Investigation Of KLD-12 Self-assembling Polypeptide TGF-β1 Nanofiber Scaffolds Effect The Proliferation, Phenotype And Function Of Rabbit Annulus Fibrosus Cell In Vitro

Objective In this study, we investigated the impact of KLD-12 polypeptide/TGF-β1scaffolds on the in vitro proliferation, phenotype, and function of rabbit annulus fibrosus cells(AFCs).Methods The rabbit annulus fibrosus cell were isolated with pancreatin and complexed with either KLD-12 polypeptide/TGF-β1(experimental group, TGF-β1) or KLD-12 polypeptide nanofiber gel(control group) scaffolds in 3D cell cultures and cell growth. The CCK-8 cell proliferation assay and Calcein-AM/PI fluorescent staining were used to show the situation of cells growth. The Alcianblue method,immunofluorescence technique, and Real-time quantitative PCR(RT-q PCR) were used to measure the expression of aggrecan, glycosaminoglycans(GAG), and collagen type II.Results The cells in the scaffolds were grew well, the circular shape was maintained on the scaffolds, and the spindle and fusiform shape of cells on the scaffolds edges The CCK-8 cell proliferation assay showed Cell proliferation increased non-linearly as measured on 2, 5, 8, 11 and 14 d in culture. The proliferation rates in the experimental group were higher than the control group from 5d to 14d(P < 0.05).Calcein-AM/PI fluorescent staining showed: extensively living cells maintaining round morphology, with small amounts of spindle and fusiform shaped cells on the gel edges. The cell survival rate of the experimental group was 97.44% ± 0.86% and94.04% ± 3.27% in control group at 14 d. There was no significant difference between the two groups(P>0.05). The production of ECM was showed the experimental group GAG content was significantly higher as compared to the control group’s GAG content(P<0.05) at 5 d and 8 d. At 11 d and 14 d, experimental GAG content was also statistically significant(P<0.01) Immunofluorescence staining of collagen type II confirmed the type II collage intensity levels were significantly greater in the experiment group than the control group(P<0.05).. Finally, Real-time PCR results revealed that the relative expression levels of collagen type II and aggrecan m RNA were higher in the experiment group than the control group in 14 days(P < 0.05).Conclusion scaffolds containing KLD-12 polypeptide/TGF-β1 nanofiber gel and AFCs produced more ECM and thus can potentially serve as vital 3D supports for the treatment of intervertebral disc degeneration.