The effect of recombinant Fibronectin on PC12 cell adhesion to PLGA nanofiber scaffold

Abstract

조직공학은 생명공학과 생체 적응 재료에 의해 발전 되어지고 있는 최신 분야이다. 조직 공학의 필수 3대 요소는 세포, scaffold, signaling molecule 이다. 특히 생분해성 다공성 scaffold는 세포 부착성 및 조직재생 능력이 우수하다. 본 연구에서는 나노섬유로 만들어진 조직공학적 PLGA scaffold를 제작하였고, 만들어진 PLGA 나노섬유 scaffold에 다양한 농도의 recombinant fibronectin을 도포하여 세포부착 및 생활성에 미치는 영향에 대해 알아보고자 하였다. PLGA를 전기회전방사법으로 나노섬유형태의 조직공학적 scaffold를 제작하였다. 제작한 PLGA scaffold를 SEM을 이용하여 형태학적인 구조와 pore size를 확인 하였다. 그리고 recombinant fibronectin을 각각 5?M, 10?M, 20?M로 희석하여 PLGA scaffold에 도포하였다. 각 그룹을 Group I, Group II, Group III로 나누었으며, 대조군에는 recombinant fibronectin을 도포하지 않았다. PC12세포를 scaffold에 이식하고 48시간 동안 배양하였다. SEM과 MTT assay을 시행하였으며 결과는 One-way ANOVA를 이용하여 통계 처리하였다(p<0.05). 1. 전기방사선법으로 제작한 PLGA 나노섬유 scaffold는 6-8μm의 여러겹의 망상구조를 보였다. 2. SEM 관찰에서는 PC 12세포들이 납작한 다각형의 형태로 분화하는 것을 관찰할 수 있었으며, 농도가 증가됨에 따라 이 현상이 두드러지는 것을 볼 수 있었다. 3. Recombinent fibronectin의 농도가 증가 됨에 따라 PC 12 세포부착도가 증가되었으며, Group III 에서는 대조군과 Group I을 비교하여 볼 때 통계적으로 유의성을 보였다(p< 0.05). 그러므로, 20μM recombinent fibronectin 농도를 최소유효농도로 사용할 수 있으며, 향후 이를 이용하여 In vivo 연구가 필요하리라 사료된다.;Current progress in biotechnology of morphogenetic factors and biomaterials has converged into the evolving field of tissue engineering. It is well known that three key components of tissue engineering are cell, scaffold, and molecule. Especially bioresorbable synthetic scaffolds are predominant in the ability of cellular attachment and tissue regeneration. It is necessary to study PC12 cell adhesion on recombinant fibronectin (FNIII 9-10) coated onto Poly (lactide-co-glycolide) (PLGA) nanofiber scaffold. The purpose of this study is to examine the Scanning Electronic Microscope (SEM) view of electrospun PLGA nanofiber scaffold and to evaluate the biological effect of different concentration of recombinant fibronectin in the aspects of SEM examination of PC 12 cell and cell viability test by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)assay. PLGA nanofiber scaffold was fabricated by electrospinning method. These PLGA nanofiber scaffolds were evaluated morphological structure and pore size by the SEM examination. And then, recombinant fibronectin were diluted respectively 5μM, 10μM, 20μM and coated onto the PLGA nanofiber scaffolds. Each group was assigned group I, group II and group III respectively. In control group, the PLGA scaffolds were not coated with recombinant fibronectin. PC 12 cell suspension was seeded onto PLGA nanofiber scaffold, incubated for 48 hours, and examined by SEM and the MTT assay. The results obtained from the samples were analyzed by one-way ANOVA (p <0.05). 1. The electrospun PLGA nanofiber scaffold was an inner and outer inter-communicated multi-layered net with many pores. The pore size was regular between 6-8 μm. 2. The SEM view of cell-PLGA scaffold complex showed that spindle shaped cells with many elongated cytoplasmic extension were seen more prominent according to the increase of the concentration of recombinant fibronectin. 3. PC 12 cells in Group III showed higher cellular adhesion to PLGA nanofiber scaffold compared to control group and Group I (p< 0.05). Conclusively, it can be recommended that 20 μM recombinant fibronectin is the minimal concentration for cellular adhesion to PLGA nanofiber scaffold.