内部结构可控的大体积三维细胞支架制备研究

Study on Inner-Structure Controllable Large Volume 3-D Scaffolds Formation Method

目的：研究一种可以控制三维细胞支架内部孔隙结构的实验技术，用于制备孔隙结构可控的三维细胞支架，以满足组织工程对支架孔隙结构的要求。方法：均匀混合粘结剂与致孔剂，在离心力作用下去除混合物中多余的粘结剂，应用溶剂浇注／颗粒沥析方法制备三维细胞支架。结果：致孔剂粘结块的结构非常均匀，粘结程度可以通过实验条件控制。例如，直径为100-220μm的致孔剂，在离心力为161g，粘结剂浓度分别为20％和40％时，颗粒间粘结程度分别为33．78±5．56（134）μm和42．89±5．87（132）μm。并且，利用该技术制备的三维多孔支架，其内部孔隙大小取决于致孔剂颗粒大小，孔隙间的通道直径取决于致孔剂的粘结程度，即离心粘结与溶剂浇注／颗粒沥析技术相结合，能够方便地控制三维支架的孔隙结构。例如，当粘结程度为33．78±5．56（134）μm时，支架的通道直径为33．34±5．21（12）μm，两者之间无显著差异。结论：利用离心粘结与溶剂浇彰颗粒沥析技术结合，获得了孔隙呈球形、孔隙间完全连通的、结构均匀的大体积三维细胞支架，并且支架的孔隙以及孔隙间通道大小均可以实现人为控制。

Objective： To develop a novel method for preparating large volume three-dimensional scaffolds, and controlling the pore structure, uniformity, and interconnectivity of the scaffolds to meet the requirement of pore structure for tissue engineering. Methods： Spherical porogen （a pore generating materials made from sodium chlorate） and bonding reagents （developed by our laboratory） were mixed uniformly, the mixture was put in a polypropylene mold （cylindrical vial with microholes on bottom for solution leaving off）, the mold containing mixture was centrifuged at a chosen force for 5min to get ride of unwanted solution, then the bonded porogen assembly was cut into halves with a razor blade after completely dried in dessicator at room temperature. The upper, middle and bottom sections of assemblies were observed by optical microscope to detect the bonding uniformity and degree. A chosen polymer （PDLLA） was dissolved in chloroform to prepare a solution of a desired concentration, the polymer solution was cast onto the assembly, and additional casting was repeated after the solvent was evaporated. The dried porogen/polymer discs were removed from the mold, and the top and bottom layers were cut away to obtain flat surfaces. The discs was immersed in distilled water to remove porogen, dried under vacuum, then the scaffolds was harvested for structure characterization. Results： Optical micrographs clearly displayed that the porogen spheres remained spherical appearance and the bonding areas between spherical particles were homogeneous in large dimensional bonded assembly, and there was no statistical difference in bonding extent. In addition, the bonding extent could be controlled by variety of bonding reagent concentration as well as centrifugal force. For instance, the bonding extent was 33.78 ± 5.56 （134） μm and 42.89 ± 5.87 （132）μm respectively, when reagent concentration was 20% and 40% with centrifugal force of 161g and porogen size range of 100 - 220μm. SEM imagines revealed that the pore size and the diameter of interconnected openings of the scaffolds equaled separately to porogen size and bonding extent in bonded assembly. For example, the diameter of openings was 33.34 ±5.21 （12）μm, when the bonding degree was 33.78 ± 5.56 （134）μm in bonded assembly. Conclusion： With the newly developed bonding reagent and bonding technique, large dimensional biodegradable polymer scaffolds with high porosity as well as with controllable and homogeneous inner-structure can be formed, the pore size of scaffolds as well as diameter of openings between pores can be controlled by adjusting the porogen size and bonding degree in bonded porogen assembly. In addition, the resulting completely interconnected scaffolds have implications for facilitating cell migration, nutrients or waste exchange , abundant cell-cell interaction, and potentially improved neural and vascular growth within tissue engineering scaffolds.