 and 3H-proline as the respective indices for the metabolism of proteoglycan and collagen. We examined in vitro the 35S and 3H rates of incorporation after IL-1 was added to free chondrocytes incubated from human and porcine joint cartilage. As a result, IL-1 suppressed dose-dependently the secretion of both 35S and 3H into the supernatant of the medium containing free human or porcine chondrocytes. There was no difference between the control and the IL-1 group in the elution pattern of proteoglycan on the column-chromatogram. These results indicated that human IL-1 did not affect the molecular weight of the proteoglycan produced by human and porcine chondrocytes.     

丝裂酶原活化蛋白激酶激酶1/2抑制对家兔关节软骨保护作用的研究

目的评价丝裂酶原活化蛋白激酶（mitogen—activated protein kinase，MAPK）信号转导通路中MAPK激酶1／2（MEKI／2）在骨关节炎发病过程中的作用。方法新西兰家兔30只，制作OA模型，随机分成3组，每组10只。从造模术后第4天开始，第1组和第2组分别注射100μmol／L和40μmol／L的PD98059，第3组注射PBS液体作为安慰剂对照组，每周2次。另取10只家兔，正常关节内注射PBS液体作为正常对照组。8周后处死动物，进行股骨髁关节软骨退变的大体评分。用Western Blot印记杂交法测定软骨组织中ERK1／2以及磷酸化ERK1／2的表达。用实时定量PCR方法测定基质金属蛋白酶-1／13（MMp-1／13）的mRNA表达水平。结果与使用PBS的安慰剂对照组相比，使用PD98059的关节软骨破坏明显减轻。磷酸化的ERK1／2在100μmol／LPD98059干预组明显低于40μmol／L干预组和安慰剂对照组。而MMP-1／13的mRNA表达在不同浓度的干预组均低于安慰剂对照组。结论MAPK信号转导通路参与了骨关节炎关节软骨破坏的病理过程。MEK1／2选择性阻滞剂PD98059可以在关节炎动物活体内有效抑制关节炎软骨破坏的程度，该作用可能与其有效抑制ERK1／2的磷酸化激活有关。     

Endogenously produced adenosine regulates articular cartilage matrix homeostasis: enzymatic depletion of adenosine stimulates matrix degradation

OBJECTIVE: Enhanced extracellular levels of adenosine have been shown to inhibit experimentally induced cartilage degradation. The objective of this study was to investigate the role of adenosine and A(2)adenosine receptors in regulating cartilage homeostasis in the absence of inflammatory stimuli. METHODS: Cartilage explants were exposed to adenosine deaminase (ADA) to deplete extracellular adenosine, and conditioned medium was collected for evaluation of glycosaminoglycan (GAG), prostaglandin E(2)(PGE(2)), nitric oxide (NO), and matrix metalloproteinases-3 and -13 (MMP-3, MMP-13) levels. In a second set of experiments, cartilage incubated with ADA was simultaneously exposed to the adenosine kinase inhibitor 5'-iodotubercidin (ITU) to inhibit adenosine breakdown, or to the A(2A)adenosine receptor agonist N(6)-[2-(3,5-dimethoxyphenyl)-ethyl]adenosine (DPMA). Finally, explants were incubated with the adenosine receptor antagonists ZM241385, CGS15943, theophylline or caffeine to block normal receptor activation by endogenous adenosine. RESULTS: Exposure to ADA induced a concentration-dependent increase in GAG release and production of total MMP-3, MMP-13, PGE(2), and NO. Both ITU and DPMA inhibited the ADA-mediated increases in GAG release and PGE(2), and NO production, but only ITU inhibited MMP-13 release. Exposure to ZM 241385 increased GAG, MMP-3 and MMP-13 release. Additionally, CGS 15943 increased MMP-3 production while theophylline increased GAG, PGE(2), and NO release. CONCLUSIONS: Endogenous adenosine levels appear to regulate cartilage matrix homeostasis even in the absence of inflammation. Regulation occurs, at least in part, through activation of cell surface receptors. This study suggests that autocrine and paracrine responses to adenosine release are important for maintenance of healthy articular cartilage.     

The effects of matrix compression on proteoglycan metabolism in articular cartilage explants

The effects of compressive stress on the rate of proteoglycan synthesis and release were determined in bovine articular cartilage from 4-5-month-old animals. Full depth cartilage explants were compressed in an unconfined configuration at various stresses ranging up to 1.0 MPa. At mechanical equilibrium (after 24 h), no significant changes were detected in the rate of [35S]-sulfate (35SO4) incorporation at the low level of compressive stresses used (less than 0.057 MPa). At an intermediate level of compressive stress (0.057, 0.1, 0.5 MPa), 35SO4 incorporation rates were reduced to approximately 60% of control values. At the highest level compressive stress (1.0 MPa) studied, 35SO4 incorporation rates were further reduced to approximately 20% that of controls. Recovery experiments at intermediate stress levels showed increased rates of 35SO4 incorporation at 24 h after compression. In explants loaded for 24 h at stresses of 0.1 MPa or higher, there was a stress-dose dependent inhibition of proteoglycan release into the media (up to 61% at 1.0 MPa), and proteoglycan release rates did not return to control values following a 24 h recovery period. While cartilage composition and biosynthetic activity were found to vary significantly with depth in control cartilage, the observed suppression (% change) in biosynthetic activity was relatively uniform with depth in both loading and recovery experiments. The study indicates that compression of the tissue to physiological strain magnitudes serves as a signal to modulate chondrocyte biosynthetic and catabolic responses through the depth of cartilage, while prolonged compression at higher strains may be responsible for tissue and cell damage.     

力学刺激对关节软骨基质代谢的影响

关节软骨主要由软骨细胞和细胞外基质组成,软骨细胞能够感受力学环境的变化而不断调整细胞外基质的代谢活动.适当的力学刺激会促进软骨细胞外基质的代谢,不适当的力学刺激有时不但会抑制细胞外基质的代谢,而且可能引起软骨细胞的变形或破坏而导致各种骨骼疾病的发生.正常的关节软骨一直处于静态压力与动态压力交替活动的力学环境中,体外构建工程化软骨过程中必须考虑这些力学因素,选择适当的力学刺激(力的作用方式、大小、频率、持续时间等)作用于软骨基质,使之形成具有体内软骨的力学特性.认识和加深理解力学刺激的作用机制,也有助于临床骨科诊疗观念和手段的发展与进步.该文就软骨基质的功能、力学刺激对软骨基质代谢的影响及在关节软骨组织工程中的应用展望,作一综述.     

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.     

Integrative articular cartilage repair: dependence on developmental stage and collagen metabolism

OBJECTIVES: The objectives of this research were to determine whether the integrative repair of bovine cartilage explants was dependent on developmental stage, and whether observed differences in integration with developmental stage were related to deposition of newly synthesized collagen and lysyl oxidase-mediated collagen cross-linking. METHODS: Pairs of fetal, newborn calf, and adult bovine cartilage blocks were cultured in partial apposition for 2 weeks in medium supplemented with serum, ascorbate, and [3H]proline. Following culture, mechanical integration between apposed cartilage blocks was assessed by measuring adhesive strength in a single-lap shear configuration. Formation and stabilization of newly synthesized protein and collagen was investigated by determination of [3H]proline and [3H]hydroxyproline in tissue digests and guanidine extracts. RESULTS: Calf cartilage exhibited a relatively high integrative repair phenotype, achieving an adhesive strength that was three--four-fold that of adult or fetal specimens. The low and high integrative repair phenotypes appeared related in part to different levels of collagen biosynthesis, which was approximately four--five-fold higher in calf cartilage samples than in the adult. However, fetal cartilage also exhibited a high level of biosynthesis. The different integrative repair phenotypes were not associated with marked differences in the kinetics of chemical stabilization of newly synthesized collagen, as the proportion of incorporated [3H]proline and newly-formed [3H]hydroxyproline that was resistant to extraction by 4M guanidine-HCl following culture was similar for cartilage from all developmental stages. Integration of calf cartilage appeared to depend on lysyl oxidase-mediated collagen cross-link formation, since inclusion of beta-aminopropionitrile (BAPN) in the culture medium completely eliminated development of adhesive strength. BAPN treatment also increased the percentage of newly synthesized protein in the guanidine extracts from 10% to 36% of the total, and that of newly synthesized collagen from 2% to 20%, while having only slight inhibitory effects on overall protein and collagen biosynthesis. CONCLUSION: The finding that cartilage exhibits enhanced integrative repair at a certain developmental stage suggests that it may ultimately be possible to enhance repair when needed in clinical situations.     

Collaborative action of M-CSF and CTGF/CCN2 in articular chondrocytes: possible regenerative roles in articular cartilage metabolism

It is known that expression of the macrophage colony-stimulating factor (M-CSF) gene is induced in articular chondrocytes upon inflammation. However, the functional role of M-CSF in cartilage has been unclear. In this study, we describe possible roles of M-CSF in the protection and maintenance of the articular cartilage based on the results of experiments using human chondrocytic cells and rat primary chondrocytes. Connective tissue growth factor (CTGF/CCN2) is known to be a potent molecule to regenerate damaged cartilage by promoting the growth and differentiation of articular chondrocytes. Here, we uncovered the fact that M-CSF induced the mRNA expression of the ctgf/ccn2 gene in those cells. Enhanced production of CTGF/CCN2 protein by M-CSF was also confirmed. Furthermore, M-CSF could autoactivate the m-csf gene, forming a positive feed-back network to amplify and prolong the observed effects. Finally, promotion of proteoglycan synthesis was observed by the addition of M-CSF. These findings taken together indicate novel roles of M-CSF in articular cartilage metabolism in collaboration with CTGF/CCN2, particularly during an inflammatory response. Such roles of M-CSF were further supported by the distribution of M-CSF producing chondrocytes in experimentally induced rat osteoarthritis cartilage in vivo.     

Inhibition of renal rho kinase attenuates ischemia/reperfusion-induced injury

The Rho kinase pathway plays an important role in dedifferentiation of epithelial cells and infiltration of inflammatory cells. For testing of the hypothesis that blockade of this cascade within the kidneys might be beneficial in the treatment of renal injury the Rho kinase inhibitor, Y27632 was coupled to lysozyme, a low molecular weight protein that is filtered through the glomerulus and is reabsorbed in proximal tubular cells. Pharmacokinetic studies with Y27632-lysozyme confirmed that the conjugate rapidly and extensively accumulated in the kidney. Treatment with Y27632-lysozyme substantially inhibited ischemia/reperfusion-induced tubular damage, indicated by reduced staining of the dedifferentiation markers kidney injury molecule 1 and vimentin, and increased E-cadherin relative to controls. Rho kinase activation was inhibited by Y27632-lysozyme within tubular cells and the interstitium. Y27632-lysozyme also inhibited inflammation and fibrogenesis, indicated by a reduction in gene expression of monocyte chemoattractant protein 1, procollagen Ialpha1, TGF-beta1, tissue inhibitor of metalloproteinase 1, and alpha-smooth muscle actin. Immunohistochemistry revealed reduced macrophage infiltration and decreased expression of alpha-smooth muscle actin, collagen I, collagen III, and fibronectin. In contrast, unconjugated Y27632 did not have these beneficial effects but instead caused systemic adverse effects, such as leukopenia. Neither treatment improved renal function in the bilateral ischemia/reperfusion model. In conclusion, the renally targeted Y27632-lysozyme conjugate strongly inhibits tubular damage, inflammation, and fibrogenesis induced by ischemia/reperfusion injury.     

组织培养法在关节软骨保存中的应用

[目的]探讨3种保存方法对关节软骨细胞活性的不同影响,寻求效果优良的软骨组织保存方法.[方法]切取成年猪骨软骨,制成约4.5 mm×5 mm大小的圆柱形骨软骨块.采用组织培养法、慢速梯度降温冷冻法、传统慢速连续降温冷冻法对软骨块进行保存处理,观察并比较保存后软骨细胞活性的变化.[结果]保存8周时,采用传统冷冻法的关节软骨细胞存活率不足50%,软骨基质成分大量丢失;采用慢速梯度降温冷冻法的细胞存活率66%,而使用组织培养法保存的关节软骨细胞存活率高达76%以上,软骨基质成分仅少量丢失.[结论]3种方法相比较,组织培养法可以长期保存关节软骨组织活性,是更为理想的软骨组织保存方法.     

Relationship between synovial fluid biomarkers of articular cartilage metabolism and the patient's perspective of outcome depends on the severity of articular cartilage damage following ACL trauma

Anterior cruciate ligament (ACL) trauma often occurs in combination with injury to the articular cartilage of the knee, this can result in earlier radiographic evidence of post traumatic osteoarthritis (OA) of the knee compared to the contralateral, ACL intact knee; however, the biomechanical and biological mechanisms associated with the onset and progression of this disease are not understood. We sought to gain insight into the mechanisms by determining the relationship between articular cartilage injury associated with ACL trauma and the expression of synovial fluid biomarkers of articular cartilage metabolism, and to evaluate the relationship between these biomarkers and the patient's perspective of the outcomes. Synovial fluid samples were acquired from 39 ACL injured subjects at an average of 10 weeks after injury, and 32 control subjects with normal knees (documented with clinical exam and MRI assessment). Subjects in the ACL‐injured group were classified as low‐risk for future OA if they displayed an International Cartilage Repair Society (ICRS) Grade 2 articular cartilage lesion or less and high‐risk for future OA if they had an ICRS Grade 3A articular cartilage lesion. The patient's perspective of the injury was evaluated with the Knee Injury and Osteoarthritis Outcomes Score (KOOS). There were no significant differences in mean concentrations of the markers of type II collagen metabolism (CPII, C2C, and C1,2C) or the aggrecan breakdown Alanine–Arginine–Glycine–Serine (ARGS) ‐fragment between control subjects and the subjects in the low‐ and high‐risk groups (p‐value range: 0.80–0.43). Associations between ARGS‐aggrecan concentration and KOOS subscales of symptoms and pain were significantly different between the low‐ and high‐risk groups (p = 0.03 and p = 0.01, respectively). Likewise, there was strong evidence in support of an association between the markers of type II collagen metabolism (C1,2C and CPII concentrations) and the KOOS subscale of pain between the low‐ and high‐risk groups (p = 0.051 and 0.077, correspondingly). In ACL injured subjects with concomitant Grade 3A articular cartilage injuries, concentrations of synovial fluid ARGS‐aggrecan were directly associated with improvements in KOOS symptoms and pain. These findings suggest the possible involvement of ARGS‐aggrecan in a localized tissue repair response involving an increase in aggrecan turnover following severe knee trauma. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:820–827, 2016.