Tara Shuhei, Kurobe Hirotsugu, Maxfield Mark W, Rocco Kevin A, Yi Tai, Naito Yuji, Breuer Christopher K, Shinoka Toshiharu
Tissue Engineering Program and Surgical Research, Nationwide Children's Hospital, Columbus, Ohio.
Department of Surgery, Yale University School of Medicine, New Haven, Conn.
J Vasc Surg. 2015 Sep;62(3):734-43. doi: 10.1016/j.jvs.2014.03.011. Epub 2014 Apr 16.
Autologous grafts are used to repair atherosclerotic cardiovascular diseases; however, many patients lack suitable donor graft tissue. Recently, tissue engineering techniques have emerged to make biologically active blood vessels. We applied this technique to produce arterial grafts using established biodegradable materials without cell seeding. The grafts were evaluated in vivo for vessel remodeling during 12 months.
Poly(L-lactide-co-ε-caprolactone) scaffolds reinforced by poly(lactic acid) (PLA) fiber were prepared as arterial grafts. Twenty-eight cell-free grafts were implanted as infrarenal aortic interposition grafts in 8-week-old female SCID/Bg mice. Serial ultrasound and micro computed tomography angiography were used to monitor grafts after implantation. Five grafts were harvested for histologic assessments and reverse transcription-quantitative polymerase chain reaction analysis at time points ranging from 4 months to 1 year after implantation.
Micro computed tomography indicated that most implanted mice displayed aneurysmal changes (three of five mice at 4 months, four of five mice at 8 months, and two of five mice at 12 months). Histologic assessments demonstrated extensive tissue remodeling leading to the development of well-circumscribed neovessels with an endothelial inner lining, a neointima containing smooth muscle cells and elastin, and a collagen-rich extracellular matrix. There were a few observed calcified deposits, located around residual PLA fibers at 12 months after implantation. Macrophage infiltration into the scaffold, as evaluated by F4/80 immunohistochemical staining, remained after 12 months and was focused mostly around residual PLA fibers. Reverse transcription-quantitative polymerase chain reaction analysis revealed that gene expression of Itgam, a marker for macrophages, and of matrix metalloproteinase 9 was higher than in native aorta during the course of 12 months, indicating prolonged inflammation (Itgam at 8 months: 11.75 ± 0.99 vs native aorta, P < .01; matrix metalloproteinase 9 at 4 months: 4.35 ± 3.05 vs native aorta, P < .05).
In this study, we demonstrated well-organized neotissue of cell-free biodegradable arterial grafts. Although most grafts experienced aneurysmal change, such findings provide insight into the process of tissue-engineered vascular graft remodeling and should allow informed rational design of the next generation of arterial grafts.
自体移植物用于修复动脉粥样硬化性心血管疾病；然而，许多患者缺乏合适的供体移植物组织。最近，组织工程技术已出现用于制造生物活性血管。我们应用该技术使用既定的可生物降解材料制备无细胞接种的动脉移植物。在12个月内对这些移植物进行体内血管重塑评估。
制备由聚乳酸（PLA）纤维增强的聚（L-丙交酯-共-ε-己内酯）支架作为动脉移植物。将28个无细胞移植物作为肾下腹主动脉间置移植物植入8周龄雌性SCID/Bg小鼠体内。植入后使用连续超声和微型计算机断层扫描血管造影术监测移植物。在植入后4个月至1年的时间点，采集5个移植物进行组织学评估和逆转录-定量聚合酶链反应分析。
微型计算机断层扫描显示，大多数植入小鼠出现动脉瘤样改变（4个月时5只小鼠中有3只，8个月时5只小鼠中有4只，12个月时5只小鼠中有2只）。组织学评估显示广泛的组织重塑，导致形成界限清晰的新生血管，其具有内皮内衬、含有平滑肌细胞和弹性蛋白的新内膜以及富含胶原蛋白的细胞外基质。在植入后12个月时，观察到一些钙化沉积物，位于残留的PLA纤维周围。通过F4/80免疫组织化学染色评估，巨噬细胞浸润到支架中在12个月后仍然存在，并且主要集中在残留的PLA纤维周围。逆转录-定量聚合酶链反应分析显示，在12个月的过程中，巨噬细胞标志物Itgam和基质金属蛋白酶9的基因表达高于天然主动脉，表明炎症持续存在（8个月时Itgam：11.75±0.99 vs天然主动脉，P<.01；4个月时基质金属蛋白酶9：4.35±3.05 vs天然主动脉，P<.05）。
在本研究中，我们展示了无细胞可生物降解动脉移植物的组织良好的新生组织。尽管大多数移植物出现动脉瘤样改变，但这些发现为组织工程血管移植物重塑过程提供了见解，并应有助于对下一代动脉移植物进行明智的合理设计。
