Biocompatibility Study Of Silk Fibroin/gelatin Composite Scaffolds For Liver Tissue Engineering

【Background】Suitable liver tissue engineering methods can be effective instrument for supporting Acute liver failure (ALF) and final stage liver diseases such as bioartificial liver which provides r?eplacement therapy. It may provides a"bridge"for patients who have severe liver disease to recovery or transplantation.Recently there has been a particular focus on tissue engineering especially targeting the liver. Biomaterial scaffolds and seed cells are two basic aspects for liver tissue engineering, and their biocompatibility should be paid close attention to. Silk fibroin, with its high histocompatibility and mechanical characteristics, has been wildly used in the engineering of series of tissues, including bone and skin. Gelatin is another favourable biomaterial derived from nature collagen which is a major part in cartilaginous tissue. As an extracellular matrix, gelatin has been shown to promotes cell adhering and proliferation, and to accelerate cell differentiation. In recent years researchers manage to manufacture silk fibroin/gelatin composite scaffolds for liver tissue engineering so as to fully utilize their good qualities. Our research mainly focused on the cytotoxicity of silk fibroin/gelatin(SF/G) composite scaffolds in vitro and their biocompatibility and degradation in vivo. Thus lay a foundation for choosing a better liver tissue engineering biomaterial scaffolds.【Objectives】To investigate normal human hepatic QZG cells adhering,proliferation and apoptosis on silk fibroin/gelatin(SF/G) composite scaffolds in vitro, to detect their biocompatibility and degradation in vivo of Sprague-Dawley (SD) rats.【Methods】(1)The proliferation and relative growth rate (RGR) of normal human hepatic QZG cells grown on four different blends of two-dimensional (2-D) SF/G scaffolds were assessed using the 3-[4, 5-dimethylthiazol -2-yl]-2,5-diphenyltetrasodium bromide (MTT ) colorimetric assay. (2) Normal human hepatic QZG cells proliferation on four different blends of two-dimensional (2-D) SF/G scaffolds was assessed cell counting. (3) Flow cytometry was used to evaluate apoptotic rate of QZG cells on four different blends of 2-D SF/G scaffolds. (4) The effect of three-dimensional (3-D) SF/G scaffolds on the growth of cells was observed by scanning electron microscopy (SEM). (5) Three-dimensional (3-D) SF/G scaffolds of four different ratios (diameter: 10 mm×thickness: 1 mm) were implanted into subcutaneous pockets on male Sprague-Dawley (SD) rats (200-250 g). On days 7, 14 and 30 post-implantation, rats were sacrificed. Scaffold area, including the surrounding tissues, was retrieved. Hematoxylin and Eosin (HE) staining was performed for observation under a light microscope.【Results】(1) We observed significant cell attachment and proliferation on SF/G scaffolds. As the gelatin concentration increased, SF/G scaffolds became more amenable to cell adhesion.MTT and cell counting results showed cell significant growth on SF/G scaffolds which of a ratio:Silk fibroin(wt%):Gelatin(wt%) =4:4. (2) The results of analysis of QZG cell apoptosis by flow cytometry showed on the first day after seeding, except pure silk material, the apoptotic rate of QZG cells cultured on three other different blends SF/G films was similar, which was not significantly different from controls. These results suggestted that except pure silk material cell viability after culture on all other ratios of SF/G films was identical. After 5 days of culture, the apoptotic rates among all five groups (including the control group) presented significant differences. There were relatively less apoptotic rates on SF/G scaffolds which of a ratio:Silk fibroin(wt%):Gelatin(wt%) =4:4. The decrease in the apoptotic rate may due to the increase of the gelatin content, which is better for cell attachment. (3) SEM results showed the spherical shape of the QZG cells and the abundant extracellular secretions. The adhering condition and proliferation of QZG cells on pure silk material 3D scaffolds were worse than other blends of scaffolds. Feelers from the QZG cells attached to the surface of the SF/G scaffold. QZG cells that were cultured for 3 days adhered to the surface of the scaffolds and had low rates of proliferation; on day 7, the cells associated with each scaffold showed excellent growth, including extracellular secretions and intercellular connections. The results also showed a better cell condition on scaffolds with higher gelatin ratios especially on SF/G scaffolds which of a ratio:Silk fibroin(wt%):Gelatin(wt%) =4:4. (4) After subcutaneous implantation of SF/G scaffolds into SD rats, immunological rejection tests showed only slight inflammation, measured by the presence of inflammatory cells on days 7 and 14. By day 30, each scaffold had been completely infiltrated and organized by fibroblasts and inflammatory cells. The greater the gelatin concentration in the scaffold, the faster the rate of degradation. SF/G scaffolds of a ratio:Silk fibroin(wt%):Gelatin(wt%) =4:4 had quickest degradation in vivo within 30 days.【Conclusion】These results suggest that composite SF/G scaffolds are a promising candidate matrix for implantable bioartificial livers. SF/G scaffolds of a ratio:Silk fibroin(wt%):Gelatin(wt%) =4:4 showed significantly superior to other ratios,which gave us new perspective for applying in liver tissue engineering.