Recombinant Spidroin Films Attenuate Individual Markers of Glucose Induced Aging in NIH 3T3 Fibroblasts

The effect of bioresorbable materials on aging in cultured mouse NIH 3T3 fibroblasts treated with elevated glucose concentration was investigated. The cells were grown on films produced from the silkworm fibroin and rS1/9, a recombinant analog of Nephila clavipes spidroin 1. Exposure to 50 mM glucose of the cells grown on uncoated glass support resulted in the cell growth retardation. The average areas of the cells and nuclei and the percentage of apoptotic cells increased, whereas the amount of soluble collagen decreased. In contrast, on the fibroin and spidroin films, the cell density and the percentage of 5-bromo-2'-deoxyuridine (BrdU)-positive cells were higher vs. the cells grown on the glass support. The films protected NIH 3T3 fibroblasts from the glucose-induced death. The most prominent effects on the cell density, BrdU incorporation, and apoptosis prevention were observed in the cells cultured on spidroin films. Unlike the cells grown on glass support (decrease in the soluble collagen production) or fibroin (no effect), production of soluble collagen by the cells grown on spidroin films increased after cell exposure to 50 mM glucose. Molecular analysis demonstrated that 50 mM glucose upregulated phosphorylation of the NFκB heterodimer p65 subunit in the cells grown on the glass support. The treatment of cells grown on fibroin films with 5.5 mM or 50 mM glucose had no effect on p65 phosphorylation. The same treatment decreased p65 phosphorylation in the cells on the spidroin films. These results demonstrate the anti-aging efficacy of biomaterials derived from the silk proteins and suggest that spidroin is more advantageous for tissue engineering and therapy than fibroin.