Preparation of lactose–silk fibroin conjugates and their application as a scaffold for hepatocyte attachment

We prepared glycoconjugates (Lac–CY–SF) by the homogeneous chemical modification of solubilized silk fibroin (SF) with lactose bearing the galactose residue using cyanuric chloride (CY) as a coupling spacer and examined the usefulness of their application as a scaffold for hepatocyte attachment. The covalent immobilization of lactose into SF was assessed by several criteria including 1H-NMR measurements and reactions with lectins such as Ricinus communis agglutinin (RCA120) and fluorescein isothiocyanate-labeled RCA120 (FITC-RCA120). The 1H-NMR spectrum of Lac–CY–SF showed new broad peaks attributed to methine and methylene protons of lactose, and by the integrated intensities of the peaks the weight ratio of lactose to SF in Lac–CY–SF was determined to be 0.20. Addition of RCA120 to Lac–CY–SF solution caused an increase in the turbidity of the Lac–CY–SF solution. Incubation of Lac–CY–SF films with FITC-RCA120 showed that the fluorescence emitted from the whole surface of Lac–CY–SF films. Furthermore, we examined the effect of the Lac–CY–SF conjugate-coating onto polystyrene culture dishes on the attachment of rat hepatocytes and their morphology. Attachment of hepatocytes onto the 0.1% (w/v) conjugate-coated dishes showed about an 8-fold increase as compared with that on uncoated dishes, being comparable to that on collagen-coated dishes, whereas the attachment on the SF-coated dishes was lower than that on uncoated dishes. Hepatocytes cultured onto the conjugate-coated dishes for 2.5 h showed smaller round-shaped morphology compared to those on collagen-coated dishes. After 2 days of culture in medium containing 100 n insulin and 100 n dexamethasone, hepatocytes on the conjugate-coated dishes formed monolayer islands with a slightly dispersed morphology, while hepatocytes cultured on collagen-coated dishes were uniformly spread flat. These results indicate that the Lac–CY–SF conjugates are useful as a scaffold for hepatocyte attachment, but the morphology of hepatocytes cultured on the conjugate-coated dishes is different from that on collagen-coated dishes.