An optimal method to seed chondrocytes into PLGA scaffolds with tiny pores

Objective To explore an optimal method for seeding chondrocytes into a poly (lactic-co-glycolic acid) (PLGA) scaffold of 50-100 μm pore size.Methods The experiment was conducted in three groups (n =9):injection,low-pressure and orbital shaker ones.Fibrin gel containing chondrocytes was seeded into PLGA scaffolds with a porosity of 92％ and pore size of 50-100 μm.Forty-eight hours later,the DNA content in each construct was analyzed by Hoechst33285 fluorometric detection.FITC (fluorescein isothiocyanate) stained fibrin was also used to check distribution of the fibrin gel (without chondrocytes) in PLGA scaffolds.The distribution of chondrocytes in the scaffolds was assessed by hard tissue slice and DAPI (4',6-diamidino-2-phenylindole) nucleus staining.Seven days later,SEM detection was used to observe the presence of chondrocytes in the outer periphery and the interior of the scaffolds.Some constructs were implanted subcutaneously into nude mice (n =3).PLGA scaffolds with no chondrocytes were set as blank control group.After 8 weeks,the slices were examined by toluidine blue and collagen type Ⅱ immunohistochemistry staining.Finally,the Image-Pro Plus 6.0 software was used to analyze the integrated optical density (IOD) of the images.Results The DNA content was 755.79 ± 80.50 ng/mg in the injection group,657.32 ± 89.68 ng/mg in the low-pressure group and 650.18 ± 106.33 ng/mg in the orbital shaker group,with no significant difference between the 3 groups (F =1.214,P =0.361).The fibrin gel was uniformly distributed in the PLGA scaffolds in all the 3 groups.DAPI staining indicated that the distribution of cells in the injection group was more uniform than in the other 2 groups.SEM detection demonstrated that more cells distributed in the outer periphery in the low-pressure and orbital shaker groups than in the injection group,and only the inside pores in the injection group had visible cell adhesion.The IOD values of toluidine blue and collagen type Ⅱ immunohistochemistry staining displayed that the injection group was better than the other 2groups (P ＜ 0.05).Conclusion For PLGA scaffolds with a pore size of 50-100 μm,injection is a quick and efficient method for cell seeding. Key words: Polymers;  Cytoskeleton;  Chondrocytes;  Seeding method