Incorporating shape-morphing capability into 3D microprinting enables the fabrication of 4D-printed microarchitectures as proof-of-concept actuators for potential use in soft robotics and microfluidic systems. The ability of these 3D microstructures to actuate rapidly and reversibly enables precise, non-invasive, and controllable deformation. In this study, we investigated the programmable shape-morphing behavior of 3D microarchitectures fabricated using two-photon polymerization (2PP) of a well-established temperature-responsive hydrogel, poly(N-isopropylacrylamide) (pNIPAM). We first systematically studied how 2PP 3D printing parameters (e.g., laser power, scanning speed) and the chemical composition of pNIPAM, including monomer and crosslinker, influence the shape morphing of bilayer microstructures within a temperature range of ~ 32 °C to 60 °C. The (thermo)mechanical properties of the hydrogels, including the Young’s modulus, thermal expansion coefficients, and angular deflection, were also measured at different laser doses and temperatures. Based on these experimental measurements, we calibrated a thermomechanical model capable of predicting the shape morphing of 4D-printed microarchitectures. These microarchitectures served as proof-of-concept actuators, demonstrating the potential of programmable microscale soft robotics and microfluidic systems. The findings provide design guidelines for engineering stimuli-responsive 3D microstructures, highlighting limitations and opportunities for future integration into functional soft robotic or microfluidic systems made of a single material.

The development of high-fidelity three-dimensional (3D) tissue models can minimize the need for animal models in clinical medicine and drug development. However, physical limitations regarding the distances within which diffusion processes are effective impose limitations on the size of such constructs. That is because larger-size constructs experience necrosis, especially in their centers, due to the cells residing deep inside such constructs not receiving enough oxygen and nutrients. This hampers the sustained in vitro growth of the tissues which is required for achieving functional microtissues. To address this challenge, we used three types of 3D printing technologies to create perfusable networks at different length scales and integrate them into such constructs. Toward this aim, networks incorporating porous conduits with increasingly complex configurations were designed and fabricated using fused deposition modeling, stereolithography, and two-photon polymerization while optimizing the printing conditions for each of these technologies. Furthermore, following network embedding in hydrogels, contrast agent-enhanced micro-computed tomography and confocal fluorescence microscopy were employed to characterize one of the essential network functionalities, namely the diffusion function. The investigations revealed the effects of various design parameters on the diffusion behavior of the porous conduits over 24 h. We found that the number of pores exerts the most significant influence on the diffusion behavior of the contrast agent, followed by variations in the pore size and hydrogel concentration. The analytical approach and the findings of this study establish a solid base for a new technological platform to fabricate perfusable multiscale 3D porous networks with complex designs while enabling the customization of diffusion characteristics to meet specific requirements for sustained in vitro tissue growth. Statement of significance: This study addresses an essential limitation of current 3D tissue engineering, namely, sustaining tissue viability in larger constructs through optimized nutrient and oxygen delivery. By utilizing advanced 3D printing techniques this research proposes the fabrication of perfusable, multiscale and customizable networks that enhance diffusion and enable cell access to essential nutrients throughout the construct. The findings highlighted the role of network characteristics on the diffusion of a model compound within a hydrogel matrix. This work represents a promising technological platform for creating advanced in vitro 3D tissue models that can reduce the use of animal models in research involving tissue regeneration, disease models and drug development.