Yamanaka Hiroki, Morimoto Naoki, Yamaoka Tetsuji
Department of Biomedical Engineering, National Cerebral and Cardiovascular Center Research Institute, 6-1 Kishibe-Shinmachi, Suita, Osaka, 564-8565, Japan.
Department of Plastic and Reconstructive Surgery, Graduate School of Medicine and Faculty of Medicine, Kyoto University, Kyoto, Japan.
J Artif Organs. 2020 Jun;23(2):156-162. doi: 10.1007/s10047-019-01152-0. Epub 2020 Jan 1.
Various decellularization methods for allogenic and xenogenic bioscaffolds have been previously reported; however, decellularization methods for very thin (submillimeter-diameter) vascular tissues have not been discussed well. In this study, rat tail arteries (inner diameter, 0.6 mm) were decellularized with peracetic acid (PAA) and DNase I. PAA treatment is expected not only to disrupt cell membranes which improves the decellularization efficiency in the subsequent DNase treatment, but also to sterilize vascular scaffolds. We succeeded in adequate cell removal by immersing in 0.3% isotonic PAA solution and subsequent washing with DNase solution. For the DNase washing process, the perfusion method was superior in terms of cell removal to the static immersion method. Graft lumen was modified with a peptide composed of a collagen binding sequence and endothelial progenitor cell-binding sequence, (Pro-Hyp-Gly)-Gly-Gly-Gly-Arg-Glu-Asp-Val, as previously reported. They were patent in rat allogeneic transplantation model for 2 weeks, but unexpectedly resulted in graft rupture or tear formation, thereafter, suggesting reduced mechanical strength of the decellularized scaffolds. Histology showed that the thickness of the extracellular matrix (ECM) was decreased by the perfusion of the DNase solution. The method of combination of PAA and DNase was not necessarily optimal for the decellularization of very thin vascular tissues. The decellularization method is a compromise between effective cell removal and maintenance of the ECM nature. Since the acceptability of ECM denaturation by the host tissue highly depends on individual cases, decellularization methods should be carefully selected according to the type of target tissue and its intended use.
此前已有多种用于同种异体和异种生物支架的脱细胞方法的报道；然而，针对非常细（直径亚毫米级）的血管组织的脱细胞方法尚未得到充分讨论。在本研究中，使用过氧乙酸（PAA）和脱氧核糖核酸酶I（DNase I）对大鼠尾动脉（内径0.6毫米）进行脱细胞处理。预计PAA处理不仅会破坏细胞膜，从而提高后续DNase处理中的脱细胞效率，还能对血管支架进行消毒。通过浸入0.3%等渗PAA溶液并随后用DNase溶液洗涤，我们成功实现了充分的细胞去除。对于DNase洗涤过程，灌注法在细胞去除方面优于静态浸泡法。如先前报道，用由胶原结合序列和内皮祖细胞结合序列（Pro-Hyp-Gly）-Gly-Gly-Gly-Arg-Glu-Asp-Val组成的肽对移植血管腔进行修饰。在大鼠同种异体移植模型中，它们在2周内保持通畅，但出乎意料的是，此后出现了移植血管破裂或撕裂形成，这表明脱细胞支架的机械强度降低。组织学显示，通过灌注DNase溶液，细胞外基质（ECM）的厚度减小。PAA和DNase联合使用的方法对于非常细的血管组织的脱细胞处理不一定是最佳的。脱细胞方法是有效去除细胞与维持ECM性质之间的一种折衷。由于宿主组织对ECM变性的可接受性高度依赖于具体情况，应根据目标组织类型及其预期用途仔细选择脱细胞方法。
