异种脐血干细胞移植修复兔全层关节软骨缺损的初步研究

A Pilot Study of Repair of Full-thickness Articular Cartilage Defect with Human Umbilical Cord Blood Stem Cells in A Rabbit Model

目的 :探讨人脐血干细胞对兔全层关节软骨缺损的修复作用及免疫反应。材料和方法 :取人脐带血中脐血干细胞及幼兔的骨髓基质细胞 ,体外分离培养 ;以聚乳酸 (PLA)为载体 ,将培养的原代细胞植入PLA支架上 ,形成细胞 -PLA复合物。于 2 0只成年新西兰大白兔的股骨滑车关节面上造成直径 4.5mm深 3 .0mm的全层关节软骨缺损 ,将两种细胞 -PLA复合物分别植入关节软骨缺损处。植入异种脐血干细胞 -PLA复合物为实验组 ,植入同种异体骨髓基质细胞 -PLA复合物为阳性对照组 ,缺损不处理为阴性对照组。术后 6周、1 2周观察缺损修复情况、新生组织类型及有无免疫反应。结果 :脐血干细胞组 6周时标本为纤维组织修复 ,内有少量软骨细胞 ;1 2周时 40 %标本为软骨样组织修复 ,较薄 ;60 %标本为纤维组织修复。移植物周围无明显淋巴细胞聚集 ,部分滑膜有炎症反应。骨髓基质细胞组(阳性对照)为软骨样组织修复 ;滑膜无明显炎症反应。阴性对照组为纤维组织修复 ,无软骨形成。结论 :异种脐血干细胞移植修复软骨缺损优于缺损不处理组 (阴性对照) ,但明显差于同种骨髓基质细胞组 (阳性对照 )。脐血干细胞有可能成为软骨修复的新的种子细胞。由于种属差异的影响 ,脐血干细胞组可能存在免疫反应 。

Objective The purpose of this study was to evaluate reparative tissues formed in full-thickness articular cartilage defects in an adult rabbit model implanted with cultured chondrogenic human umbilical cord blood stem cells seeded in PLA (polylactic acid) matrices and to probe if immunological response against transplanted cells exists. Materials and methods: Full-thickness articular cartilage defects (4.5mm in diameter and 3mm in depth) were produced in trochlea grooves of both knees of 20 adult New Zealand White rabbits. Human umbilical cord blood stem cells were isolated from the umbilical cord blood of newborn children. Mesenchymal stem cells (MSCs) were isolated from the 6-week-old New Zealand rabbits. The two kinds of cells were cultured in vitro and embedded in PLA matrices. Total three groups were made according to the implanted cell type: Group A, umbilical cord blood stem cells; Group B, MSCs (positive control group); Group C, untreated defects (negative control group). These cell-PLA composites were transplanted into the cartilage defects. The repair tissues and synovium were evaluated grossly, histologically, biochemically and by a semiquantitative scoring system at 6 weeks and 12 weeks after implantation. Results: In Group A, the defects were mainly repaired with fibrous tissues and scattered chondrocytes at 6 weeks after implantation, which was negative for typeⅡcollagen and toluidine blue staining. At 12 weeks after transplantation,forty percent of specimens were repaired with a thin-layer hyaline-like cartilage, which was positive for typeⅡcollagen and toluidine blue staining;but sixty percent of specimens were repaired with fibrous tissues,which was negative for type Ⅱ collagen and toluidine blue staining. There was no obvious lymphocytes infiltration in repair tissues, but inflammatory reaction was observed in synovium tissues. In Group B, the defects were repaired with hyaline-like cartilage, which was positive for typeⅡcollagen and toluidine blue staining. The subchondral bone and tidemark was well remodeled. The repair tissue was similar to normal cartilage. There was no obvious immunological reaction. In Group C, the defects were repaired with fibrous tissues. The surrounding cartilage showed obviously degenerative changes. Conclusions: Full-thickness articular cartilage defects treated with umbilical cord blood stem cells transplantation were better than the untreated defects,but significantly worse than the repair tissues treated with MSCs transplantation. The infiltration of lymphocytes in synovium in experiment group might be the immunological response to the transplantation between different species. Human umbilical cord blood stem cells might be the new choice of seeded-cell for tissue-engineered cartilage,but need further investigation.