藻酸钠复合自体软骨细胞修复猪关节软骨缺损的近期效果

Short-term effect of sodium alginate solution plus autologous chondrocytes on repairing porcine cartilage defects

目的:采用藻酸钠自体软骨细胞混合物修复猪的关节软骨缺损,观察近期成骨效应。方法:实验于2004-03/2005-04在中山大学附属第二医院实验中心完成。3个月龄的小型猪8只,取其关节软骨进行细胞原代培养,获取原代细胞后,分别进行免疫组织化学法检测细胞Ⅱ型胶原表达。然后对8只小型猪的32个关节进行动物关节缺损的模型建立,每个关节共造3个人工缺损,选择关节左右随机对照。将已经培养好的软骨细胞与已经配制好并消毒的藻酸钠混合,移植到猪的关节软骨缺损处。每只猪关节内注射3个实验点,1个用藻酸钠/细胞混合液为实验组,1个单纯支架材料为单纯材料对照组,1个缺损做空白对照组,模型中缺损区均为关节负重区,胶原纤维绿色(亮绿复染)。修复所用细胞为自体软骨体外培养后,进行移植修复,采用3代体外培养软骨细胞,均匀混和藻酸钠后,行自体移植。8周后取实验关节进行大体观察、苏木精-伊红染色、Masson染色、免疫组织化学法染色观察及O’driscoll组织学评分。结果:纳入小型猪8只,除其中1例因手术切断伸直肌腱而使猪关节活动障碍,其余均未见手术后关节活动障碍,所有手术无术后感染,无关节内感染,无动物死亡。移植的软骨细胞在藻酸钠载体中生长良好,形成透明软骨。根据O’driscoll组织学评分标准,实验组32个猪关节缺损修复的组织学评分为(20.25±1.64)分,高于单纯材料对照组的(7.46±1.29)分及空白对照组的(6.00±2.09)分。免疫组织化学染色可见修复软骨以表达Ⅱ型胶原为主,无Ⅰ型胶原。结论:藻酸钠复合自体软骨细胞作为软骨移植的替代物是可行的,远期效果还有待进一步研究。

AEH： To observe the short -term bone formation effect of the reparation of porcine arthrodial cartilage defect with alginate gels and chondrocytes mixture.. METHODS： The experiment was conducted in the Experiment Center of Second Hospital affiliated to Sun Yat-sen University from March 2004 to April 2005. Chondrocytes were harvested from 8 swine of 3-month old for primary culture. Then the expression of type Ⅱ collagen in cells were detected with immunohistomedical method. The models of arthrodial cartilage defects were made in the 32 joints of 8 swine with 3 defects in each joint, and one side of the joint served as the control. Cultivated chondrocytes were mixed with disinfected alginate gels, and transplanted to the defects. Each joint made 3 experimental points, in which one served as the experiment group with sodium alginate solution mixture, one as simple control group with simple sodium alginate solutions and the last was blank control. The defect regions of the models were loaded part of joint with green collagen fibers （light green re-stained）. The chondrocytes were selected from the third passage cells, which were autografted after mixed with sodium alginate solution. Eight weeks later, the experiment joints were observed by naked eyes, HE strainidg, Masson straining, immunohistochemical straining and O＇driscoll histology evaluation. RESULTS： Among the eight swine, there were no postoperative infection, intra-articular infection or death except joint activity disturbance appeared in one swine as the extensor tendon was cut off. Transplanted chondrocytes grew well in alginate gels carrier, and formed hyaline cartilage. According to the standard in O＇driscoll histology, the scores of the arthrodial cartilage defects in 32 swine of the experiment were （20.25±1.64） points, which were higher than the simple control group （7.46±1.29） points and the blank group （6.00±2.09） points. Immunohistochemical observation suggested that the recovery cartilage only expressed type Ⅱ collagen, but not type Ⅰ collagen. CONCLUSION： It is feasible to regard the sodium alginate solution mixed with chondrocytes as the substitutes in cartilage transplantation; however, its long-term effect needs further study.