We present a 3D-printing technology allowing free-form fabrication of centimetre-scale injectable structures for minimally invasive delivery. They result from the combination of 3D printing onto a cryogenic substrate and optimisation of carboxymethylcellulose-based cryogel inks. The resulting highly porous and elastic cryogels are biocompatible, and allow for protection of cell viability during compression for injection. Implanted into the murine subcutaneous space, they are colonized with a loose fibrovascular tissue with minimal signs of inflammation and remain encapsulation-free at three months. Finally, we vary local pore size through control of the substrate temperature during cryogenic printing. This enables control over local cell seeding density in-vitro and over vascularization density in cell-free scaffolds in-vivo. In sum, we address the need for 3D-bioprinting of large, yet injectable and highly biocompatible scaffolds and show modulation of the local response through control over local pore size.

Statement of significance

This work combines the power of 3D additive manufacturing with clinically advantageous minimally invasive delivery. We obtain porous, highly compressible and mechanically rugged structures by optimizing a cryogenic 3D printing process. Only a basic commercial 3D printer and elementary control over reaction rate and freezing are required. The porous hydrogels obtained are capable of withstanding delivery through capillaries up to 50 times smaller than their largest linear dimension, an as yet unprecedented compression ratio. Cells seeded onto the hydrogels are protected during compression. The hydrogel structures further exhibit excellent biocompatibility 3 months after subcutaneous injection into mice. We finally demonstrate that local modulation of pore size grants control over vascularization density in vivo. This provides proof-of-principle that meaningful biological information can be encoded during the 3D printing process, deploying its effect after minimally invasive implantation.

我们提出了一种3D打印技术，允许以自由形式制造厘米级的可注射结构，以实现微创递送。它们是由3D打印到低温基材上以及基于羧甲基纤维素的冷冻凝胶油墨的优化组合而成的。所得的高度多孔和弹性的冷冻凝胶具有生物相容性，可在注射压缩过程中保护细胞活力。植入鼠的皮下空间后，它们被疏松的纤维血管组织定植，发炎的迹象极少，并且在三个月内保持无囊化状态。最后，我们通过控制低温印刷过程中基材的温度来改变局部孔径。这样可以控制体外局部细胞接种密度体内无细胞支架中的血管生成密度和过度血管生成密度。总而言之，我们满足了对大型但可注射且具有高度生物相容性的支架进行3D生物打印的需求，并通过控制局部孔径显示了局部响应的调节。

重要声明

这项工作将3D增材制造的功能与临床上有利的微创递送相结合。通过优化低温3D打印过程，我们获得了多孔，高度可压缩且机械坚固的结构。仅需要基本的商用3D打印机以及对反应速度和冻结的基本控制。所获得的多孔水凝胶能够承受比最大线性尺寸小50倍的毛细管输送，这是前所未有的压缩比。压在水凝胶上的细胞在压缩过程中受到保护。皮下注射入小鼠3个月后，水凝胶结构进一步表现出优异的生物相容性。我们最终证明，孔径的局部调节在体内可以控制血管密度。