Silk Fibroin and Gelatin Immobilization on Poly(Caprolactone) Nonwoven Mats via Carbodiimide Coupling and Electrostatic Layer-By-Layer Deposition: Characterization and Cell Growth

Abstract

Surface modification of scaffolds is a topic of great interest in tissue engineering due to the crucial role of scaffold surface in interactions with cells. In this study, we characterize electrospun poly(caprolactone) (PCL) nonwoven mats coated with silk fibroin (SF) protein, gelatin, and combined SF-gelatin coating in the context of material properties and response of human osteoblast-like (MG-63) cells. We compare two methods of protein attachment to aminolyzed PCL fibers: chemical one, namely carbodiimide coupling, and electrostatic layer-by-layer deposition. Five different types of coatings were studied, including separate coatings of SF and gelatin, as well as combined coatings. To enable both types of modification, additional –COOH and –NH₂ groups were introduced into the SF by chemical modification. The protein immobilization processes did not change the morphology and diameter of the PCL fibers. As a result of all modifications, hydrophobic PCL scaffolds became wettable. Colorimetric bicinchoninic acid (BCA) assay on PCL fibers detected the immobilized proteins and revealed that carbodiimide coupling and layer-by-layer deposition of SF coatings resulted in a similar level of absorbance, indicating no difference in protein content. However, the chemically bound SF coating exhibited greater stability in phosphate-buffered saline (PBS) solution. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy analysis confirmed the immobilization of SF, gelatin, and SF-gelatin coatings on PCL fibers by the presence of amide I, amide II, and amide A bands. Analysis of the morphology and metabolic activity of osteoblast-like MG-63 cells on the modified scaffolds indicated that the SF coatings without the addition of gelatin provided the best outcomes in terms of cell adhesion and growth. These results indicate the predominance of SF properties over the effect of the bioactive RGD-sequence of gelatin.