Changes in the production and the effect of nitric oxide with aging in articular cartilage: An experimental study in rabbits

We studied the production and the effect of nitric oxide (NO) in articular cartilage from rabbits of various ages. 40 New Zealand white rabbits in 4 age groups (1 month, 6 months, 1 year, and 3 years of age with 10 rabbits in each group) were used. Basal and induced levels of NO were lower in cultured chondrocytes from older rabbits. Exogenous NO administration suppressed the proliferative activity of chondrocytes to a greater degree in younger rabbits. Immunohistochemistry showed that older rabbits had fewer eNOS positive chondrocytes. Our findings imply that the age-related change in NO in articular cartilage does not have a relevance to increased NO production in osteoarthrosis.     

一氧化氮合酶抑制剂对关节软骨修复的影响

目的观察一氧化氮合酶(iNOS)抑制剂S-甲基异硫脲(S-methylisothiourea,SMT)对关节软骨修复的影响,探讨NO在软骨修复过程中的作用。方法36只新西兰大白兔双侧股骨髁间关节面制成全层软骨缺损,随机分为3组:(1)对照组12只,软骨缺损区旷置;(2)rhBMP组12只,缺损区充填rhBMP纤维蛋白凝胶复合物;(3)SMT组12只,缺损区充填rhBMP纤维蛋白凝胶复合物后,皮下注射iNOS抑制剂SMT。术后16周处死动物,按组织形态学分级标准,作修复组织质量评价;天狼猩红-苦味酸染色观察修复组织内Ⅰ、Ⅱ型胶原分布;化学比色法检测NO释放量和NOS活性;35S掺入法检测蛋白多糖合成率;RT-PCR检测iNOS和MMP9mRNA的表达情况。结果术后16周,形态学评分表明,SMT组和rhBMP组在缺损区充填程度方面与对照组差异无显著性意义(F=2.32,P >0.05),在边缘修复结合度、细胞形态和缺损区结构以及软骨下骨修复等方面均优于对照组(P<0.05),其中SMT组优于rhBMP组(P<0.05)。化学比色法显示SMT组NO释放量(24.97±3.95)μmol/L和NOS活性(1.17±0.31)U/ml显著低于rhBMP组和对照组(F=21.287,F=15.932,P<0.05)。SMT组Ⅰ型胶原明显少于rhBMP组和对照组,Ⅱ型胶原多于rhBMP和对照组。SMT组35S掺入量(48276±5791.58)cpm明显高于对照组(10285±867.5)cpm和rhBMP组(37624±5     

透明质酸促进关节软骨缺损修复的实验研究

[目的]观察透明质酸对关节软骨损伤修复的作用,并探讨其机制。[方法]经手术建立兔股骨内髁软骨2.5 mm圆形缺损模型,术中于缺损处局部注射、术后每周关节内注射透明质酸0.5 m1。4周后组织学和免疫组织化学分析缺损处软骨修复情况及凋亡细胞发生率;并分析关节液中Ⅱ型胶原、粘多糖和肿瘤坏死因子含量。[结果]透明质酸对关节软骨缺损的修复有明显的促进作用,4周时组织学上可见纤维软骨修复为主,其间散在少量Ⅱ型胶原阳性细胞,部分纤维软骨向透明软骨转化。并且关节液中Ⅱ型胶原和粘多糖增多,TNF-α含量减少,同时软骨和软骨下骨中凋亡细胞发生率降低。[结论]局部注射透明质酸可能通过影响骨髓干细胞增殖与分化,自骨髓和软骨下骨向缺损处诱发新生软骨组织的形成,从而促进软骨损伤的修复。     

Effect of nitric oxide synthase inhibitor on proteoglycan metabolism in repaired articular cartilage in rabbits

Nitricoxide (NO )isanimportantregulatoryfactorandasignal transductionmoleculeintheprocessofcellgrowthanddifferentiation .Recentreportshaveindicatedthatnitricoxideiscloselyrelatedtothedisintegrationofarticularcartilage,becausehighexpressionofinducednitricoxidesynthase (iNOS)inhibitormRNAisdetectedinthearticularcartilageandiNOScanamelioratethecartilagemetabolisminpatientswithosteoarthritisorrheumaticarthritis .1,2 OurpreviousstudyshowedthatiNOScanamelioratethe qualityofrepairedcartilages ,t…     

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.     

修复关节软骨大面积缺损的实验研究

目的 比较和评价筋膜软骨细胞和骨膜、关节软骨移植修复关节软骨大面积缺损的能力和生物特性。方法 用冻存和新鲜的异体筋膜上培养的软骨细胞、骨膜和关节软骨移植修复大面积关节软骨缺损,通过大体标本、光学显微镜、扫描和透射电镜、放射身显影、微量元素和柱层析氨基酸定量测定、一氧化氮含量测定等多种观察方法进行评价。结果 新鲜和冻存的筋膜软骨细胞移植在结构、形成新的软骨细胞能力、代谢活性方面均优于游离软骨细胞移植     

The effects of static and intermittent compression on nitric oxide production in articular cartilage explants.

Nitric oxide (NO) production and NO synthase (NOS) expression are increased in osteoarthritis and rheumatoid arthritis, suggesting that NO may play a role in the destruction of articular cartilage. To test the hypothesis that mechanical stress may increase NO production by chondrocytes, we measured the effects of physiological levels of static and intermittent compression on NOS activity, NO production, and NOS antigen expression by porcine articular cartilage explants. Static compression significantly increased NO production at 0.1 MPa stress for 24 h (P < 0.05). Intermittent compression at 0.5 Hz for 6 h followed by 18 h recovery also increased NO production and NOS activity at 1.0 MPa stress (P < 0.05). Intermittent compression at 0.5 Hz for 24 h at a magnitude of 0.1 or 0.5 MPa caused an increase in NO production and NOS activity (P < 0.05). Immunoblot analysis showed stress-induced upregulation of NOS2, but not NOS1 or NOS3. There was no loss in cell viability following any of the loading regimens. Addition of 2 mM 1400 W (a specific NOS2 inhibitor) reduced NO production by 51% with no loss of cell viability. These findings indicate that NO production by chondrocytes is influenced by mechanical compression in vitro and suggest that biomechanical factors may in part regulate NO production in vivo.     

Repairing articular cartilage defects with tissueengineering cartilage in rabbits

Objective: To investigate the effect of cancellous bone matrix gelatin ( BMG ) engineered with allogeneic chondrocytes in repairing articular cartilage defects in rabbits. Methods: Chondrocytes were seeded onto three-dimensional cancellous BMG and cultured in vitro for 12 days to prepare BMG-chondrocyte complexes. Under anesthesia with 2.5% pentobarbital sodium (1ml/kg body weight), articular cartilage defects were made on the right knee joints of 38 healthy New Zealand white rabbits (regardless of sex, aged 4-5 months and weighing 2. 5-3 kg) and the defects were then treated with 2. 5% trypsin. Then BMG-chondrocyte complex ( Group A, n = 18 ), BMG (Group B, n = 10), and nothing (Group C, n = 10) were implanted into the cartilage defects, respectively. The repairing effects were assessed by macroscopic, histologic, transmission electron microscopic ( TEM ) observation, immunohistochemical examination and in situ hybridization detection, respectively, at 2, 4, 8, 12 and 24 weeks after operation. Results: Cancellous BMG was degraded within 8 weeks after operation. In Group A, lymphocyte infiltration was observed around the graft. At 24 weeks after operation, the cartilage defects were repaired by cartilage tissues and the articular cartilage and subchondral bone were soundly healed. Proteoglycan and type II collagen were detected in the matrix of the repaired tissues by Safranin-O staining and immunohistochemical staining, respectively. In situ hybridization proved gene expression of type II collagen in the cytoplasm of chondrocytes in the repaired tissues. TEM observation showed that chondrocytes and cartilage matrix in repaired tissues were almost same as those in the normal articular cartilage. In Group B, the defects were repaired by cartilage-fibrous tissues. In Group C, the defects were repaired only by fibrous tissues. Conclusions: Cancellous BMG can be regarded as the natural cell scaffolds for cartilage tissue engineering. Articular cartilage defects can be repaired by cancellous BMG engineered with allogeneic chondrocytes. The nature of repaired tissues is closest to the normal cartilage. Local administration of trypsin can promote the adherence of repaired tissues to host tissues. Transplantation of allogeneic chondrocytes has immunogenicity, but the immune reaction is weak.     

Effects of various irrigating solutions on articular cartilage. An experimental study in rabbits

The effect of six different solutions (normal saline, ringer's lactate, chlorhexidine, povidone iodine, ceftriaxone, chloramphenicol) on articular cartilage was investigated in an in vivo rabbit model study. The right knees were aspirated and injected with one of these solutions for five days and, three days later, the patellae of the rabbits were excised and investigated histologically. Left knees were used as controls. There was no difference between the groups and the controls with respect to structure, cell density, and nuclei-to-lacunae ratio. These results suggest that, these solutions have no noxious effects on articular cartilage when used as irrigating fluids in orthopaedic practice.     

高纯度猪软骨Ⅱ型胶原修复兔膝关节软骨缺损的实验研究

目的 研制高纯度猪软骨Ⅱ型胶原海绵,探讨其修复关节软骨缺损的可行性.方法 将40只成年雄性新西兰兔制成膝关节软骨缺损模型,随机分成两组.A组20只兔的缺损区植入Ⅱ型胶原海绵,B组20只兔的缺损区旷置,不植入胶原作为对照.于术后2、4、8、12、24周分别做HE染色、Masson染色、Safranin O染色及Ⅱ犁胶原免疫组化检测.结果 ①HE染色及Masson染色显示A组在术后2周即出现新生软骨细胞,细胞以胶原纤维为支架迁移进入缺损区,并与胶原纤维生长融合;12周后新生骨和软骨组织完全填满缺损区并与周围组织整合,而B组直至术后24周仍由纤维组织所填充;②免疫组化检测结果 显示A组新生软骨细胞具有正常兔软骨细胞的表型;③Safranin O染色结果 显示A组新生软骨细胞具有分泌软骨基质的功能.结论 Ⅱ型胶原具有较强的诱导软骨细胞生长的能力,其诱导生长的新生软骨细胞具有正常透明软骨细胞的表型和功能,是良好的软骨缺损修复的生物填充材料.     

Free periosteal autografting in repair of articular cartilage: an experimental study and clinical application

38 white rabbits in 4 groups were used in this experiment to investigated the metaplastic process and the outcome of the autografted periosteum on cartilage defect created artificially on acetabulum and femoral condyle. The specimens were scrupulously surveyed with conventional methods and analysis of their trace elements and amino acids, which was the means first used in evaluating the nature of metaplastic tissue. Conclusions drown from this experiment are as follows: (1) The autografted periosteum processes a metaplastic potential of forming articular cartilage under certain conditions. (2) Continuous passive movement of the grafted joint accelerate the metaplastic process, forming cartilage. (3) Synovial fluid plays certain role in survival of the transplanted periosteum and its metaplasia. However, excision of the synovium did not affect the result much, probably due to its rapid and powerful regeneration.     

一氧化氮合酶抑制剂对关节软骨缺损修复影响的形态学研究

目的　探讨诱导型一氧化氮合酶 (iNOS)抑制剂S 甲基异硫脲 (SMT)对关节软骨修复的影响。方法　将 2 0只新西兰大白兔双侧股骨髁关节面造成全层软骨缺损。随机分为 2组 :对照组 10例 ,缺损软骨面用纤维蛋白凝胶BMP复合物充填 ;给药组 10例 ,缺损软骨面用纤维蛋白凝胶BMP复合物充填后 ,皮下注射SMT(5mg·kg-1·12h-1)。术后 8周、16周、1年处死动物 ,按组织形态学分级标准 ,双盲法行 16周和 1年修复组织评价 ;化学比色法检测修复组织NO释放量和NOS活性。结果　形态学观察证实 ,术后 8周、16周及 1年后 ,给药组软骨缺损修复在缺损区结构、细胞形态和基质染色等评分方面优于对照组 (P <0 0 5 )。术后 16周及 1年对照组NO释放量和NOS活性明显高于给药组 (P <0 0 5 )。结论　iNOS抑制剂SMT可减少NO释放 ,降低iNOS活性 ,提高软骨修复质量。     

Repair of articular cartilage defects with cultured chondrocytes on polysulphonic membrane: experimental studies in rabbits

INTRODUCTION: Autologous osteochondral transplantation is one method that can be used to create hyaline or hyaline-like repair in a defect area. The purpose of the present study was to repair full-thickness articular cartilage defects in 9 rabbit knee joints with autologous cultured chondrocytes. METHODS: An articular cartilage defect was created on the patellar groove of the femur. The defect was filled with chondrocytes cultured in vitro and placed into the knee on a polysulphonic membrane. At 8 weeks after the operation, the reparative tissue was analyzed macroscopically and histologically. RESULTS: At 8 weeks after the operation, the surfaces of the reparative tissue were smooth, and the defects were filled with mature hyaline cartilage in 5 cases. In 2 cases, the reparative hyaline cartilage was immature and there was worse integration of grafted tissue into the adjacent normal cartilage. In 2 cases, the surface of the grafted area was irregular, and the reparative tissue was disintegrated and incompletely differentiated. CONCLUSION: The results suggest that transplantation of autologous chondrocytes cultured in vitro and placed into the knee on polysulphonic membrane is effective in repairing an articular cartilage defect.     

Long-lasting tolerance of articular cartilage after experimental intraoperative radiation in rabbits.

In experimental intraoperative irradiation, 18 adult rabbits received a single, 50-Gy dose of x-radiation at a unilateral knee joint, and subsequent changes in the articular cartilage were examined over a 15-month period by histology, scanning electron microscopy, and autoradiography. Although the subchondral bone showed histologically typical findings of osteonecrosis three to nine months postirradiation, the articular cartilage revealed no obvious degenerative changes during the entire study period. Scanning electron microscopy revealed normal collagen architecture in the irradiated cartilage for as long as 15 months postirradiation. Autoradiography demonstrated active RNA synthesis by the irradiated chondrocytes during the same period. These results indicate that articular cartilage tissue tolerates intraoperative radiotherapy without sustaining serious degenerative changes, unless possible collapse or contracture disturbs its biomechanical integrity. The survival of articular cartilage can be advantageous for this type of limb-salvage surgery in the treatment of malignant bone tumors around a synovial joint.     

Ultrastructural study of articular cartilage in experimental pyogenic arthritis

Ultrastructural changes of the articular cartilage at the early stage of experimental pyogenic arthritis were studied by electron microscopy. The superficial zone of the cartilage at the site of the cartilage-synovium junction demonstrated far more changes than those at the site of the free surface of the articular cartilage. Changes were observed at the cartilage-synovium junction as early as four hours after the intraarticular injection of the infecting organism. The most interesting finding was migration of erythrocytes and polymorphonuclear leucocytes into the superficial zone of the cartilage at the early stage. The pericellular matrix in the middle zone became so loose in some areas that collagen fibrils were partially exposed after three days. The naked collagen fibrils were tapered, nicked and irregular in size after seven days.