生物材料之壓縮/彈性模數對於大鼠肝細胞行為與功能性之影響

Abstract

在臨床醫學上，肝癌與肝硬化一直在國人十大死因中名列前茅，對於重症病患而言，肝器官移植仍是臨床上最有效的方法，然而，器官捐贈的不足仍是主要障礙。同時等待器官捐贈的時間是漫長的，加上當肝臟衰竭時，體內的毒素會不斷的累積於體內，並且影響其他器官的功能；或是接受肝臟移植後，若併發感染或是出現排斥反應時，則必須接受洗肝替代性治療來恆定病人的生命。目前肝替代性治療的方式是使用洗肝機來移除毒素，但洗肝機僅能短暫的維持病患的生命，其因是受損的肝臟其肝功能無法維持一定程度，所以開發具可應用於病患並且有機能性的人工肝臟是目前醫學工程領域正在發展的 重要議題之一。 具有機能性的人工肝臟(Functional BAL)的設計概念，其方式是保留分子吸附再循環系統的基礎設計概念，將血漿分離出來後，並且將毒素清除之後的血漿接引導入含有肝實質細胞(Primary hepatocyte)貼附於支架(Scaffold)的肝細胞生物反應器(Hepatocyte bioreactor)，經由自體灌流培養後，反應器內部的肝實質細胞所合成出來的產物會回流入病患體內，進而提升患者的生存機會。然而生物反應器內部的支架會隨著環境與酵素的影響，造成降解的情形，使其結構一直無法長時間維持並且影響肝實質細胞的貼附與生長。為此，本研究透過不同固含量(g/g)的戊二醛與幾丁質聚醣/明膠溶液(C/G Solution)進行交聯反應，並經由冷凍乾燥程序製做出不同壓縮模數的肝臟支架，並將肝實質細胞培養於處理過後的肝臟支架，用來觀察肝實質細胞與壓縮模數之間的關聯性。 同時能夠用於移植的人工肝臟，內部的肝細胞數量至少也是人體的35%，因此，如何填塞等量的肝細胞到肝臟支架是ㄧ大挑戰，勢必考驗肝臟支架的空間整體尺寸，肝臟支架的厚度與幾何尺寸也是扮演養分/代謝物傳輸重要的因素，所以本篇論文也會探討厚度改變與其細胞活性的影響。

Functional artificial livers (FALs), with embedded hepatocytes that perform the functions of a normal liver, have been developed for liver diseases for decades. It is important to note that the liver scaffold, which is a biologically functional core of the artificial livers, plays a vital role in bio-cartridge within artificial liver. In this study, a three-dimensional (3D) liver scaffold for in vitro cultures was fabricated by freezing-drying chitosan/gelatin (C/G) solution. CG liver scaffold has some advantages such as (1): Inexpensive and easy-to-make; (2) Easy to fabricate CG liver scaffold with varying compressive tangent modulus by changing the concentration of glutaraldehyde; (3) Non-cytotoxicity; (4) The porous structure of CG scaffold is similar to extracellular matrix (ECM) and therefore helpful for hepatocyte adhesion. In the result, we found that the compressive tangent modulus and maintability of CG liver scaffold correlated to the glutaraldehyde increase, these results were also consistent with the results determined by the dynamic mechanical analysis (DMA) analysis. Furthermore, hepatocyte viability and albumin synthesis shows the best performances in 0.61% glutaraldehyde-CG scaffold. This CG scaffold has not only higher hepatocyte biocompatibility and mechanical strength, but can also maintain hepatocyte function and viability in vitro cultures, we believe that CG scaffold as liver scaffold may have high potential for further artificial liver design in the near future.