Static Compressive Loading Reduces the mRNA Expression of Type II and X Collagen in Rat Growth-Plate Chondrocytes During Postnatal Growth

The mechanisms by which chondrocytes modulate longitudinal bone growth are not well understood. This in vitro study investigated the effects of loading on the mRNA expression pattern of key molecular components of the growth-plate related to the extracellular matrix (type II and type X collagen) and the PTH-PTHrP feedback loop. Short-term static compressive loading was applied to rat proximal tibial growth-plate explants. Four age groups at specific developmental stages were investigated. The spatial variation in the mRNA expression was compared among loaded explants, their contralateral sham controls, and uncultured growth plates from normal animals. Basic cell metabolism (18S rRNA) was unaffected by load. Results indicated a narrower spatial distribution of mRNA expression of type II collagen throughout the growth plate; similarly, a narrowed distribution of expression of type X collagen was noted in the lower hypertrophic zone of the growth-plate. This suggests that mechanical compression influences chondrocytes of the hypertrophic zone to alter their expression of specific genes encoding proteins of the extracellular matrix, while PTH-PTHrP receptor mRNA, a regulatory protein, remained unaffected by loading. The effects of compression were similar at the different stages of growth, suggesting that additional factors may be involved in the clinical progression of skeletal deformities observed during growth spurts. Although this study was done in vitro and limited to static loading, it furthers our understanding of growth-plate mechanobiology as a first step toward providing a scientific rationale for treating progressive musculoskeletal deformities.     

骨关节炎软骨细胞凋亡调控基因的研究

目的　比较分析正常人及老年性骨关节炎患者软骨细胞bax和bcl 2的表达及细胞凋亡状况。　方法　取 9例骨关节炎患者的关节软骨做实验标本 ,以 6例无骨关节炎病史的意外死亡者关节软骨作为正常对照 ;采用逆转录 /聚合酶链反应 (RT PCR)方法检测bax和bcl 2mRNA表达 ,免疫组化检测bax和bcl 2蛋白 ;应用TUNEL方法进行凋亡细胞原位检测。　结果　骨关节炎患者和正常对照软骨细胞都能表达bax和bcl 2mRNA ;骨关节炎关节软骨细胞baxmRNA表达量较正常对照显著增高 (P <0 0 1) ,bcl 2mRNA表达量也高于正常对照组 (P <0 0 5 ) ,两组间bax/bcl 2表达量的比值差异无显著性意义 (P >0 0 5 ) ;免疫组化可检测到相应表达水平的蛋白 ;骨关节炎软骨细胞凋亡 (4%～ 14% )多于正常对照 (0～ 2 % )。　结论　软骨细胞凋亡受bax和bcl 2共同调节 ;bax和bcl 2的共同调节结果可能是OA患者软骨细胞凋亡增加 ,但凋亡率不高、病理过程进展缓慢的一个重要的原因     

Membrane-seeded autologous chondrocytes: cell viability and characterization at surgery

The implantation of chondrocytes, seeded on matrices such as hyaluronic acid or collagen membranes, is a method that is being widely used for the treatment of chondral defects. The aim of the present study was to evaluate the distribution, viability and phenotype expression of the cells seeded on a collagen membrane just at the time of the implantation. Twelve patients who were suffering from articular cartilage lesions were treated by the MACI^® procedure. The residual part of each membrane was tested by colorimetric assay (MTT) and histochemical and ultrastructural analyses were carried out. In all of the samples a large number of viable cells, quite homogenously distributed, was detected. The cells expressed the markers of the differentiated hyaline chondrocytes. These data reassure in that the MACI procedure provides a suitable engineered tissue for cartilage repair, in line with the clinical evidences emerging in the literature.     

共培养诱导小鼠ES细胞向软骨细胞分化的初步研究

目的 探讨软骨共培养体系诱导小鼠ES细胞向软骨细胞分化的可行性.方法 GFP标记的小鼠ES细胞初步分化为EB后,将EB消化为单个细胞,同猪关节软骨细胞按一定比例(1∶3)昆合后接种于PGA材料,体外培养1周后植入裸鼠皮下3周取材.对照组为EB细胞接种组及软骨细胞接种组.取材后行连续冰冻切片,切片分别做荧光拍照,HE染色及甲苯胺蓝染色.结果 组织学结果显示,EB细胞接种组形成畸胎瘤;软骨细胞对照组形成软骨组织;实验组形成软骨组织和畸胎瘤的混合体.甲苯胺蓝染色结果和荧光照片对照结果显示,部分软骨组织GFP阳性,由小鼠ES细胞分化而来.结论 软骨共培养体系可以诱导小鼠ES细胞向软骨细胞分化,但得到的软骨组织不纯,混有畸胎瘤组织.     

正钒酸钠对椎间盘软骨细胞功能的抑制作用

目的探讨正钒酸钠（Sodium Orthovanadate,Na3VO4）对椎间盘软骨终板软骨细胞的作用。方法使用酶消化法培养大鼠椎间盘软骨终板软骨细胞,以10、20及30umol／L不同浓度Na3VO4干预第三代软骨细胞。培养7d后,用MTT法检测软骨细胞增殖率,RT-PCR检测Ⅱ型、Ⅸ型胶原和蛋白聚糖（Aggrecan）mRNA的表达,定量RT—PCR检测PTPN1、IGFRmRNA的表达。结果与对照组比较：（1）10umol／L Na3VO4组上述各项检测指标变化不明显,差异无统计学意义;（2）20及30umol／L Na3VO4组软骨细胞增殖率下降（q=31．51,81．42,P〈0．01）、Aggrecan（q=52．09,102．55,P〈0．01）和PTPN1（q=7．67,4．74,P〈0．01）mRNA表达降低;（3）30umol／L Na3VO4组Ⅱ型胶原（q=51．46,P〈0．01）、Ⅸ型胶原（q=8．62,P〈0．01）mRNA表达降低;（4）各浓度Na3VO4组IGFRmRNA均增加（q=13．96,7．67,4．74,P〈0．01）。结论20及30umol／L Na3VO4可抑制椎间盘软骨细胞增殖,降低Ⅱ、Ⅸ型胶原、Aggrecan以及PTPN1的mRNA表达,说明Na3VO4对软骨细胞中PTPs的活性有抑制作用,从而降低了软骨细胞生物学功能。但Na3Vo4对IGFR mRNA无抑制作用,并可能有一定的促表达作用。     

周期性应力下胰岛素样生长因子1受体对大鼠软骨细胞功能的影响

目的探讨周期性机械应力条件下胰岛素样生长因子1型受体(IGF1R)对大鼠软骨细胞增殖及细胞外基质合成的影响。 方法体外分离培养大鼠软骨细胞,随机分为4组：加压0 h组、加压0.5 h组、加压1 h组、加压2 h组,采用Western Blot法检测各组细胞IGF1R的表达及磷酸化水平,并在此基础上将同代无处理的软骨细胞在加压各组检测的同时给予同样检测即为静态组,应用Gel-Pro Analyzer软件进行半定量灰度分析比较各时间段静态和加压两两各组磷酸化IGF1R/总IGF1R水平差异。另取大鼠软骨细胞随机分为静态组、加压对照组、IGF1R阻断组、加压后IGF1R阻断组,IGF1R阻断采用顺式－3－[8－胺基－1－(2－苯基－喹啉－7－基)－咪唑并[1,5－a]吡嗪－3－基]－1－甲基－环丁醇(OSI－906)或者以IGF1R shRNA两种方式。阻断IGF1R 1 h后Western Blot法检测各组细胞磷酸化细胞外信号调节激酶1/2(ERK 1/2)表达及磷酸化水平;8 h后实时荧光定量PCR检测各组2型胶原(collagen Ⅱ)、蛋白聚糖(aggrecan)表达;3 d后采用直接细胞计数方式及细胞计数试剂盒(CCK-8)对软骨细胞增殖状况进行测定。应用SPSS 18.0软件进行相关统计学分析,两组间差异采用t检验比较。 结果磷酸化IGF1R/总IGF1R蛋白水平在加压0 h组与静态组之间差异无统计学意义(t＝0.255, P＝0.811),而在0.5 h组,1 h组,2 h组较相对应的静态组增高,差异具有统计学意义(t＝－5.881、－6.172、－10.518,P均小于0.05)。IGF1R被OSI-906或shRNA抑制后,加压处理的ERK 1/2磷酸化水平显著降低(OSI-906阻断后t＝3.074,shRNA阻断后t＝3.990,P均小于0.05),细胞外基质collagen Ⅱ(OSI-906阻断后t＝3.243, shRNA阻断后t＝3.621,均为P<0.05)、aggrecan(OSI-906阻断后t＝3.128,shRNA阻断后t＝3.608,P均小于0.05)基因表达水平显著降低,大鼠软骨细胞增殖能力减弱,差异均具有统计学意义(OSI-906阻断后t＝2.835、shRNA阻断后t＝3.467,均为P<0.05)。 结论周期性机械应力通过压力感受器IGF1R将机械信号转变为生物化学信号,激活ERK 1/2信号通路促进软骨细胞增殖和细胞外基质合成。     

软骨细胞三维支架短期固定培养实验研究

目的 :观察兔肋软骨细胞聚羟基乙酸 (polyglycolic acid,PGA)三维支架短期固定培养对软骨细胞利用率、软骨细胞长满支架所需时间及软骨细胞支架复合体厚度等影响。方法 :设 PGA支架固定组和非固定组 ,固定组 :先用鼠尾胶把 PGA支架粘附在盖玻片上 ,再往固定后的 PGA支架上滴加软骨细胞悬液 ,3～ 4天后支架与盖玻片分离。非固定组 :把软骨细胞悬液直接滴加到 PGA支架上进行复合培养。固定组和非固定组软骨细胞 PGA支架复合体培养 2周后分别行同种异体皮下移植 ,3月后取出标本 ,光镜观察软骨形成情况。结果 :固定组中 PGA支架在加培养液过程中 ,不会在培养液内漂动 ,支架内软骨细胞无大量流失现象 ;相反 ,非固定组中 PGA支架在加培养液和移动培养皿过程中 ,会在培养液内飘动或晃动 ,支架内很多软骨细胞重新漂浮至培养液中。固定组 PGA支架内软骨细胞分裂、增殖 1周时 ,软骨细胞已充满 PGA网眼 ;在非固定组 ,2周时 ,软骨细胞才长满 PGA支架内网眼。固定组软骨细胞支架复合体同种异体移植后能形成成片软骨 ;非固定组软骨细胞支架复合体移动后无软骨形成。结论 :软骨细胞 PGA三维支架短期固定培养 ,能有效减少支架内软骨细胞流失 ,缩短软骨细胞长满 PGA支架网眼的时间 ,增加软骨细胞支架复合体厚度 ,有利于组     

In vitro evaluation of (S)-ibuprofen toxicity on joint cells and explants of cartilage and synovial membrane

Intra-articular drug delivery systems (DDSs) are envisaged as interesting alternative to locally release nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen to reduce pain in patients with osteoarthritis. The present study examines the toxicity of (S)-ibuprofen on chondrocytes and synoviocytes isolated from sheep shoulder joint and cultured in monolayers during 72 h, and on joint explants (cartilage and capsule) cultured in mono- or in co-culture for 13 days. (S)-ibuprofen (5 μM up to 1 mM) did not reduce the cell viability and protein content when added on chondrocyte monolayers, while at 1 mM (S)-ibuprofen reduced (by 8%, p = 0.01) the synoviocytes viability compared to untreated cells. During co-culture of joint explants, (S)-ibuprofen at 50 μM significantly reduced by 35% the spontaneous release of glycosaminoglycans (GAGs) from cartilage (p = 0.0065) whereas in monoculture, (S)-ibuprofen was inactive on GAG metabolism. (S)-ibuprofen at 1 mM significantly reduced cell lysis (lactate dehydrogenase leakage) by 74% during monoculture of capsule explants (p = 0.0136) and by 35% during co-culture of explants (p = 0.0013). Our findings demonstrate that the active isomer of ibuprofen at micro- and millimolar levels was not toxic for chondrocytes and synoviocytes and may reduce at 1 mM the cell lysis during culture of joint explants. The limited toxicity of (S)-ibuprofen at low and high concentration in sheep joint shoulder makes this enantiomer a promising drug candidate for the loading of intra-articular DDS.     

Osteoarthritic cartilage fibrillation is associated with a decrease in chrondrocyte adhesion to fibronectin

OBJECTIVE: Cartilage destruction in osteoarthritis (OA) is generally accepted as a failed repair process. Cell adhesion is implicated in tissue repair. Therefore, adhesion of OA chondrocytes to extracellular matrix proteins was investigated. DESIGN: Using chondrocytes from human OA femoral head cartilage, adhesion to fibronectin and type II collagen of cells from distinct areas showing an intact cartilage surface or a fibrillated cartilage surface was studied. Modulation of chondrocyte adhesion by both protein kinase C (PKC) inhibitors and glucosamine sulfate (GS) was also investigated. RESULTS: A significant (P < 0.05) decrease in adhesion to fibronectin of chondrocytes from fibrillated cartilage, relative to those from grossly normal OA cartilage, was demonstrated. Adhesion to type II collagen was not modified by the chondrocyte origins (either from normal or fibrillated OA cartilage). Adhesion to fibronectin of cells from grossly intact cartilage was decreased by the addition of PKC and calmodulin-dependent kinase inhibitors, W7 and sphingosine, to the cell culture. Adhesion to fibronectin of chondrocytes from fibrillated cartilage was significantly (P < 0.05) increased after glucosamine sulfate treatment. CONCLUSION: Fibrillation of cartilage from OA femoral head is associated with a defective adhesion of chondrocytes to fibronectin. The process is suggested to be dependent of PKC and/or calmodulin-dependent kinases and potentially reversible. Conceivably, it could play a role in OA cartilage destruction.