Silk fibroin-hydroxyapatite scaffolds promote the proliferation of adipose-derived mesenchymal stem cells by activating the ERK signal

Abstract

Adipose-derived mesenchymal stem cell (Ad-MSC) with capacities of releasing trophic factors and chondrogenic differentiation was a promising candidate for tracheal reconstruction. Silk fibroin (SF)- hydroxyapatite (HA) scaffolds were fabricated by the freeze-drying method. And Ad-MSCs were co-cultured on the scaffolds for 14 days in vitro. The role of the SF-HA scaffold in regulating the adhesion, growth, and proliferation of Ad-MSCs, and its potential mechanisms were investigated. The identity of Ad-MSCs was confirmed by cell morphology, surface markers, and differentiation characteristics. Cell proliferation, viability, and morphology were observed via CCK-8, live/dead assay, and scanning electron microscopy (SEM). Gene mRNA and protein levels were examined using quantitative real-time polymerase chain reaction and western blotting, respectively. SF-HA scaffolds showed excellent properties of promoting Ad-MSCs adhesion, growth, and proliferation for at least 14 days. In the CCK-8 assay, the relative OD value of Ad-MSCs cultured on SF-HA scaffolds increased (p < 0.001). Furthermore, live/dead staining showed that the fluorescent coverage increased with time (p < 0.05). SEM also showed that 3 days after inoculation, the coverage of Ad-MSCs on the SF-HA scaffolds was 78.15%, increased to 92.91% on day 7, and reached a peak of 94.38% on day 14. Extracellular signal-regulated kinase (ERK) mRNA and phosphorylated ERK (pERK) protein expression increased at day 3 (p < 0.05), followed by a significant decline at day 7 (p < 0.05). And ERK mRNA expression was positively correlated with Ad-MSCs proliferation (p < 0.05). In summary, the SF-HA scaffold co-cultured with Ad-MSCs is a promising biomaterial for tracheal repair by activating the ERK signal pathway.