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 (RCA(120)) and fluorescein isothiocyanate-labeled RCA(120) (FITC-RCA(120)). 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 RCA(120) 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-RCA(120) 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 nM insulin and 100 nM 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.