纳米P(LLA-b-CL)与犬关节软骨细胞体外生物相容性实验研究

Study on biocompatibility of the nano-PLLA-b-PCL with canine chondrocytes in vitro

[目的]研究具有纳米结构二嵌段共聚物左旋聚乳酸/聚已内酯(PLLA-b-PCL)与犬关节软骨细胞的体外生物相容性,探讨其作为软骨组织工程支架的可行性。[方法]开环聚合制备PLLA-b-PCL,液-液相分离制备PLLA-b-PCL纳米支架,扫描电镜观察材料结构:分离培养犬膝关节软骨细胞,取第3代软骨细胞接种于PLLA-b-PCL膜进行复合二维培养,MTT法检测细胞毒性;通过倒置显微镜、Hoechst33342荧光法观察细胞的形态与粘附状况;另取第3代软骨细胞与纳米PLLA-b-PCL(实验组)、PLLA-b-PCL(对照组)支架材料上进行三维培养3周,扫描电镜观察软骨细胞的形态、粘附、生长状况,Hoechst33258荧光法检测复合物中细胞DNA含量、BCA法测定蛋白质含量。[结果]PLLA-b-PCL无细胞毒性,软骨细胞在纳米PLLA-b-PCL支架上粘附、增殖良好,随时间延长软骨细胞在支架材料上DNA和蛋白质含量逐渐增加,DNA和蛋白质含量均明显高于对照组(P<0.05)。[结论]PLLA-b-PCL纳米支架能为软骨细胞的生长分化提供较好的环境,具有良好的生物相容性,有望成为一种较好的软骨组织工程支架材料。

[ Objective ] To evaluate the cellular biocompatibility of the nano poly （L -lactic acid） -block - poly （ε - eaprolactone） （Nano -PLLA -b -PCL） with canine articular cells and its feasibility as a scaffold for the cartilage tissue engineering. [ Methods ] Nano - PLLA - b - PCL was made by liquid - liquid phase separation. Canine articular cells were isolated and multiplied in vitro. The passage 3 cells were seeded onto the PLLA - b - PCL films and cultured in the 2 - dimensional environment. The cytotoxicity was measured with MTT assay. Cellular Morphological changes were observed by phase - contrast microscopy and Hoechst33342 fluorometric method. Another passage 3 cells were seeded onto the Nano - PLLA - b - PCL scaffolds （ experiment group）, PLLA - b - PCL scaffolds （ control group） and cultured in the 3 - dimensional environment for 3 weeks. The ratio of cell adhesion was detected by cell counting method. The morphological changes of cells were observed by scanning electron microscopy. The protein content in seeded cells were determined by bioinchoninie acid assay .（ BCA ） . The content of DNA was quantified using Hoechst33258 assay. [ Results ] MTT assay showed the PLLA - b - PCL had no cytotoxicity. The seeded cells adhered and proliferated well into the Nano -PLLA -b -PCL scaffolds, and they maintained good cell phenotype. After 21 -day cell culture within the Nano -PLLA -b -PCL scaffolds, the chondrocyte DNA and protein contents increased with time. Moreover, the content of DNA and protein was higher in the experiment group than that in the control group, respectively （ P 〈 0. 05 ） . [ Conclusion] The Nano - PLLA - b - PCL scaffold has a good cellular compatibility. It can provide a suitable environment for chondrocytes, and will be an ideal scaffold for the cartilage tissue engineering.