Leibniz Research Laboratory for Biotechnology and Artificial Organs, Department of Thoracic and Cardiovascular Surgery, Hanover Medical School, Carl-Neuberg-Str. 1, Hanover 30625, Germany.
Cardiovasc Res. 2010 Mar 1;85(4):806-13. doi: 10.1093/cvr/cvp370. Epub 2009 Nov 17.
A problem in generating artificial tissues is supplying nutrients to cells within 3D constructs. The use of a decellularized biological vascularized matrix with preserved pedicles (BioVaM), as a scaffold, appears to aid the generation of perfusable tissue constructs in vitro. To prevent vessel occlusion upon implantation, a functional endothelium must line the graft vessel bed. Here we tested whether the pro-angiogenic factor CCN1 could improve the re-endothelialization of BioVaM in vitro.
BioVaM vessel beds were incubated with 100 ng/mL recombinant human CCN1. Human cord blood endothelial cells (hCBEC) were analysed with respect to adhesion behaviour upon CCN1 exposure and seeded onto vessel structures of CCN1 exposed BioVaM (cBioVaM). BioVaMs were fixed in a bioreactor and perfusion cultured for 4 and 14 days (d). BioVaM without CCN1 treatment served as controls. Initial seeding success and endothelialization progression were monitored by fluorescence-labelled hCBEC. During construct cultivation, pH and lactate production were measured. Degree of endothelialization and characterization of seeded cells, with respect to endothelial markers, were investigated histologically. BioVaM vessel structures showed a 78 +/- 17% increase of attached cells when pre-treated with CCN1. Evaluation of re-endothelialization (arbitrary units) was 4.0 +/- 0.8 and 2.6 +/- 0.8 after 4 d, and 5.0 +/- 0.0 and 3.0 +/- 0.5 after 14 d in cBioVaM vs. BioVaM, respectively. On day 14, lactate concentration, an indicator of metabolic activity, was increased 12-fold in cBioVaM relative to BioVaM. A preserved endothelial phenotype of seeded cells was verified in all cultures by acetylated low density lipoprotein uptake and positive immunohistochemistry against von Willebrand factor, endothelial nitric oxide synthase, and CD31.
Coating of decellularized vessel structures with CCN1 supports adhesion of hCBEC and enhances re-endothelialization of BioVaM. Perfusable, endothelialized constructs may aid in solving the problem of nourishing cells inside 3D tissue-engineered constructs.
在生成人工组织时，一个问题是为 3D 构建物中的细胞提供营养。使用具有保留蒂（BioVaM）的去细胞化生物血管基质作为支架，似乎有助于在体外生成可灌注的组织构建物。为了防止植入后的血管阻塞，移植物血管床必须有功能的内皮细胞覆盖。在这里，我们测试了促血管生成因子 CCN1 是否可以改善 BioVaM 的体外再内皮化。
用 100ng/ml 重组人 CCN1 孵育 BioVaM 血管床。分析了 CCN1 暴露后人脐血内皮细胞（hCBEC）的粘附行为，并将其接种到 CCN1 暴露的 BioVaM（cBioVaM）的血管结构上。BioVaM 在生物反应器中固定并进行 4 和 14 天（d）的灌注培养。未用 CCN1 处理的 BioVaM 作为对照。通过荧光标记的 hCBEC 监测初始接种成功率和内皮化进展。在构建物培养过程中，测量 pH 值和乳酸盐的产生。通过组织学研究，评估内皮化程度以及种子细胞的内皮标记物特征。当用 CCN1 预处理时，BioVaM 血管结构的附着细胞增加了 78% +/- 17%。cBioVaM 的再内皮化评估（任意单位）分别为 4.0 +/- 0.8 和 2.6 +/- 0.8 培养 4 天后，以及 5.0 +/- 0.0 和 3.0 +/- 0.5 培养 14 天后。在第 14 天，cBioVaM 中的乳酸盐浓度，代谢活性的一个指标，比 BioVaM 增加了 12 倍。所有培养物中的乙酰化低密度脂蛋白摄取和 von Willebrand 因子、内皮型一氧化氮合酶和 CD31 的免疫组织化学阳性均证实了种子细胞的保存内皮表型。
用 CCN1 包被去细胞化的血管结构可支持 hCBEC 的粘附，并增强 BioVaM 的再内皮化。可灌注的内皮化构建物可能有助于解决 3D 组织工程构建物内部细胞营养的问题。
