壳聚糖/亚磷酸化壳聚糖复合海绵复合人脐带间充质干细胞用于异位成骨的实验研究

EXPERIMENTAL STUDY ON ECTOPIC BONE FORMATION OF CHITOSAN/PHOSPHONIC CHITOSAN SPONGE COMBINED WITH HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS

目的通过构建壳聚糖/亚磷酸化壳聚糖复合海绵,与人脐带间充质干细胞(human umbilical cordmesenchymal stem cells,hUCMSCs)复合构建组织工程骨并行成骨诱导培养,探讨其异位成骨效果,为骨组织工程寻找理想支架材料。方法在壳聚糖分子中引入亚磷酸根基团,制备亚磷酸化壳聚糖,并进行表征。分别将浓度为2%的壳聚糖和亚磷酸化壳聚糖溶液,按1∶1体积比混合均匀,构建壳聚糖/亚磷酸化壳聚糖复合海绵;然后在模拟体液中进行原位矿化,通过扫描电镜观察矿化前后复合海绵的结构。用酶消化法分离培养hUCMSCs,取生长良好的第3代细胞与壳聚糖/亚磷酸化壳聚糖复合海绵复合培养,构建细胞-支架复合物,并行成骨诱导培养。成骨诱导培养1、2周,分别于光镜及扫描电镜下观察细胞黏附情况;培养1、2、3、4、5、6 d时,MTT法分析细胞增殖情况。取3～4月龄新西兰大白兔40只,雌雄不限,体重2.1～3.2 kg,平均2.5 kg;制备双侧竖脊肌肌袋,在每只兔右侧肌袋内植入细胞-支架复合物(A组,n=40),于其中20只兔左侧肌袋植入壳聚糖/亚磷酸化壳聚糖复合海绵(B组,n=20),剩余20只兔左侧肌袋不植入材料(C组,n=20)。术后观察动物一般情况,4周后处死动物取材,行大体及组织学观察。结果亚磷酸化壳聚糖分析表明亚磷酸化反应主要发生在羟基上,其质子类型和化学位移强度与化学结构一致。扫描电镜观察到壳聚糖/亚磷酸化壳聚糖复合海绵孔洞均匀,孔壁较薄;原位矿化后孔壁表面有钙磷涂层,晶体颗粒生成;细胞在壳聚糖/亚磷酸化壳聚糖复合海绵支架上黏附、生长良好。体内实验大体观察A组可见细胞-支架复合物大小、形态基本保持原状,质地有所增韧,材料周围有薄层结缔样组织;B组复合海绵体积缩小,质地松软;C组肌袋手术创口已愈合。组织学观察A组支架材料部分吸收,边缘有均质类骨物质出现,并见成骨细胞;B组植入区形成圆形空腔,残留壳聚糖支架网络;C组创面基本愈合,肌肉纤维间可见少量淋巴细胞浸润。结论壳聚糖/亚磷酸化壳聚糖复合海绵是一种具有良好生物相容性的支架材料,与hUCMSCs复合构建的组织工程骨在兔体内可异位成骨。

Objective To investigate the ectopic bone formation of the chitosan/phosphonic chitosan sponge combined with human umbilical cord mesenchymal stem cells（hUCMSCs） in vitro.Methods Phosphorous groups were introduced in chitosan molecules to prepare the phosphonic chitosan;2% chitosan and phosphonic chitosan solutions were mixed at a volume ratio of 1 ∶ 1 and freeze-dried to build the complex sponge,and then was put in the simulated body fluid for biomimetic mineralization in situ.The hUCMSCs were isolated by enzyme digestion method from human umbilical cord and were cultured.The chitosan/phosphonic chitosan sponge was cultured with hUCMSCs at passage 3,and the cell-scaffold composite was cultured in osteogenic medium.The growth and adhesion of the cells on the scaffolds were observed by light microscope and scanning electron microscope（SEM） at 1 and 2 weeks after culturing,respectively.The cell proliferation was detected by MTT assay at 1,2,3,4,5,and 6 days,respectively.Bilateral back muscles defects were created on 40 New Zealand rabbits（3-4 months old,weighing 2.1-3.2 kg,male or female）,which were divided into groups A,B,and C.In group A,cellscaffold composites were implanted into 40 right defects;in group B,the complex sponge was implanted into 20 left defects;and in group C,none was implanted into other 20 left defects.The gross and histological observations were made at 4 weeks postoperatively.Results The analysis results of phosphonic chitosan showed that the phosphorylation occurred mainly in the hydroxyl,and the proton type and chemical shifts intensity were conform to its chemical structure.The SEM results showed that the pores of the chitosan/phosphonic chitosan sponge were homogeneous,and the wall of the pore was thinner;the coating of calcium and phosphorus could be observed on the surface of the pore wall after mineralized with crystal particles;the cells grew well on the surface of the chitosan/phosphonic chitosan sponge.The MTT assay showed that the chitosan/phosphonic chitosan sponge could not inhibit the proliferation of hUCMSCs.The gross observation showed that the size and shape of the cell-scaffold composite remained intact and texture was toughened in group A,the size of the complex sponge gradually reduced in group B,and the muscle defects wound healed with a little scar tissue in group C.The histological observation showed that part of the scaffold was absorbed and new blood vessels and new bone trabeculae formed in group A,the circular cavity and residual chitosan scaffolds were observed in group B,and the wound almost healed with a small amount of lymphocytes in group C.Conclusion The chitosan/phosphonic chitosan sponge has good biocompatibility,the tissue engineered bone by combining the hUCMSCs with chitosan/phosphonic chitosan sponge has the potential of the ectopic bone formation in rabbit.