玻璃化冷冻保存组织工程肌腱大鼠体内植入对血液免疫及生化指标的影响

Influence of implantation of tissue engineered tendon of vitreous cryopreservation in vivo on blood immune and biochemical indexes in rats

目的研究组织工程肌腱体内植入修复跟腱缺损的同时,根据细胞与支架材料的选择、工程化组织构建与保存方式所引起的体内毒性反应特征,从血液免疫和生化学指标方面评价玻璃化冷冻保存组织工程肌腱材料对机体的安全性影响。方法随机将72只SD大鼠分为3组,A、B两组各32只,C组8只。手术造成A、B两组大鼠一侧跟腱0.5cm缺失,分别植入1cm长的玻璃化冷冻保存与非玻璃化冷冻保存组织工程肌腱材料,C组不经任何手术。三组大鼠分批于术后第2、4、6、8周股动脉放血处死,收集全血分离血清,采用双抗体夹心ELISA、速率、双缩脲、溴甲酚绿、尿酶紫外速率、苦味酸、已糖激酶、氧化酶、DMSO、Diaso法测定血液相关免疫及生化指标。结果 A、B两组各项指标在相同植入期比较,差异无统计学意义(P>0.05);A、B两组与C组比较,除A组4周、B组4周和6周AST值及A组2周ALB值、B组2周CREA值明显低于C组相应指标外(P<0.05),其他指标差异均无统计学意义(P>0.05)。结论玻璃化冷冻保存组织工程肌腱大鼠体内植入后血液免疫及生化各项指标与非手术大鼠基本一致。证实了玻璃化冷冻保存组织工程肌腱用于肌腱缺损修复是安全可靠的,未发现免疫排斥反应,对肝肾功能无明显影响。

Objective To study the repair ofachilles tendon defect by implantation in vivo,as well as value the safety effect of tissue engineered tendon materials of vitreous cryopreservation on organism with blood immune and biochemical indexes according to selection of the cell and scaffold,the feature of toxic reaction in vivo induced by the engineered tissue construction and conservation.Methods Seventy-two SD rats were randomly assigned to three groups A,B and C.There were 32 rats,respectively,in groups A and B caused by a 0.5 cm in length of lateral achilles tendon defect.Tissue engineered tendon materials of vitreous cryopreservation were used in groupA,and the same tissue engineered tendon materials of non-vitreous cryopreservation were used in group B.A1 cm in length of material was implanted,respectively,into the defect site.There were eight rats in group C without any operation.Two,four,six and eight weeks after the operation,rats were sacrificed to obtain serum separated from whole blood by femoral arterial bloodletting.Blood immune and biochemical indexes,e.g.,immunoglobulin G（IgG）,immunoglobulin M（IgM）,alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,alkaline phosphomonoesterase（ALP）,total protein（TP）,albumin（ALB）,blood urea nitrogen（BUN）,creatinine（CREA）,glucose（GLU）,triglyceride（TG）,totalcholesterol（TC）,total bilirubin（TBIL）,direct bilirubin（DBIL）,were detected by double-antibody sandwich,enzyme-linked immunosorbent assay（ELISA）,rate,biuret,Bromocresol Green,urinary enzyme UV rate,picric acid,hexose kinase,oxidase,dimethyl sulfoxide（DMSO）and Diaso methods.Results There was no significant difference in all indexes of groups A and B at the same implantation stage（P〉0.05）.AST of group A at the 4th week,AST of group B at the 4th and 6th weeks,ALB of group A and CREA of group B at the 2nd week were all significantly lower when compared to those of group C respectively.There was no significant difference in other indexes of groups A and B（P〉0.05）when compared to group C.Conclusions All blood immune and biochemical indexes of tissue engineered tendon of vitreous cryopreservation implanted were accordant with the control group,proving that to repair tendon defectwith tissueengineered tendon of vitreous cryopreservation is safeandreliable without any immune rejection and obvious influences on hepatic,renal function.