人骨软骨组织冷冻保存效果及组织库建立

发布时间：2018-06-03 11:18

本文选题：关节软骨 + 骨软骨　；参考：《泰山医学院》2011年硕士论文

【摘要】：目的按照AATB标准,应用慢速梯度降温冷冻保存骨软骨标本,观察保存后软骨细胞存活率及细胞活性的变化,并建立骨软骨组织库,初步应用于临床,为骨软骨移植物临床应用奠定基础。方法年龄在15-45岁之间的捐献者(山东省千佛山医院临床脑死亡的病人,无关节病损),死前做血尿及其他体液常规检查符合捐献条件,于死亡后12小时内,无菌条件下应用Arthrex关节软骨移植器械垂直于膝关节股骨髁与胫骨平台关节面切取直径为6mm、8mm、10mm,骨柱长约10-15mm的圆柱形骨软骨块,共200枚。经10%DMSO处理后无菌独立包装,行慢速梯度降温至-80℃,保存入-80℃深低温冰箱,建立骨软骨组织库。分别在保存1个月、3个月、6个月、1年时随机抽取10枚标本行胎盘蓝染色检测软骨细胞存活率,MTT法检测软骨细胞活性;10枚做组织学和组织化学染色观察软骨细胞组织形态和代谢活性变化。将各时间段保存标本的检测结果与新鲜组对照,评价保存效果,并在冷冻保存早期选取6枚标本应用于临床治疗3例患者膝关节软骨缺损,平均随访2个月,评价临床效果。结果 1、软骨细胞存活率的变化新鲜对照组软骨细胞存活率为94.80±4.59,应用梯度降温冷冻保存1个月、3个月、6个月、1年时,细胞存活率分别为66.95±9.98、58.11±9.11、43.74±8.37、29.21±5.03,实验组与新鲜对照组比较均有统计学差异(P0.05),各保存时间点实验组组间两两比较均有统计学差异(P0.05)。即深低温冷冻保存对软骨细胞存活率的影响具有动态时间依从性。 2、关节软骨PG的变化新鲜对照组关节软骨PG含量丰富,番红O染色为红橙色,经梯度降温冷冻保存1个月、3个月、6个月、1年时,番红O染色逐渐变浅,各组浅、中、深层平均IOD值(表1),各实验组与新鲜对照组比较均有统计学差异(P0.05),各保存时间点实验组组间两两比较均有统计学差异(P0.05),即深低温冷冻保存对软骨PG的影响有时间依从性。 3、软骨细胞活性新鲜对照组OD值为0.65±0.03,经阶梯降温冷冻保存1个月、3个月、6个月、1年时,OD值分别为0.42±0.04、0.29±0.02、0.21±0.03、0.16±0.04,各实验组与新鲜对照组比较均有统计学差异(P0.05),各保存时间点实验组组间两两比较均有统计学差异(P0.05),即深低温冷冻保存对软骨细胞活性的影响有时间依从性。 4、无菌操作取材的骨软骨标本经慢速梯度降温至-80℃冷冻保存1月-1年,包装完好,各保存时间点随机抽取标本行细菌培养检测结果均为阴性;临床应用冷冻保存的6枚标本治疗3例关节软骨缺损,术后MRI检查均可见移植骨与宿主骨融合,Brittberg-Peterson功能评分较术前明显降低,显示早期移植效果满意。结论 1、深低温保存对关节软骨活性的影响具有动态的时间依从性。随着保存时间的延长,关节软骨活性会逐渐降低;低温导致软骨被冻伤是客观存在的。 2、梯度降温深低温冷冻保存可操作性强,能使骨软骨移植物细胞存活时间延长,建立骨软骨组织库具有可行性。 3、应用组织库保存的骨软骨移植物治疗关节软骨缺损手术取得了初步临床效果。意义:青少年运动伤导致关节软骨损伤的治疗一直是个难题,骨软骨移植是公认的较为理想的治疗方法。由于供体来源的有限,如何长时间的保存有活性的软骨移植物成为研究者关注的焦点。目前国外已开展了冷冻保存骨软骨组织方法与临床应用研究,国内对关节软骨保存方法的研究还处于探索阶段,软骨组织库基本空白。本课题以人关节软骨为标本,应用10%DMSO预处理后慢速梯度降温冷冻法保存建立实验用软骨组织库,并阶段性研究冻存软骨细胞的存活率及细胞活性,初步应用于临床行关节软骨缺损的治疗,可为软骨组织库建立和临床应用奠定基础。
[Abstract]:objective
According to the AATB standard, the bone cartilage specimens were preserved by slow gradient cooling, and the survival rate of cartilage cells and the changes of cell activity after preservation were observed, and the tissue Library of bone and cartilage was established. It was preliminarily applied to clinical application and laid the foundation for the clinical application of bone and cartilage graft.
Method
A donor between 15-45 years of age (clinical brain death in Qianfo Hill hospital in Shandong Province, no joint lesion), hematuria and other routine physical examination before death conforms to donor conditions. Within 12 hours after death, the application of Arthrex articular cartilage transplantation instruments to the articular surface of the knee joint of the femoral condyle and the tibial plateau under the aseptic condition The diameter of 6mm, 8mm, 10mm, a total of 200 cylindrical bone cartilage blocks with a bone column of about 10-15mm, was treated by 10%DMSO, and then aseptic and independently packaged and cooled to -80 C by slow gradient, and stored in -80 C deep cryogenic refrigerator to establish a bone cartilage tissue library. 10 specimens were randomly selected to detect cartilage by staining the placental blue for 1 months, 3 months, 6 months and 1 years respectively. Cell viability, MTT method was used to detect the activity of chondrocytes. 10 tissue and histochemical staining were used to observe the changes of tissue morphology and metabolic activity of chondrocytes. The results were compared with those in the fresh group to evaluate the preservation effect, and 6 specimens were selected for clinical treatment of the knee joint in 3 patients. Cartilage defects were followed up for an average of 2 months to evaluate the clinical effect.
Result
1, the survival rate of chondrocytes in the fresh control group was 94.80 + 4.59, and the cell survival rate was 66.95 + 9.98,58.11 + 8.37,29.21 + 8.37,29.21 + 5.03, respectively, with gradient cooling for 1 months, 3 months, 6 months and 1 years, respectively. There was a statistical difference between the experimental group and the fresh control group (P0.05), and the preservation time points were compared. There was statistical difference between the 22 groups in the experimental group (P0.05), that is, cryopreservation had a dynamic time dependence on the survival rate of chondrocytes.
2, the changes of articular cartilage PG in the fresh control group were rich in articular cartilage PG content, red O staining was red orange, frozen for 1 months, 3 months, 6 months, and 1 years, the red O staining gradually became shallow, each group was shallow, middle, and deep mean IOD value (table 1). The experimental groups were statistically different from those in the fresh control group (P0.05), each preservation time There was statistical difference between the 22 groups in point test group (P0.05), that is, cryopreservation had time dependence on cartilage PG.
3, the OD value of the chondrocyte active fresh control group was 0.65 + 0.03. After 1 months, 3 months, 6 months and 1 years, the OD values were 0.42 + 0.04,0.29 + 0.03,0.16 + + 0.04 respectively, and the experimental groups were statistically different from those of the fresh control group (P0.05), and 22 of the experimental groups of each preservation time were statistically poor. The effect of cryopreservation (P0.05) on chondrocyte viability is time dependent.
4, the osteochondral specimens obtained by the aseptic operation were cooled to -80 C for -1 year in January, and the packing was intact. The results of the bacterial culture test were negative in the specimens. 6 specimens of frozen preserved specimens were used to treat 3 cases of articular cartilage defect, and the fusion of bone graft and host bone was found after MRI examination, Br The ittberg-Peterson function score was significantly lower than that before operation, indicating that the early transplant effect was satisfactory.
conclusion
1, the effect of deep cryopreservation on the activity of articular cartilage has a dynamic time dependence. With the prolongation of the preservation time, the activity of articular cartilage will gradually decrease, and the cold injury of cartilage is an objective existence at low temperature.
2, gradient cooling and cryopreservation are feasible and can prolong the survival time of osteochondral allograft cells. It is feasible to establish osteochondral tissue bank.
3, the application of osteochondral grafts preserved in tissue bank for the treatment of articular cartilage defects has achieved initial clinical results.
Significance: the treatment of articular cartilage injury caused by sports injuries in adolescents has been a difficult problem. Osteochondral transplantation is an accepted ideal treatment. Because of the limited source of donor, how to preserve active cartilage graft for a long time has become the focus of attention. At present, the methods of cryopreservation of osteochondral tissue have been developed abroad. The research of articular cartilage preservation methods in China is still in the exploration stage, and the cartilage tissue library is basically blank. In this study, human articular cartilage was used as a specimen. The experimental cartilage tissue library was established by 10%DMSO pretreatment slow gradient cooling freezing method, and the survival rate and cell of frozen cartilage cells were studied step by step. The preliminary application of this method in the treatment of articular cartilage defects can lay a foundation for the establishment and clinical application of cartilage tissue bank.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R361

【参考文献】