Leibniz Research Laboratories for Biotechnology and Artificial Organs, Hannover Medical School, Carl-Neuberg-Strasse 1, Hannover, Germany.
Eur J Cardiothorac Surg. 2010 Oct;38(4):450-5. doi: 10.1016/j.ejcts.2010.02.009. Epub 2010 Mar 23.
A persistent problem in generating a functional myocardial patch is maintaining contractions in a thicker construct. Thus far, we have successfully created contracting constructs with a defined directionality by seeding neonatal rat cardiomyocytes (CMs) on decellularised porcine small-intestinal submucosa (SIS). Here, we report our efforts in generating a thicker contracting construct by combining CM cell sheets with CM-seeded SIS.
Porcine SIS was decellularised, opened along the longitudinal axis, fixed in a metal frame (45 mm × 25 mm) and seeded onto the submucosal side with neonatal rat CMs at a density of 1.8 × 10(5) cells cm(-2). CM sheets were prepared using temperature-responsive dishes by seeding CMs at a density of 4.0 × 10(5)cells cm(-2). Three days after CM seeding, one- or three-layered CMs sheet(s) were stacked onto seeded SIS. Construct contraction was observed for an additional 10 days followed by histological analysis.
Stacked CM sheets contracted spontaneously and synchronously with seeded SIS after adherence. A large portion of analysed constructs showed a defined contraction direction, parallel to the longitudinal axis (seeded SIS: 83%, seeded SIS+1 sheet: 70%, seeded SIS+3 layered sheets: 71%). This finding was in agreement to the histological finding of aligned CMs parallel to the longitudinal axis. The thickness of seeded SIS with and without three-layered sheets was approximately 800 μm and 500 μm, respectively.
By combining layered CM sheets with CM-seeded SIS, a three-dimensional myocardial patch with contraction in a defined direction was successfully generated. This may represent an intermediate step to a multiple layered, vascularised contractile myocardial graft.
在构建具有功能的心肌贴片时，一个持续存在的问题是如何维持较厚结构中的收缩。到目前为止，我们已经通过将新生大鼠心肌细胞（CMs）接种到脱细胞化的猪小肠黏膜下层（SIS）上来成功地创建了具有定向收缩的构建体。在这里，我们报告了通过将 CM 细胞片与 CM 接种的 SIS 结合来生成较厚的收缩构建体的努力。
将猪 SIS 脱细胞化，沿纵轴打开，固定在金属框架（45mm×25mm）中，并在黏膜下层侧以 1.8×10（5）个细胞/cm（2）的密度接种新生大鼠 CMs。CM 片通过温度响应培养皿制备，以 4.0×10（5）个细胞/cm（2）的密度接种 CMs。CM 接种后 3 天，将一层或三层 CM 片叠放在接种 SIS 上。在附加的 10 天内观察构建体的收缩情况，然后进行组织学分析。
在附着后，堆叠的 CM 片会自发且与接种 SIS 同步收缩。分析的大部分构建体显示出定义明确的收缩方向，与纵轴平行（接种 SIS：83%，接种 SIS+1 片：70%，接种 SIS+3 层片：71%）。这一发现与纵轴上对齐的 CM 的组织学发现一致。有和没有三层片的接种 SIS 的厚度分别约为 800μm 和 500μm。
通过将层状 CM 片与 CM 接种的 SIS 结合，成功生成了具有定义收缩方向的三维心肌贴片。这可能代表着向多层、血管化收缩性心肌移植物的一个中间步骤。
