小肠黏膜下层作为肌腱组织工程支架的评价

Evaluation of the use of small intestinal submucosa as a scaffold in tendon tissue engineering

目的评价脱细胞小肠黏膜下层(SIS)的细胞毒性、亲水性、细胞黏附性等组织工程支架性能,为其作为肌腱组织工程支架材料提供实验依据。方法制备猪小肠黏膜下层,并进行光镜和扫描电镜观察。以不同浓度(100%、50%、25%、12.5%、0)的SIS浸提液培养兔肌腱细胞,采用MTT法测定1、3、5、7、9d时肌腱细胞的增殖率以评价SIS的细胞毒性。测定SIS的吸水率以评价其亲水性,并测定SIS与兔肌腱细胞黏附率以评价其细胞黏附性。结果光镜观察显示,SIS结构疏松,无细胞碎屑残留;扫描电镜见两个表面的纤维直径不同。MTT法检测显示,各浓度SIS浸提液的细胞毒性均为0～1级。冻干SIS吸水率在6h达饱和,饱和吸水率498.2%±15.0%;兔肌腱细胞在SIS光面和糙面的黏附率分别为53.49%±5.46%和61.45%±7.84%,两者比较差异无统计学意义(P>0.05)。肌腱细胞在SIS上生长良好,部分细胞长入SIS内。结论SIS对肌腱细胞无毒性,细胞黏附性良好,适合作为组织工程肌腱的支架材料。

Objective To fabricate the cell-free porcine small intestinal submucosa （SIS）,and study the quality of SIS （cytotoxicity,hydrophilicity,cell adhesion,and so on） for providing a scientific basis for the use of SIS as a tendon tissue engineering scaffold.MethodsThe porcine SIS was prepared,and then observed with light microscope and scanning electron microscope.The tenocytes of rabbit were cultured in extract of SIS in different concentrations （100%,50%,25%,12.5%,0%） in vitro,and the proliferation rates were calculated with MTT assay at the time points of 1st,3rd,5th,7th and 9th day to evaluate the cytotoxicity of SIS extract.The water absorbency of SIS was measured to evaluate its hydrophilicity,and the adhesion rate of SIS to tenocytes of rabbit was measured to evaluate its cell adhesion.ResultsThe light microscopic observation revealed a loose structure with no cell debris in SIS,and scanning electron microscope images showed that the fiber diameter of the two surfaces was different （the fibre diameters on the mucasal surface were slender than that on the serosal surface）.The MTT assay displayed that the cytotoxicity of different concentrations of SIS extraction ranged from 0 to 1 grade.The lyophilized SIS reached its saturate absorption rate （498.2%±15.0%） after dipped in the PBS for 6 hours;the adhesion rate of rabbits＇ tenocytes on the smooth and rough surfaces was 53.49%±5.46% and 61.45%±7.84%,respectively （P0.05）.After co-cultured with SIS for 7 days,the tenocytes grew well on the surfaces of SIS,and a great deal of them grew deeply into the SIS.ConclusionSIS has no cytotoxicity and shows a well adhesion ability to tenocytes,thus it seems to be suitable for use as a scaffold in tendon tissue engineering.