Lee Yong-Ung, Yi Tai, Tara Shuhei, Lee Avione Y, Hibino Narutoshi, Shinoka Toshiharu, Breuer Christopher K
Tissue Engineering Program and Surgical Research, Nationwide Children's Hospital;
Tissue Engineering Program and Surgical Research, Nationwide Children's Hospital.
J Vis Exp. 2014 Jun 4(88):51632. doi: 10.3791/51632.
Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with ε-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (>90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.
接种骨髓单个核细胞(BMCs)的可生物降解支架常用于重建手术,以治疗先天性心脏畸形。长期临床结果显示通畅率极佳,然而,狭窄发生率也很高。为了研究组织工程血管移植物(TEVG)中血管新组织形成的细胞和分子机制,并预防狭窄的发展,我们建立了一种内径约为1毫米的移植物小鼠模型。首先,TEVG由可生物降解的管状支架组装而成,该支架由涂有ε-己内酯和L-丙交酯共聚物的聚乙醇酸非织造毡网制成。然后将支架放入冻干机中,抽真空24小时,并储存在干燥器中直至接种细胞。其次,从供体小鼠中采集骨髓,并通过密度梯度离心分离单个核细胞。第三,将大约一百万个细胞接种到支架上,并孵育过夜。最后,将接种后的支架作为肾下腔静脉间置移植物植入C57BL/6小鼠体内。植入的移植物显示出极佳的通畅率(>90%),没有血栓栓塞并发症或动脉瘤形成的迹象。这种小鼠模型将有助于我们理解和量化TEVG中新组织形成的细胞和分子机制。