激光微孔气管脱细胞基质的制备及构建兔组织工程气管管状软骨的实验研究

TTo repair the tracheal long defect has not been clinically resloved for a long time. Compare with other methods, tissue-engineered trachea is the most ideal tracheal substitute. Compared with biodegradable synthetic scaffolds, natural matrices have strong potential advantages . They maintain the natural extracellular matrix composition, do not release toxic biodegradable products nor induce inflammation, and play an active part in the regulation of cell behavior affecting cell proliferation, migration, differentiation, and, as a consequence, tissue regeneration. Starting from these considerations, decellularized matrices could be an ideal scaffold for tissue-engineered trachea. However, acellular cartilage matrix has not been widely used due to its' unique tissue structure. It is known that decellularized matrices is a compact tissue, which is difficult for the chondrocytes to grow into the matrix. If cells do not efficiently migrate into the decellularized matrix, the structure may eventually collapse in the body with degradation of the matrix.. We prepare laser miropore decellularized matrices in order to overcome the cell-seeding problems while maintain the original structure of the matrix. Pre-test proved that chondrocytes could migrate into the decellularized matrix along the miropore. We make futhrer study to detect the fundetmental properties of the laser miropore decellularized matrices so as to select the optimal procedure and paramaters of laser micropore. To test the efficiency of the laser micropore decellularized matrices, we re-seeded with chondrocytest on the surface of decellularized matrices in vtiro and implantation in a nude mouse. Then we construct a tubular tissue-engineered cartilage and repair the segmental tracheal defec in situ. We hope that laser micropore decellularized matrices could overcome the problems of the decellularized matrices and make a cornerstone for further constructing an ideal tissue-engineered trachea.

长段气管缺损的重建是临床尚待解决的难题，而组织工程气管是最符合生理要求的气管替代物。气管脱细胞基质支架具有组织相容性好，力学性能佳及促进组织再生的优点，作为组织工程气管支架具有很强的优势和应用前景。其缺陷是组织结构致密，软骨细胞无法在基质内部深入生长导致气管管壁软骨再生不全。随着基质的降解，移植段气管软化塌陷而导致长期组织修复失败。本课题在不破坏气管细胞外基质结构的基础上对其进行激光微孔处理，预实验结果表明软骨细胞可沿激光微孔深入到基质内部。本课题进一步通过对脱细胞基质的各项重要性能和指标进行检测，筛选出最佳的激光微孔处理方式和参数，从而制备出具有合适孔隙率的激光微孔脱细胞基质支架，通过将软骨细胞与该管状支架体外复合并植入动物皮下，构建组织工程化气管管状软骨并原位移植修复气管节段性软骨缺损。以期解决脱细胞基质结构致密致使气管管壁软骨化不全的难题，为下一步构建理想的组织工程气管打下坚实的基础

组织工程气管为长段气管缺损的重建提供了希望。然而由于缺乏一种理想的生物支架，从而限制了其临床应用。脱细胞气管基质由于其理想的管状结构，天然的软骨基质成分以及良好的生物降解性，从而被认为是一种理想的组织工程气管再生支架。然而，由于其软骨结构致密，导致很难将细胞脱干净，同时接种的细胞也无法长入基质内部，最终会引起炎症反应和再生软骨不全。为了解决这个问题，我们引入激光打孔技术，它可以将脱细胞基质变成多孔结构，这样就既利于脱细胞过程，也利于细胞长入基质内部。我们在脱细胞过程中发现激光处理过的气管基质更容易将细胞脱干净。更重要的是，在我们将激光打孔以及脱细胞过程参数优化之后，可以使激光打孔脱细胞基质只有轻微损害的情况下保证其原始的管状结构。随后我们将软骨细胞与激光打孔脱细胞基质体外复合并且培养8周后，发现细胞可以均匀地长在支架内外表面以及微孔内，并且形成很好的管状软骨。随后我们将该复合物植入裸鼠和兔子体内，进一步证明该复合物可以形成更加成熟的管状软骨，包括DNA数量和基质含量的增多，力学强度的增强。随后，我们将体外培养8周后的新生气管软骨植入兔子气管表面的左侧胸骨舌骨肌，经过4周在兔子体内的培养，实现了气管的预血管化。最后我们在保护血管的前提下充分游离肌肉形成双蒂肌瓣，然后对气管进行原位移植。该实验目前兔子原位回植存活时间达到8周。总的来说，我们的实验结果证明激光打孔脱细胞基质是一个理想的组织工程气管再生支架，为功能性管状气管重建提供了新的思路。

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