骨关节炎患者滑液中COMP水平与病情严重程度的相关性研究

背景：软骨寡聚基质蛋白（cartilage oligomeric matrix protein,COMP）是软骨中非胶原蛋白的主要成分,软骨的损伤、修复和代谢变化均可能影响COMP的表达水平。目的：研究骨关节炎（osteoarthritis,OA）患者关节滑液中COMP水平与病变严重程度的相关性,探讨注射玻璃酸钠对关节滑液中COMP的影响。方法：52例膝关节0A患者接受关节内注射玻璃酸钠治疗,治疗前、治疗后6个月行X线片检查,按Kellgren放射学诊断标准评级,记录治疗前和治疗开始后5周、6个月时患者膝关节的WOMAC评分。采用ELISA方法测定在接受透明质酸钠治疗前、治疗开始后5周关节液中的COMP水平。19例因半月板或韧带损伤接受关节镜手术的患者作为正常对照组。结果：OA组患者滑液中COMP水平明显高于对照组,有统计学差异（P=0．036）。OA组患者滑液中COMP水平与OA严重程度（WOMAC评分）呈正相关（P=0．001）,与影像学Kellgren分级标准无相关性（P=0．12）。结论：滑液中COMP水平与OA病变严重程度呈正相关,测定OA患者血中COMP水平对进一步研究OA发病机理、早期发现并采取有效防治策略及监测防治效果具有极其重要的意义。     

血清COMP在骨关节炎早期诊断中的作用

目的:运用血清COMP含量检测从有临床症状而未出现影像学改变的人群中筛选出骨关节炎(os-teoarthritis,OA)亚临床患者,从而开展OA的早发现、早治疗。方法:收集2007年8月至2009年9月入院诊断为膝骨关节炎患者或骨关节炎高危人群组115例(OA组),体检健康对照人群35例(健康组)。OA组男55例,女60例;年龄39～76岁,平均(55±13.32)岁;体重指数15.1～29.8;病程6～60个月。健康组男16例,女19例;年龄36～77岁,平均(53±12.53)岁;体重指数14.8～29.2。按照国际骨关节炎学会制定的诊断分类标准以及Kellgren和Lawrence衡量标准,确诊OA并分为Ⅰ-Ⅳ级,采用口服塞来昔布胶囊治疗,检测血清COMP含量并分析与OA级别的相关性。OA亚临床组患者随访调查24～38个月,平均33.4个月,检测随访前后的血清COMP含量。结果:健康组血清COMP含量受年龄影响较大(t=2.50,P=0.02),但健康组和OA组均不受性别(t=0.98,P=0.34;t=0.18,P=0.86),体重指数(t=0.56,P=0.92;t=0.17,P=0.85)和吸烟史(t=1.89,P=0.08;t=0.70,P=0.49)影响。随着OA分级的升高,血清COMP含量逐渐升高(F=15.56,P=0.001)。即使针对未出现影像学改变的OA亚临床患者,也可以在该患者确诊的2年前检测出显著的血清COMP含量升高,并能有效地将其与其他相关疾病的亚临床患者区分(t=2.55,P=0.03)。结论:血清COMP可作为潜在的生物学标记物为膝关节OA的亚临床和早期患者提供新的诊断指标。     

Impact of knee joint loading on fragmentation of serum cartilage oligomeric matrix protein

The aim of the study was to examine the effect of mechanical knee joint loading on the fragmentation pattern of serum cartilage oligomeric matrix protein (COMP). Ten healthy men ran with knee orthoses that were passive or active (+30.9 N·m external flexion moments) on a treadmill (30 minute; v = 2.2 m/s). Lower-limb mechanics, serum COMP levels, and fragmentation patterns (baseline; 0, 0.5, 1, 2 hours postrunning) were analyzed. Running with active orthoses enhanced knee flexion moments, ankle dorsiflexion, and knee flexion angles (P < .05). There was an increase in serum COMP (+25%; pre: 8.9 ± 2.4 U/l; post: 10.7 ± 1.9 U/l, P = .001), COMP pentamer/tetramer (+88%; 1.88 ± 0.81, P = .007), trimer (+209%; 3.09 ± 2.65, P = .005), and monomer (+78%; 1.78 ± 0.85, P = .007) after running with passive orthoses and in serum COMP (+41%; pre: 8.5 ± 2.7 U/l; post: 11.3 ± 2.1 U/l, P < .001), COMP pentamer/tetramer (+57%; 1.57 ± 0.39, P = .007), trimer (+86%; 1.86 ± 0.47, P = .005), and monomer (+19%; 1.19 ± 0.34, P = .114) after running with active orthoses. Increased fragmentation might indicate COMP release from cartilage while running. Interestingly, 0.5 h up to 2 hours after running with passive orthoses, trimer (0.5 hour: 2.73 ± 3.40, P = .029; 2 hours: 2.33 ± 2.88, P = .037), and monomer (0.5 hour: 2.23 ± 2.33, P = .007; 1 hour: 2.55 ± 1.96, P = .012; 2 hours: 2.65 ± 2.50, P = .009) increased while after running with active orthoses, pentamer/tetramer (1 hour: 0.79 ± 0.28, P = .029), and trimer (1 hour: 0.63 ± 0.14, P = .005; 2 hours: 0.68 ± 0.34, P = .047) decreased. It seems that COMP degradation and clearance vary depending on joint loading characteristics.     

Bone vitamin D-dependent calcium-binding protein is localized in chondrocytes of growth-plate cartilage

Summary The cellular distribution of vitamin D-dependent calcium-binding protein (CaBP) was examined in rat and chicken bone by immunocytochemical methods using an antiserum raised against purified chicken intestinal CaBP. In EDTA-decalcified, Vibratome sections of growing rat long bones, specific CaBP immunostaining was observed in cytoplasm of chondrocytes of the growth plate, particularly in regions of calcification. In undecalcified, frozen sections from neonatal rat, positive staining was seen in chondrocytes of tibial growth plate and also in chondrocytes of the long bones of the skull. No specific immunostaining was observed in osteoblasts, osteocytes or osteoclasts in mineralized bone. In frozen sections of tibias from 19-day-old chick embryos specific immunostaining was again confined to dividing chondrocytes of the growth plate and was much less intense in “resting” cartilage. The finding of CaBP in chondrocytes, cells known to possess specific receptors for 1,25-dihydroxyvitamin D₃ and to respond to the hormone, suggests a possible functional role for CaBP in chondrocyte maturation, differentiation and/or cartilage calcification.     

软骨寡聚基质蛋白在慢性胰腺炎损伤中退变腺泡细胞内的表达

目的 研究正常胰腺、慢性胰腺炎与胰腺癌组织中软骨寡聚基质蛋白(cartilage oligomeric matrix protein,COMP)mRNA和蛋白表达水平的差异,揭示COMP在慢性胰腺炎样损伤中的意义。方法 采用Northern印迹法、Western印迹法、原位杂交法与免疫组化方法对14例慢性胰腺炎、14例胰腺癌及15例正常胰腺组织进行分析。结果 在慢性胰腺炎组织中和胰腺癌组织中类似慢性胰腺炎损伤的退变腺泡细胞胞浆内,存在高水平的COMP mRNA信号与免疫反应;而在胰腺癌细胞、正常胰腺组织的导管细胞与胰岛细胞的胞浆内,COMP mRNA信号与免疫反应微弱或缺如。结论 COMP在慢性胰腺炎及胰腺癌中类似慢性胰腺炎损伤的退变腺泡细胞内高表达,可能与慢性胰腺炎中腺泡细胞功能异常有关。     

Effect of increased mechanical knee joint loading during running on the serum concentration of cartilage oligomeric matrix protein (COMP)

The purpose of the study was to investigate the effect of an increase in mechanical knee joint loading during running on the serum COMP level. On two different test days, 20 healthy men ran with knee orthoses for 30 min on a treadmill (v = 2.2 m/s). On day 1, the orthoses were passive, whereas on day 2 they were pneumatically driven (active) and thus increased the external knee flexion moments (+30.9 Nm) during stance phase. Lower‐limb mechanics and serum COMP levels (baseline; 0, 0.5, 1, 2 h post running) were analyzed. COMP levels increased immediately after running with passive (+35%; pre: 7.5 U/l, 95%CI: 6.4, 8.7, post: 9.8 U/l, 95%CI: 8.8, 10.8, p < 0.001) and active orthoses (+45%; pre: 7.6 U/l; 95%CI: 6.4, 8.8, post: 10.3 U/l, 95%CI: 9.2, 11.5, p < 0.001), but they did not differ between interventions. While running with active orthoses, greater ankle dorsiflexion angles, knee flexion angles, and moments occurred (p < 0.05). Comparing both interventions, the Δ COMP pre–post, meaning the difference (Δ) between running with active and passive orthoses in pre to post COMP level change (=level after (post) running minus level before (pre) running), correlated negatively with Δ COMP baseline (difference between the baseline COMP level before running with active and passive orthoses, r = ?0.616; p = 0.004), and with a positive tendence with the Δ maximum knee flexion (r = 0.388; p = 0.091). Therefore, changes in COMP concentration after physical activity seem to be highly influenced by the COMP baseline level. In addition, correlation analysis indicates that modifications in knee joint kinematics have a greater effect on cartilage metabolism than an increase in joint moments. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1937–1946, 2018.

     

终末期肾病血液透析患者血浆COMP表达与动静脉内瘘失功及预后的相关性研究

目的 检测终末期肾病(end-stage renal disease,ESRD)血液透析患者血浆软骨寡聚基质蛋白(cartilage oligomeric matrix protein,COMP)表达,并分析COMP与动静脉内瘘(arteriovenous fistula,AVF)失功及预后的相关性.方法 选取2015...     

膝骨关节炎患者关节滑液中骨桥蛋白和软骨寡聚基质蛋白水平与疾病严重程度相关性

目的:探讨膝骨关节炎（knee osteoarthritis,KOA）患者关节滑液中骨桥蛋白（osteopontin,OPN）和软骨寡聚基质蛋白（cartilage oligomeric matrix protein,COMP）水平与疾病严重程度的相关性。方法：选取2018年2月至2020年5月收治的59例KOA患者作为KOA组,其中男25例,女34例;年龄60~75（65.57±1.56）岁;体质量指数（body mass index,BMI）21.4~30.7（26.12±1.54） kg/m²。采用Kellgren-Lawrence（K-L）分级对X线结果进行评估,其中Ⅱ级（K-L2组）14例,Ⅲ级（K-L3组）27例,Ⅳ级（K-L4组）18例。另选取18例因韧带或半月板疾病进行关节镜检查且无软骨损伤患者作为对照组,男7例,女11例;年龄61~78（64.88±1.60）岁;BMI 22.8~29.9（25.89±1.49） kg/m²。治疗前采集研究对象关节滑液样本,采用酶联免疫吸附试验检测关节滑液OPN、COMP水平,比较KOA组与对照组关节滑液OPN、COMP水平。比较不同K-L分级KOA患者性别、年龄、BMI等临床资料,采用酶联免疫试验检测其关节滑液中白细胞介素-1β（interleukin -1β,IL-1β）,OPN,COMP,基质金属蛋白酶3（matrix metalloproteinase -3,MMP-3）水平,比较不同K-L分级KOA患者临床资料和生化指标,采用Logistic回归分析影响KOA患者K-L分级的因素,采用ROC曲线下面积（area under the curve,AUC）预测KOA疾病严重程度。结果：59例KOA患者获得随访,时间8~27（15.75±3.27）个月。KOA组关节滑液OPN、COMP水平高于对照组（P<0.001）。K-L2组、K-L3组、K-L4组IL-1β、OPN、COMP、MMP-3水平比较差异有统计学意义（P<0.001）;与K-L2组比较,K-L3、K-L4关节滑液关节滑液IL-1β、OPN、COMP、MMP-3 水平均升高（P<0.05）;与K-L3组比较,K-L4关节滑IL-1β、OPN、COMP、MMP-3水平均升高（P<0.05）。多因素Logistic回归分析结果显示：关节滑液OPN、COMP、MMP-3水平是影响KOA患者K-L分级的独立危险因素（OR=6.653, 4.229,1.579,P<0.001）。关节滑液OPN预测K-L4级KOA的AUC为0.720[95%CI（0.588-0.851）],灵敏度为94.4%,特异度为65.9%;关节滑液COMP预测K-L4级KOA的AUC为0.731[95%CI（0.592-0.870）],灵敏度为88.9%,特异度为63.4%;关节滑液OPN联合COMP预测K-L4级KOA的AUC为0.839[95%CI（0.724-0.953）],灵敏度为94.4%,特异度为51.2%;OPN联合COMP预测K-L4级KOA的AUC大于单独OPN、COMP的AUC（Z=4.037,3.540,P<0.05）。结论：KOA患者关节滑液OPN、COMP水平升高,并随着K-L分级增加而升高。关节滑液OPN、COMP是影响KOA患者K-L分级的独立危险因素,二者预测K-L4级KOA 的AUC、灵敏度、特异度高,可用于评估KOA疾病进展。     

Enhanced concentration of COMP (cartilage oligomeric matrix protein) in osteochondral fractures from racing Thoroughbreds.

The aim of the present study was to correlate the levels of COMP and aggrecan as indicators of tissue damage, in synovial fluid (sf) from carpal joints of acutely lame racehorses, with macroscopical lesions of articular cartilage (OA), osteochondral fractures and ligament tears found at arthroscopy. Sixty-three lame horses [49 Standardbred trotters (STB) and 14 Thoroughbreds (TB)] in conventional training and racing that underwent arthroscopy of their middle carpal or radiocarpal joints were included in the study. Intact as well as fragmented COMP and aggrecan released into the synovial fluid were quantified by western blot analyses and ELISA. The expression of COMP in tissues was estimated by mRNA in situ hybridisation and protein immunolocalisation in cartilage and osteochondral fractures. The concentration of sf-COMP was higher in TB with an osteochondral fracture than in STB with osteochondral fractures and TB and STB with OA. The chondrocytes in middle and deep zones of the articular cartilage of the osteochondral fragments (from a TB) expressed COMP mRNA, in contrast to the cartilage on the opposite side of the fracture where no expression was detected. In the synovial fluid from a joint (TB) with osteochondral fractures only intact COMP was present, whereas, fragmented COMP was more prominent in synovial fluid from a joint with OA. The concentration of sf-aggrecan did not differ between the two breeds, or between different lesions. The increased concentration of sf-COMP in TB with osteochondral fractures, but not in synovial fluid from equine joints with OA, is a novel finding. The results from this study indicate that elevated sf-COMP concentration in the joints of Thoroughbreds may be a useful marker for carpal joint osteochondral fragments.     

A critical role for collagen II in cartilage matrix degradation: Collagen II induces pro‐inflammatory cytokines and MMPs in primary human chondrocytes

We report a process that results in the acceleration of matrix degradation in human articular cartilage, a phenomenon commonly observed in osteoarthritis (OA). The study was conducted by (1) examining the potential of collagen II in modulating the gene expression profile of primary human chondrocytes (PHCs), and (2) investigating the involvement of pro‐inflammatory signaling cascades. We first tested the collagen II‐dependent induction of pro‐inflammatory cytokines and matrix metalloproteinases (MMPs) in PHCs. PHCs were incubated with or without monomeric (i.e., nonfibrillar) collagen II. Cells were then analyzed by RT‐PCR for the expression of MMP1, MMP3, MMP13, MMP14, and IL‐1β. ELISA was used to quantify IL‐6 and IL‐8 release. To examine the influence of collagen II signaling, specifically the role of MAPK p38, a p38‐inhibitor was added prior to collagen treatment. Changes in IκB concentration were monitored by immunoblot analysis to detect NFκB signaling. Results indicated that incubation of PHCs with collagen II did produce a dose‐dependent induction of MMP1, MMP3, MMP13, MMP14, as well as cytokines IL‐1β, IL‐6, and IL‐8. At the same time, inhibition of p38 and IκB degradation revealed that collagen II‐dependent gene induction also involves MAPK p38 and NFκB signaling. Thus, we provide evidence for a collagen II‐dependent feed‐forward mechanism whereby collagen II induces first MMPs and pro‐inflammatory cytokines and then release of collagen II fragments from mature collagen II fibers. This, in turn, induces more pro‐inflammatory cytokines and MMPs, and the process is repeated, which results in the acceleration and perpetuation of cartilage matrix degradation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:65–70, 2009     

Locomotion replacement exercise cannot counteract cartilage biomarker response to 5 days of immobilization in healthy adults

Biomarkers of cartilage metabolism are sensitive to changes in the biological and mechanical environment and can indicate early changes in cartilage homeostasis. The purpose of this study was to determine if a daily locomotion replacement program can serve as a countermeasure for changes in cartilage biomarker serum concentration caused by immobilization. Ten healthy male subjects (mean ± 1 standard deviation; age: 29.4 ± 5.9 years; body mass: 77.7 ± 4.1 kg) participated in the crossover 5-day bed rest study with three interventions: control (CON), standing (STA), and locomotion replacement training (LRT). Serum samples were taken before, during, and after bed rest. Biomarker concentrations were measured using commercial enzyme-linked immunosorbent assays. Cartilage oligomeric matrix protein (COMP) levels after 24 hours of bed rest decreased independently of the intervention (−16.8% to −9.8%) and continued to decrease until 72 hours of bed rest (minimum, −23.2% to −20.6%). LRT and STA did not affect COMP during bed rests (P = .056) but there was a strong tendency for a slower decrease with LRT (−9.4%) and STA (−11.7%) compared with CON (−16.8%). MMP-3 levels decreased within the first 24 hours of bed rest (CON: −22.3%; STA: −14.7%; LRT: −17%) without intervention effect. Both COMP and MMP-3 levels recovered to baseline levels during the 6-day recovery period. MMP-1, MMP-9, and TNF-α levels were not affected by immobilization or intervention. COMP and MMP-3 are mechano-sensitive cartilage biomarkers affected by immobilization, and simple interventions such as standing upright or LRT during bed rest cannot prevent these changes. Clinical significance: simple locomotion interventions cannot prevent cartilage biomarker change during bed rest.     

人关节软骨脱细胞基质的制备

目的制备人关节软骨脱细胞基质。方法切取人关节软骨,对之冷冻干燥后,采取化学去污剂TritonX100及DNA酶和RNA酶等试剂制备脱细胞的人关节软骨。做HE、番红0及软骨蛋白聚糖(aggrecan)免疫组化染色等方法进行检测。结果HE染色、番红0染色均显示细胞陷窝内己无细胞结构番红花0染色阳性;软骨蛋白聚糖免疫组化染色阳性。结论人关节软骨冻干后,经去污剂酶等处理方法可脱去软骨的细胞成分,并且保留了软骨细胞外基质,成功制备了人关节软骨脱细胞基质。     

Differences in the secretome of cartilage explants and cultured chondrocytes unveiled by SILAC technology

The main goal of our study was to analyze and compare the profiles of secreted proteins from adult human articular chondrocytes in monolayers, and cartilage explants in culture, using a de novo protein labeling approach. Stable isotope labeling of proteins in culture was used to differentiate between chondrocyte‐derived proteins and other preexisting matrix‐derived components, or proteins coming from serum or synovial fluids. Proteins in culture supernatants were resolved by one‐dimensional SDS‐PAGE electrophoresis, and analyzed in tandem with LC/MS‐MS (liquid chromatography/double mass spectrometry). Results from stable isotope labeling with amino acids in cell culture (SILAC) were validated by specific immunoblotting of four relevant proteins identified in the secretion media. After 8–10 days of culture, over 90% of proteins secreted during monolayer growth contained ¹³C₆‐Arg and ¹³C₆‐Lys. Nonlabeled proteins corresponded mostly to plasma‐associated proteins, indicating background contamination of medium with serum remnants. The majority of the secreted proteins in 2D cultures were extracellular matrix components and matrix regulators, along with some inflammatory agents and metabolic enzymes. In explants, only 25%–30% of proteins were labeled with heavy amino acids, corresponding to matrix regulators and carrier molecules. Nonlabeled proteins corresponded primarily to structural matrix components. In qualitative terms, all labeled proteins coming from cartilage explants were also found in chondrocytes supernatants. In summary, our results show differences in the labeling pattern of proteins found in supernatants from explants and monolayers. Most proteins found in the media of explants were subproducts of matrix turnover rather than newly synthesized. To our knowledge, this study is the first one so far applying SILAC technology in the context of cartilage and chondrocytes physiology. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1040–1049, 2010     

Serum cartilage oligomeric matrix protein (COMP) in knee osteoarthritis: A novel diagnostic and prognostic biomarker

A case–control study was conducted to estimate the association of cartilage oligomeric matrix protein (COMP) with knee osteoarthritis (OA) and to examine the potential utility of COMP as a diagnostic and prognostic biomarker in early knee OA. The COMP levels were estimated in the blood sera of 150 subjects belonging to study group (n = 100) and control one (n = 50). Patients with confirmed clinical isolated knee OA diagnosed through American College of Rheumatology criteria were included and were without any other cause of knee pain. ELISA was used to determine the levels of COMP, interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α). The median (range) serum COMP levels were observed to be 1117.21 ng/ml (125.03–4209.75 ng/ml) in OA patients and 338.62 ng/ml (118–589 ng/ml) in control subjects with p < 0.001. The COMP levels of study group were negatively correlated (correlation factor ?0.88) with disease duration and positively correlated with age, BMI, pain score and IL‐1β with correlation factors 0.86, 0.63, 0.76, and 0.79, respectively with p < 0.001. Gender differentiation was found in study group with 52% higher COMP level in males as compared to that of females. There was no significant correlation of COMP levels with radiological grading, erythrocyte sedimentation rate (ESR), hemoglobin (Hb), and TNF‐α. The serum COMP levels may be used as a diagnostic OA marker along with prognostic value in determining the patients at risk of rapidly progressing this debilitating joint disease. The serum COMP level remains significantly high in first 3 years of disease duration. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:999–1006, 2013

     

Cryopreserved articular chondrocytes grow in culture, maintain cartilage phenotype, and synthesize matrix components

For osteochondral allograft transplantation to be successful, chondrocytes must survive preservation and retain their capacity to produce normal matrix components: proteoglycans and Type II collagen. Clinical success with osteochondral allograft transplantation has created an increased demand for supplies of suitable cartilage-bearing grafts. This demand has stimulated attempts to find successful methods for low temperature storage of cartilage for "banking" and heightened interest in cartilage cryobiology. In order to achieve the maximum viability of cryopreserved articular cartilage, previous comprehensive studies have focused on rates and temperatures of freezing, cryoprotective agents, and methods and influences of thawing. This study presents evidence that cryopreserved articular chondrocytes maintain their ability to grow in tissue culture following thawing and to produce normal matrix components. Chondrocytes isolated from Japanese white rabbits were divided into groups of fresh controls and experimental cryopreserved cells. Cells were incubated in dimethylsulfoxide, frozen at a rate of -1 degrees C/min, stored at -79 degrees C, rapidly thawed, and plated for culture. Growth rates were comparable in all groups. In all groups, typical chondroid characteristics were maintained throughout 14 days of culture. Typical cartilage phenotypic characteristics included maintenance of polygonal and rhomboidal cells, cell aggregation, proteoglycan production, and Type II collagen synthesis. This investigation strongly indicates that articular chondrocyte cryopreservation yields viable, functional cells and although these results cannot be directly extrapolated to intact adult articular cartilage, they do give further support for low temperature banking of cartilage-bearing allografts for transplantation.     

The role of muscle cells in regulating cartilage matrix production

Muscle is one of the tissues located in close proximity to cartilage tissue. Although it has been suggested that muscle could influence skeletal development through generating mechanical forces by means of contraction, very little is known regarding whether muscle cells release biochemical signals to regulate cartilage gene expression. We tested the hypothesis that muscle cells directly regulate cartilage matrix production by analyzing chondrocytes cocultured with muscle cells in 2D or 3D conditions. We found that chondrocytes cultured with C2C12 muscle cells exhibited enhanced alcian blue staining and elevated expression of collagen II and collagen IX proteins. Although nonmuscle cells did not promote cartilage matrix production, converting them into muscle cells enhanced their pro‐chondrogenic activity. Furthermore, muscle cell‐conditioned medium led to increased cartilage matrix production, suggesting that muscle cells secrete pro‐chondrogenic factors. Taken together, our study suggests that muscle cells may play an important role in regulating cartilage gene expression. This result may ultimately lead to the discovery of novel factors that regulate cartilage formation and homeostasis, and provide insights into improving the strategies for regenerating cartilage. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:529–536, 2010     

Platelet rich plasma (PRP) enhances anabolic gene expression patterns in flexor digitorum superficialis tendons.

Platelet rich plasma (PRP) has recently been investigated for use in tissue regeneration studies that seek to utilize the numerous growth factors released from platelet alpha-granules. This study examined gene expression patterns, DNA, and collagen content of equine flexor digitorum superficialis tendon (SDFT) explants cultured in media consisting of PRP and other blood products. Blood and bone marrow aspirate (BMA) were collected from horses and processed to obtain plasma, PRP, and platelet poor plasma (PPP). IGF-I, TGF-beta1, and PDGF-BB were quantified in all blood products using ELISA. Tendons were cultured in explant fashion with blood, plasma, PRP, PPP, or BMA at concentrations of 100%, 50%, or 10% in serum-free DMEM with amino acids. Quantitative RT-PCR for expression of collagen type I (COL1A1), collagen type III (COL3A1), cartilage oligomeric matrix protein (COMP), decorin, matrix metalloproteinase-3 (MMP-3), and matrix metalloproteinase-13 (MMP-13) was performed as were DNA and total soluble collagen assays. TGF-beta1 and PDGF-BB concentrations were higher in PRP compared to all other blood products tested. Tendons cultured in 100% PRP showed enhanced gene expression of the matrix molecules COL1A1, COL3A1, and COMP with no concomitant increase in the catabolic molecules MMP-3 and MMP-13. These findings support in vivo investigation of PRP as an autogenous, patient-side treatment for tendonitis.     

Transient expression of the diseased phenotype of osteoarthritic chondrocytes in engineered cartilage

Due to the degradation of osteoarthritic (OA) cartilage in post‐traumatic OA (PTOA), these tissues are challenging to study and manipulate in vitro. In this study, chondrocytes isolated from either PTOA (meniscal‐release (MR) model) or normal (contralateral limb) cartilage of canine knee joints were used to form micropellets to assess the maintenance of the OA chondrocyte phenotype in vitro. Media samples from the micropellet cultures were used to measure matrix metalloproteinase (MMP), chemokine, and cytokine concentrations. Significant differences in matrix synthesis were observed as a function of disease with OA chondrocytes generally synthesizing more extracellular matrix with increasing time in culture. No donor dependent differences were detected. Luminex multiplex analysis of pellet culture media showed disease and time‐dependent differences in interleukin (IL)‐8, keratinocyte chemoattractant (KC)‐like protein, MMP‐1, MMP‐2, and MMP‐3, which are differentially expressed in OA. This memory of their diseased phenotype persists for the first 2 weeks of culture. These results demonstrate the potential to use chondrocytes from an animal model of OA to study phenotype alterations during the progression and treatment of OA. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:829–836, 2017.

     

The influence of mechanical compression on the induction of osteoarthritis-related biomarkers in articular cartilage explants

OBJECTIVE: Macromolecules of the articular cartilage extracellular matrix released into synovial fluid, blood, or urine can serve as potentially useful biomarkers of the severity of osteoarthritis (OA). Biomechanical factors play an important role in OA pathogenesis, yet their influence on biomarker production is not well understood. The goal of this study was to examine the hypothesis that dynamic mechanical stress influences the release of these biomarkers from articular cartilage. METHODS: Explants of porcine cartilage were subjected to dynamic compression at 0.5 Hz for 24h at stresses ranging from 0.006 to 0.1 MPa. The concentrations of cartilage oligomeric matrix protein (COMP), keratan sulfate (KS measured as the 5 D 4 epitope), total sulfated glycosaminoglycan (S-GAG), and the KS (keratanase-digestible) and chondroitin sulfate (CS) (chondroitinase-digestible) fractions of S-GAG were measured. Radiolabel incorporation was used to determine the rates of proteoglycan and protein synthesis. RESULTS: The magnitudes of mechanical stress applied in this study induced nominal tissue strains of 4-23%, consistent with a range of physiological to hyperphysiologic strains measured in situ. COMP release increased in proportion to the magnitude of dynamic mechanical stress, while KS, CS and total S-GAG release increased in a bimodal pattern with increasing stress. Protein and proteoglycan synthesis were significantly decreased at the highest level of stress. CONCLUSION: Mechanical stress differentially regulates the turnover of distinct pools of cartilage macromolecules. These findings indicate that mechanical factors, independent of exogenous cytokines or other stimulatory factors, can influence the production and release of OA-related biomarkers from articular cartilage.     

Ultrastructural Immunolocalization of Cartilage Oligomeric Matrix Protein (COMP) in Porcine Growth Cartilage

Cartilage oligomeric matrix protein (COMP) is a macromolecule of yet unknown function with restricted distribution among tissues. In the present study, the ultrastructural localization of COMP in porcine immature joint cartilage and growth plate cartilage was semiquantitatively delineated. Tissues were fixed in a mixture of low concentration glutar- and paraformaldehyde, embedded at low temperature, and subjected to immunocytochemistry using polyclonal antibodies raised against bovine COMP. Protein A-coated colloidal gold was used for detection. The most intense immunolabeling for COMP was noted in the proliferative zones of the growth cartilages. Here the concentration of immunomarker was higher in the territorial compartment than in the pericellular and interterritorial areas. A low concentration of COMP was observed in the resting and hypertrophic zones. The immunolabeling for COMP did not differ between the three matrix compartments of these zones. Supported by previous data obtained by in situ hybridization, the concentration of immunolabeling in the proliferative zone indicates a high rate of COMP synthesis in proliferative chondrocytes. Hence, COMP may be considered as a marker for normal differentiation into proliferative chondrocytes. Received: 28 November 1995 / Accepted: 15 October 1996