Design, physical prototyping and initial characterisation of 'lockyballs' This paper reports the fabrication of interlockable microscale scaffolds using two photon polymerization (2PP) and proposes a lockyball'' approach for tissue self-assembly for biofabrication

Directed tissue self-assembly or bottom-up modular approach in tissue biofabrication is an attractive and potentially superior alternative to a classic top-down solid scaffold-based approach in tissue engineering. For example, rapidly emerging organ printing technology using self-assembling tissue spheroids as building blocks is enabling computer-aided robotic bioprinting of three-dimensional (3D) tissue constructs. However, achieving proper material properties while maintaining desirable geometry and shape of 3D bioprinted tissue engineered constructs using directed tissue self-assembly, is still a challenge. Proponents of directed tissue self-assembly see the solution of this problem in developing methods of accelerated tissue maturation and/or using sacrificial temporal supporting of removable hydrogels. In the meantime, there is a growing consensus that a third strategy based on the integration of a directed tissue self-assembly approach with a conventional solid scaffold-based approach could be a potential optimal solution. We hypothesise that tissue spheroids with 'velcro((R))-like' interlockable solid microscaffolds or simply 'lockyballs' could enable the rapid in vivo biofabrication of 3D tissue constructs at desirable material properties and high initial cell density. Recently, biocompatible and biodegradable photosensitive biomaterials could be fabricated at nanoscale resolution using two-photon polymerisation (2PP), a development rendering this technique with high potential to fabricate 'velcro((R))- like' interlockable microscaffolds. Here we report design studies, physical prototyping using 2PP and initial functional characterisation of interlockable solid microscaffolds or so-called 'lockyballs'. 2PP was used as a novel enabling platform technology for rapid bottom-up modular tissue biofabrication of interlockable constructs. The principle of lockable tissue spheroids fabricated using the described lockyballs as solid microscaffolds is characterised by attractive new functionalities such as lockability and tunablematerial properties of the engineered constructs. It is reasonable to predict that these building blocks create the basis for a development of a clinical in vivo rapid biofabrication approach and form part of recent promising emerging bioprinting technologies.