[Preparation of three-dimensional porous scaffold of PLGA-silk fibroin-collagen nanofiber and its cytocompatibility study].
OBJECTIVE
To develop three-dimensional (3D) porous nanofiber scaffold of PLGA-silk fibroin-collagen and to investigate its cytocompatibility in vitro.


METHODS
Method of electrostatic spinning was used to prepare 3D porous nanofiber scaffold of PLGA-silk fibroin-collagen (the experimental group) and 3D porous nanofiber scaffold of PLGA (the control group). The scaffold in each group was observed by scanning electron microscope (SEM). The parameters of scaffold fiber diameter, porosity, water absorption rate, and tensile strength were detected. SC harvested from the bilateral brachial plexus and sciatic nerve of 8 SD suckling rats of inbred strains were cultured. SC purity was detected by S-100 immunohistochemistry staining. The SCs at passage 4 (5 x 10(4) cells/mL) were treated with the scaffold extract of each group at a concentration of 25%, 50%, and 100%, respectively; the cells treated with DMEM served as blank control group. MTT method was used to detect absorbance (A) value 1, 3, 5, and 7 days after culture. The SC at passage 4 were seeded on the scaffold of the experimental and the control group, respectively. SEM observation was conducted 2, 4, and 6 days after co-culture, and laser scanning confocal microscope (LSCM) observation was performed 4 days after co-culture for the growth condition of SC on the scaffold.


RESULTS
SEM observation: the scaffold in two groups had interconnected porous network structure; the fiber diameter in the experimental and the control group was (141 +/- 9) nm and (205 +/- 11) nm, respectively; the pores in the scaffold were interconnected; the porosity was 87.4% +/- 1.1% and 85.3% +/- 1.3%, respectively; the water absorption rate was 2 647% +/- 172% and 2 593% +/- 161%, respectively; the tensile strength was (0.32 +/- 0.03) MPa and (0.28 +/- 0.04) MPa, respectively. S-100 immunohistochemistry staining showed that the SC purity was 96.5% +/- 1.3%. MTT detection: SC grew well in the different concentration groups and the control group, the absorbance (A) value increased over time, significant differences were noted among different time points in the same group (P < 0.05), and there was no significant difference between the different concentration groups and the blank control group at different time points (P > 0.05). SEM observation: in the experimental group, SC grew well on the scaffold, axon connection occurred 4 days after co-culture, the cells proliferated massively and secreted matrix 6 days after co-culture, and the growth condition of the cells was better than the control group. The condition observed by LSCM 4 days after co-culture was the same as that of SEM.


CONCLUSIONS
The 3D porous nanofiber scaffold PLGA-silk fibroin-collagen prepared by the method of electrostatic spinning is safe, free of toxicity, and suitable for SC growth, and has good cytocompatibility and proper aperture and porosity. It is a potential scaffold carrier for tissue engineered nerve.
Wu
Gang
G
Department of Orthopaedics, West China Hospital, Sichuan University, Chengdu Sichuan 610041, PR China.
Dong
Changchao
C
Wang
Guanglin
G
Gao
Weiqiang
W
Fan
Hongsong
H
Xiao
Wenqian
W
Zhang
Li
L
D000818:Animals; D001672:Biocompatible Materials / Q000032:analysis*; D001917:Brachial Plexus / Q000166:cytology; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D003094:Collagen; D005260:Female; D005349:Fibroins / Q000138:chemical synthesis*; D019344:Lactic Acid / Q000138:chemical synthesis*; D008297:Male; D008422:Materials Testing; D057139:Nanofibers; D011100:Polyglycolic Acid / Q000138:chemical synthesis*; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D051381:Rats; D017207:Rats, Sprague-Dawley; D012584:Sciatic Nerve / Q000166:cytology; D047011:Silk; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds
D001672:Biocompatible Materials; D047011:Silk; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D003094:Collagen; D005349:Fibroins