Potere Federica, Belgio Beatrice, Croci Giorgio Alberto, Tabano Silvia, Petrini Paola, Dubini Gabriele, Boschetti Federica, Mantero Sara
Laboratory of Biological Structure Mechanics (LaBS), Politecnico di Milano, Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Milan, Italy.
Division of Pathology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.
Front Bioeng Biotechnol. 2022 Aug 19;10:918690. doi: 10.3389/fbioe.2022.918690. eCollection 2022.
3D-Bioprinting leads to the realization of tridimensional customized constructs to reproduce the biological structural complexity. The new technological challenge focuses on obtaining a 3D structure with several distinct layers to replicate the hierarchical organization of natural tissues. This work aims to reproduce large blood vessel substitutes compliant with the original tissue, combining the advantages of the 3D bioprinting, decellularization, and accounting for the presence of different cells. The decellularization process was performed on porcine aortas. Various decellularization protocols were tested and evaluated through DNA extraction, quantification, and amplification by PCR to define the adequate one. The decellularized extracellular matrix (dECM), lyophilized and solubilized, was combined with gelatin, alginate, and cells to obtain a novel bioink. Several solutions were tested, tuning the percentage of the components to obtain the adequate structural properties. The geometrical model of the large blood vessel constructs was designed with SolidWorks, and the construct slicing was done using the HeartWare software, which allowed generating the G-Code. The final constructs were 3D bioprinted with the Inkredible + using dual print heads. The composition of the bioink was tuned so that it could withstand the printing of a segment of a tubular construct up to 10 mm and reproduce the multicellular complexity. Among the several compositions tested, the suspension resulting from 8% w/v gelatin, 7% w/v alginate, and 3% w/v dECM, and cells successfully produced the designed structures. With this bioink, it was possible to print structures made up of 20 layers. The dimensions of the printed structures were consistent with the designed ones. We were able to avoid the double bioink overlap in the thickness, despite the increase in the number of layers during the printing process. The optimization of the parameters allowed the production of structures with a height of 20 layers corresponding to 9 mm. Theoretical and real structures were very close. The differences were 14% in height, 20% internal diameter, and 9% thickness. By tailoring the printing parameters and the amount of dECM, adequate mechanical properties could be met. In this study, we developed an innovative printable bioink able to finely reproduce the native complex structure of the large blood vessel.
3D生物打印有助于实现三维定制构建体，以再现生物结构的复杂性。新的技术挑战集中在获得具有多个不同层的3D结构，以复制天然组织的层次结构。这项工作旨在结合3D生物打印、脱细胞处理的优势，并考虑不同细胞的存在，来重现与原始组织相符的大型血管替代物。脱细胞处理过程在猪主动脉上进行。通过DNA提取、定量以及PCR扩增对各种脱细胞方案进行测试和评估，以确定合适的方案。将冻干并溶解的脱细胞细胞外基质（dECM）与明胶、藻酸盐和细胞混合，以获得一种新型生物墨水。测试了几种溶液，调整各成分的百分比以获得合适的结构特性。使用SolidWorks设计大型血管构建体的几何模型，并使用HeartWare软件进行构建体切片，该软件可生成G代码。最终构建体使用Inkredible +双打印头进行3D生物打印。对生物墨水的成分进行了调整，使其能够承受长达10毫米的管状构建体片段的打印，并重现多细胞复杂性。在测试的几种成分中，由8% w/v明胶、7% w/v藻酸盐、3% w/v dECM和细胞组成的悬浮液成功地制造出了设计的结构。使用这种生物墨水，可以打印出由20层组成的结构。打印结构的尺寸与设计尺寸一致。尽管在打印过程中层数增加，但我们能够避免生物墨水在厚度上的双重重叠。参数的优化使得能够生产出高度为20层（相当于9毫米）的结构。理论结构和实际结构非常接近。高度差异为14%，内径差异为20%，厚度差异为9%。通过调整打印参数和dECM的用量，可以满足适当的机械性能要求。在本研究中，我们开发了一种创新的可打印生物墨水，能够精细地重现大型血管的天然复杂结构。
