骨髓间充质干细胞与仿生纳米壳聚糖-胶原复合物修复大鼠胫骨缺损的实验研究

Repairment of segmental tibial defects by composite grafts of bone marrow mesenchymal stem cells with bionic nano chitosan-sodium collagen in SD rats

目的:4周龄SD大鼠骨髓间充质干细胞离体培养后与仿生纳米壳聚糖-胶原(nano chitosan-so-dium collagen,nano-CS-COL)形成复合支架,植入SD大鼠胫骨缺损处,比较修复节段性胫骨缺损的效果,初步探讨治疗骨缺损的组织工程学方法。方法:4周龄SD大鼠骨髓间充质干细胞(BMSCs)离体培养、纯化、鉴定、扩增后在等同于细胞培养的条件下与nano-CS-COL复合培养,制成MSCs/nano-CS-COL复合物并经电镜扫描证实复合物有细胞生长。制成同种异体SD大鼠胫骨干5mm节段性动物模型,MSCs/nano-CS-COL复合物通过手术移植入动物模型胫骨缺损处,通过大体观察、X线放射学、组织学对比实验组与对照组、空白组骨缺损修复情况。结果:术后6、12周实验组与实验对照组放射学检查评价新骨生成有显著差异(P<0.05)。术后组织学检查新骨生成速度、生成量均有显著差异。实验组标本与正常组基本一致。空白对照组不能自行修复骨缺损,最后缺损由纤维组织充填。结论:BMSCs是骨组织工程中适宜的种子细胞。Nano-CS-COL复合支架能够与SD大鼠骨髓间充质干细胞体外复合培养,移植入异体SD大鼠后未见明显免疫排斥反应,是可以选择的细胞载体。MSCs/nano-CS-COL复合物植入修复鼠胫骨缺损能够加速新骨形成,修复效果明显优于单纯CS植入组。

AIM： To repair the segmental tibial defects in SD rats with bionic nano chitosan -sodium collagen （ nano - CS - COL） co - cultured with bone marrow mesenchymal stem cells （BMSCs）. METHODS： BMSCs were isola- ted, purified, cultured, identified and observed continually. Scaffold materials were cut into blocks with the size of Ф3 mm×5 mm. Cells of the 3rd generations were co -cultured with nano -CS -COL in vitro. The composite grafts were test- ed by scanning electron microscope. A bone defect （5 mm in length） was created at two tibial in each mouse. The composite grafts were implanted into segmental tibial defects in rats through open operation. The curative effect was evaluated by general observation, radiographic examination, histologic analysis at 6 and 12 weeks. RESULTS ： In experimental groups, the bone defects were repaired completely. Roentgenographically, the bone defects in the experimental groups exhibited new bone formation increased with time, which were apparently superior to that in control group at 6 and 12 weeks after operation （P 〈 0. 05 ）. The quality of new bone formation was significandy different between the experimental and the control groups by histologic analysis. In blank groups, bone defect could not be repaired without any proper treatment, finally filling only with fibrous tissue. CONCLUSION ： BMSCs are ideal seeded cells for bone tissue engineering. The nano - CS - COL is compatible with BMSCs. The composite grafts cause no obvious immune rejection after transplanted into allogenic rats. The nano - CS - COL scaffold may be used as an alternative carrier for seeded cells. The composite grafts of nano -CS - COL/MSCs quickly promote the new bone formation with better curative effect on tibial defects than other materials.