Biomedical Engineering, Northwestern University, Evanston, IL, United States; Simpson/Querrey Institute, Northwestern University, Chicago, IL, United States.
Simpson/Querrey Institute, Northwestern University, Chicago, IL, United States; Surgery - Transplant Division, Northwestern University, Chicago, IL, United States; Materials Science and Engineering, Northwestern University, Evanston, IL, United States.
Acta Biomater. 2019 Feb;85:84-93. doi: 10.1016/j.actbio.2018.12.039. Epub 2018 Dec 24.
Three-dimensional (3D) printing of decellularized extracellular matrix (dECM) hydrogels is a promising technique for regenerative engineering. 3D-printing enables the reproducible and precise patterning of multiple cells and biomaterials in 3D, while dECM has high organ-specific bioactivity. However, dECM hydrogels often display poor printability on their own and necessitate additives or support materials to enable true 3D structures. In this study, we used a sacrificial material, 3D-printed Pluronic F-127, to serve as a platform into which dECM hydrogel can be incorporated to create specifically designed structures made entirely up of dECM. The effects of 3D dECM are studied in the context of engineering the intrahepatic biliary tree, an often-understudied topic in liver tissue engineering. Encapsulating biliary epithelial cells (cholangiocytes) within liver dECM has been shown to lead to the formation of complex biliary trees in vitro. By varying several aspects of the dECM structures' geometry, such as width and angle, we show that we can guide the directional formation of biliary trees. This is confirmed by computational 3D image analysis of duct alignment. This system also enables fabrication of a true multi-layer dECM structure and the formation of 3D biliary trees into which other cell types can be seeded. For example, we show that hepatocyte spheroids can be easily incorporated within this system, and that the seeding sequence influences the resulting structures after seven days in culture. STATEMENT OF SIGNIFICANCE: The field of liver tissue engineering has progressed significantly within the past several years, however engineering the intrahepatic biliary tree has remained a significant challenge. In this study, we utilize the inherent bioactivity of decellularized extracellular matrix (dECM) hydrogels and 3D-printing of a sacrificial biomaterial to create spatially defined, 3D biliary trees. The creation of patterned, 3D dECM hydrogels in the past has only been possible with additives to the gel that may stifle its bioactivity, or with rigid and permanent support structures that may present issues upon implantation. Additionally, the biological effect of 3D spatially patterned liver dECM has not been demonstrated independent of the effects of dECM bioactivity alone. This study demonstrates that sacrificial materials can be used to create pure, multi-layer dECM structures, and that strut width and angle can be changed to influence the formation and alignment of biliary trees encapsulated within. Furthermore, this strategy allows co-culture of other cells such as hepatocytes. We demonstrate that not only does this system show promise for tissue engineering the intrahepatic biliary tree, but it also aids in the study of duct formation and cell-cell interactions.
三维（3D）打印脱细胞细胞外基质（dECM）水凝胶是再生工程的一种很有前途的技术。3D 打印可在 3D 中对多种细胞和生物材料进行可重复且精确的图案化，而 dECM 具有高度的器官特异性生物活性。然而，dECM 水凝胶本身往往打印性能不佳，需要添加物或支撑材料才能形成真正的 3D 结构。在这项研究中，我们使用牺牲材料 3D 打印的 Pluronic F-127 作为平台，将 dECM 水凝胶纳入其中，以创建完全由 dECM 制成的特定设计结构。研究了 3D dECM 在工程肝内胆管树中的作用，这是肝组织工程中一个经常被忽视的课题。在体外，将胆管上皮细胞（胆管细胞）包埋在肝 dECM 中已被证明可导致复杂的胆管树形成。通过改变 dECM 结构几何形状的几个方面，例如宽度和角度，我们可以显示出可以指导胆管树的定向形成。这通过对导管对准的计算 3D 图像分析得到了证实。该系统还可以制造真正的多层 dECM 结构，并形成可以在其中接种其他细胞类型的 3D 胆管树。例如，我们表明可以很容易地将肝细胞球体纳入该系统中，并且在培养 7 天后，播种顺序会影响最终结构。研究意义：在过去的几年中，肝组织工程领域取得了重大进展，但是工程肝内胆管树仍然是一个重大挑战。在这项研究中，我们利用脱细胞细胞外基质（dECM）水凝胶的固有生物活性和牺牲生物材料的 3D 打印来创建空间定义的 3D 胆管树。过去，只能通过向凝胶中添加可能抑制其生物活性的添加剂或通过可能在植入时出现问题的刚性和永久性支撑结构来实现图案化的 3D dECM 水凝胶的创建。此外，3D 空间图案化肝 dECM 的生物学效应尚未证明与 dECM 生物活性本身无关。这项研究表明，可以使用牺牲材料来创建纯的多层 dECM 结构，并且可以改变支柱宽度和角度以影响包裹在其中的胆管树的形成和对准。此外，该策略允许共培养其他细胞，例如肝细胞。我们证明，该系统不仅有望用于组织工程肝内胆管树，而且还有助于研究导管形成和细胞-细胞相互作用。
