新型脱细胞脑组织修复支架的制备及其细胞相容性

 【目的】研制天然脱细胞脑组织修复支架，并对其形态结构、主要成分和细胞相容性进行分析，为研制出理想的脑损伤修复支架提供初步的实验依据。【方法】取成年Sprague-Dawley （SD）大鼠脑皮质，运用冻融、Triton X-100、脱氧胆酸钠、DNA/RNA酶等进行脱细胞处理并予京尼平（Genipin,GP）交联，对处理后的支架材料进行形态结构观察、主要成分分析和细胞相容性观察。【结果】经脱细胞处理后，脑组织细胞成分已去除，形态上具有高度仿生的三维立体空间网状结构。经GP交联后脱细胞支架空间网状结构变得更为明显，无菌保留3个月未见明显降解。平均孔径（14.9±2.5）μm，孔隙率达（90.4±2.2）％。脱细胞支架细胞外基质成分层粘连蛋白(Laminin,LN)强阳性表达、纤维粘连蛋白（Fibronectin,FN）和Ⅳ型胶原（Collagen Ⅳ,Col Ⅳ）弱阳性表达。神经干细胞与脱细胞支架共培养，HE可见细胞外基质支架间大量细胞粘附； SEM下可见大量神经干细胞粘附在支架材料表面，部分细胞表面有突起。【结论】初步研制的脱细胞脑组织支架呈立体空间网状结构，部分保留了细胞外基质成分，并具有良好的细胞相容性。

Preparation and Cytocompatibility of Acellular Rat Brain Matrix for Tissue Engineering Scaffolds

【Objective】 To prepare the acellular rat brain matrix scaffold and detect its cytocompatibility with neural stem cells.【Methods】 After anesthesia with 10% chloral hydrate,Sprague Dawley rats’ brain was removed,and then cerebral cortex was isolated.Being made use of freeze-thaw,Triton X-100,sodium deoxycholate,DNA enzyme,and RNA enzyme,the acellular scaffold of rat brain tissue was prepared,and after being cross-linked with Genipin,an initial acellular rat brain matrix scaffold was made.Then,Haematoxylin and Eosin staining(HE) was used to evaluate its content.Scanning elextron microscopy(SEM) was used to view the ultrastructure of the samples and measure the pore size of specimens.Immunohistochemical analysis of acellular scaffold was carried out to seek the distribution and content of the three major extracellular matrix-FN,LN and Col Ⅳ.The cytocompatibility of the acellular scaffold was assessed by observing NSCs’ growth,proliferation,and adhesion when co-cultured with the scaffold.【Results】 After being treated by a variety of reagents,cells within rat brain tissue had been largely removed.The acellular scaffold had forms of three-dimensional network structure,good hydrophilicity,and a high degree of bionic.After being cross-linked by Genipin,the three-dimensional scaffold had become more porous,with an average pore size(14.9±2.5) μm,porosity was(90.4±2.2)%.The degradation of scaffold,and no significant residual cell debris within scaffold was found.LN expression in the acellular scaffold was strong positive,while FN and Col IV expression were weakly positive.Had been cultured onto cross-linked scaffolds for 7 days,the NSCs had a pretty proliferation and adhesion to the scaffold.HE staining showed that there were a large number of cells in extracellular matrix.SEM showed that a large number of NSCs adhere to the surface of material,and some cells grew processes.【Conclusion】 The initial development of the acellular rat brain matrix scaffold has the structure of three-dimensional network,partly retains the extracellular matrix and has good cytocompatibility.