扫描电镜观察组织工程神经支架与骨髓间充质干细胞的共同培养

Culture of bone marrow stromal cells on tissue engineered nerve scaffold evaluated by scanning electron microscope

目的:扫描电镜观察骨髓间充质干细胞在组织工程神经支架中生长、附着状态,探讨骨髓间充质干细胞与该支架细胞的亲合性。方法:实验于2006-02/2006-12在中国医科大学神经解剖研究室完成。实验材料:180～200g成年清洁级健康雄性wistar大鼠,用于制备无细胞神经移植物,100～120g成年清洁级健康雄性wistar大鼠,用于制备骨髓间充质干细胞,均由中国医科大学实验动物部提供(SCXK(辽)2003-0009)。实验方法:①无细胞神经移植物的制备:参考刘承吉等组织工程化天然神经支架制备方法制备无细胞神经移植物。②骨髓间充质干细胞的体外培养与鉴定:无菌条件下分离wistar大鼠股骨、胫骨,用低糖DMEM培养液反复冲洗骨髓腔,收集冲洗液,1000r/min4℃离心10min,去上清液,DMEM培养液重悬细胞,置于37℃、体积分数为0.05的CO2孵箱内培养,24h半量换液,48h全量换液,每二三天换液1次,5～7d后达到80%融合,0.25%胰酶消化,离心,按1∶2传代培养,留取3代备用。采用免疫组织化学染色法鉴定。③骨髓间充质干细胞植入并与无细胞神经支架联合培养:手术显微镜下将1×107L-1骨髓间充质干细胞多点注入无细胞神经支架内,见支架膨胀隆起后,将支架重新放入培养液内培养7d。④实验评估:将制备的无细胞神经移植物横断面进行扫描电镜观察,骨髓间充质干细胞接种于支架上混合培养7d后,对移植物支架进行扫描电镜观察。结果:①免疫组织化学法鉴定骨髓间充质干细胞:培养的细胞不表达CD34、CD14,表达CD44、CD29,说明实验中分离、培养的细胞不是造血干细胞而是骨髓间充质干细胞。②扫描电镜观察无细胞神经移植物与移植物支架:无细胞异体神经支架在扫描电镜下见正常神经的髓鞘、轴突均消失,仅剩下空虚呈网格状的施万细胞基底膜管。联合培养7d后骨髓间充质干细胞在基底膜管内均匀分布,在管腔内贴附生长,细胞呈椭圆形或多突起形。骨髓间充质干细胞在该支架内均匀分布,并伸出伪足贴附于该支架内。结论:骨髓间充质干细胞在组织工程神经支架内生长良好,形态正常,并伸出伪足与管壁粘连紧密,该支架对骨髓间充质干细胞具有良好的细胞亲合性。

AIM： To study the morphology of bone marrow stromal cells （BMSCs） cultured on tissue engineered nerve scaffold using scanning electron microscopy （SEM）, and investigate the affinity of BMSCs to nerve scaffold. METHODS： The experiment was conducted at the Neureanatomy Laboratory of China Medical University from February to December in 2006. Adult male Wistar rats of cleaning grade, weighing 180-200 g, were used to prepare acellular nerve graft, while those weighing 100-120 g were used for BMSCs. All the animals were offered by the Experimental Animal Department of China Medical University （No.SCXK2003-0009）.（1）Acellular nerve graft was prepared according to Liu et als method for tissue engineered nerve scaffold.Gin vitro culture and identification of BMSCs： The femur and tibia were separated from Wistar rats under sterile condition. Medullary canal was repeatedly rinsed with low-glucose DMEM solution, then the rinse solution was collected and centrifuged at 4 ℃ for 10 minutes, 1 000 r/min, followed by the removal of supematant. Cells were re-suspended in DMEM solution in the incubator containing 0.05 volume fraction of CO2 at 37 %. Culture medium was changed half 24 hours later and changed completely 48 hours later, then once 2-3 days. Cells reached confluence 5-7 days later, digested with 0.25% pancreatic enzyme, centrifuged, and cultured. BMSCs at passage 3 were identified using immunohistochemistry method. （3）BMSCs at the density of 1 ×10^7 L^-1 were implanted and cultured in acellular nerve graft at multiply sites, and then cultured in the medium for 7 days after the graft appeared to expand and swell up.（4）The cell morphology of two groups was investigated by SEM. RESULTS： （1）The immunohistochemical result showed that BMSCs were negative for CD34, CD14, but positive for CD44, CD29, which indicated that the isolated and cultured cell in this study was not hemopoietic stem cells but BMSCs. （2）SEM result showed that, normal myelin sheath and axon of the nerve were absent in acellular nerve graft, remaining only the vessel of basilar membrane of Schwann cells, which was vacant and mesh-like. After 7-day co-culture, BMSCs distributed evenly in the vessel of basilar membrane, grew in adherent way in the lumina, and characterized in ellipse or multi-prominent shape. Additionally pseudopodium was found to be adhesive to the scaffold. CONCLUSION： BMSCs grow well and shape normally on tissue engineered nerve scaffold. The close adhesion of pseudopodium with vessel wall indicates the good affinity of this scaffold to BMSCs.