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Biocompatibility Study of Silk Fibroin/gelatin Composite Scaffolds for Liver Tissue Engineering

Author: YangZhao
Tutor: ZhouXinMin
School: Fourth Military Medical University
Course: Internal Medicine
Keywords: MTT assay Silk protein Gelatin Liver tissue engineering Cytocompatibility Histocompatibility
CLC: R318.08
Type: Master's thesis
Year: 2011
Downloads: 55
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Abstract

[Background] the ideal liver tissue engineering can be used as an effective bioartificial liver alternative support systems for acute liver failure and end-stage liver disease patients provide an effective alternative treatment for the patient's own liver regeneration and waiting for a liver source for their time. Liver tissue engineering in recent years in the development of tissue engineering background gained tremendous results, the carrier material and seed cells for liver tissue engineering, both foundations, both compatibility is a key link in building the ideal liver tissue engineering . The silk protein is a better biocompatible material with high strength and toughness, is widely used in bone tissue engineering and skin tissue engineering. Gelatin from natural collagen, collagen is the major structural protein in cartilage tissue, cartilage tissue has its unique mechanical properties. Collagen favor cell adhesion, proliferation and differentiation, biodegradable and provide sufficient space for the new tissue. In recent years, researchers try silk protein and collagen composite scaffold used in liver tissue engineering, in order to give full play to the characteristics of the two. In this paper, different proportions silk protein / gelatin composite vitro line establishment liver cell compatibility histocompatibility in rats and degradation, choose a better carrier for liver tissue engineering material lay the foundation. [Objective] To study silk protein / gelatin three-dimensional scaffolds immortalized human liver cell lines QZG cell adhesion, proliferation and apoptosis of silk protein / gelatin 3D scaffolds in SD rats induced immune rejection, and with the the degradation with time. [Methods] (1) using the tetrazolium salt colorimetric (MTT) detection QZG cell proliferation in four proportion of silk protein / gelatin composite film material. (2) cell count assay QZG of cell proliferation in four proportion of silk protein / gelatin composite membrane materials. (3) flow cytometry QZG cell apoptosis in four proportion of silk protein / gelatin composite film material. (4) use scanning electron microscope QZG cell attachment and proliferation on four proportion of silk protein / gelatin three-dimensional biological material. (5) four different proportions total of 180 silk protein / gelatin 3D scaffolds were implanted into three time groups of 60 male SD rats (200-250g) subcutaneously in each group each time the proportion of scaffolds the 5 rat dorsal subcutaneous implantation, each rat evenly implanted three the same proportion of material, 7 days respectively after implantation, 14 days, were sacrificed on day 30 group proportion silk protein / gelatin three-dimensional material stents implanted in rats, to remove subcutaneous fibers wrapped materials, formalin-fixed and evacuation HE sections staining observed inflammatory exudate and material degradation. 【Results】 (1) QZG cell in silk protein biological materials has proliferative activity, MTT and cell counting experiments With the increase in the proportion of gelatin ingredient in composite materials, composite materials, cell proliferation activity was significantly enhanced and significant , wherein proliferation more significant proportion of the material for the silk protein (wt%): gelatin (wt%) = 4:4. (2) Flow cytometry showed that first day other than pure silk protein QZG apoptosis in various scales silk protein / gelatin composite film with the control group no significant difference in cell activity; fifth day of a variety of the proportion of silk protein / the the gelatin composite membrane materials QZG cells apoptosis rate significantly different a QZG apoptosis rate increased with gelatin ratio reduced apoptosis less material proportion of the silk protein (wt%): gelatin (wt% ) = 4:4. (3) the sweep surface electron microscopy results show pure silk protein material on the general state of QZG cell adhesion, proliferation is not obvious; the other groups QZG cells scaffolds microporous attached close, culture third day cell proliferation is not obvious, a small amount of secretion extracellular matrix; seventh day of cell proliferation significantly, increased secretion of extracellular matrix, silk protein (wt%): gelatin (wt%) = 4:4 ratio of complex three-dimensional scaffolds cells seventh day of a large number of secretion of extracellular matrix, cell proliferation Obviously, the best condition attached. (4) various proportions of material on the 7th day HE sections were stained oozing fewer monocytes and neutrophils and other inflammatory cells; 14 days HE sections were stained monocytes and neutrophils and other inflammatory fibroblasts cells with a small amount of exudate material began to appear degradation, 30 days most of the groups material is organized machine, degradation of a large number of fibroblasts surrounding material fragments; three ratios, 30 days degradation slowest material pure silk protein, the degradation of the fastest material for the silk protein (wt.%): gelatin (wt%) = 4:4. [Conclusion] has a good performance of the cell-attached and good histocompatibility and some ability to degrade silk protein and gelatin composite scaffolds, silk protein / gelatin ratio, 4:4 ratio of in vitro cell proliferation and apoptosis, immune rejection in vivo experiments reflect the advantage of improvement in liver tissue engineering applications with a certain application prospects.

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