(J-384) 4D Printing Self-Morphing Smart Constructs for Modulating Neural Stem Cell Behaviors

Introduction:: Introduction: To date, various 3D cell culture systems have been investigated to represent the native tissue microenvironment to improve cellular behaviors when compared to traditional 2D cell cultures. However, it is still challenging to fully mimic the native dynamic microenvironment due to the static nature of the currently available systems. The emergence of 4D printing allows for programmable spatial and temporal control, enabling the recapitulation of the dynamic microenvironment in vitro. Herein, this study focused on creating 4D smart programmable constructs with a self-morphing capability that can regulate neural stem cell (NSC) behaviors in a spatiotemporal manner and enhance neural differentiation. Specifically, 4D printing of biocompatible shape memory polymers (SMPs) was performed using multiple printing techniques to fabricate surficial 100 μm microgrooves. The 4D effect refers to the ability of post-compressed microwells on the printed construct to recover their original shape at temperatures under 37°C. The effects of a variety of pattern designs on NSC behavior were also investigated.


Materials and Methods:: Materials and Methods: To fabricate self-morphing constructs for NSC culture, three different printing techniques, fused deposition modeling (FDM), extrusion, and digital light processing (DLP) printing, were used. A cylindrical sacrificial template with 100 µm aligned pattern was fabricated using polylactic acid (PLA) via FDM printing. Then, the sacrificial template was coated with polydimethylsiloxane (PDMS) to generate a mold with a 100 µm microgroove on the bottom. A preset amount of the 4D ink was extruded into the PDMS mold and cured to obtain the SMP substrates. The 6 x 6 micropillar arrays were designed in 5 different patterns, including column, triangle, anti-triangle, square, and anti-square. The micropillar arrays were printed via DLP printing using commercial photocurable resin. Finally, the micropillars were imprinted onto the aligned surface of the SMP substrate at 60 °C, generating the 4D printed self-morphing constructs. To exhibit the shape memory effect of the SMP, two different shapes, cherry blooms and the U.S. flag, were designed. Representative tensile and compressive stress-strain testing of the SMP material were studied at room temperature and 37 °C. NSCs were cultured under standard culture conditions and seeded into 4D printed dynamic constructs with varied pattern designs and static control constructs. The proliferation rates of NSCs cultured on the SMP scaffolds was tested using CCK-8 assays.


Results, Conclusions, and Discussions:: Results and Discussion: The SMP exhibited an excellent shape-changing effect as shown in Figure 1A. By applying multiple printing techniques, a series of SMP substrates with and without aligned surface patterns (Figure 1B) were successfully fabricated. As the glass transition temperature (Tg) of the photocurable resin is higher than that of the SMP, the microwells were successfully imprinted onto SMP substrates at 60 °C (Figure 1C). By varying the ratios of the 4D ink components, we obtained SMP substrates whose microwells are able to slowly disappear in 7 days as shown in Figure 1D. When compared to static controls with an aligned pattern, NSCs seeded on the 4D constructs exhibited a significantly higher rate of proliferation as exhibited in Figure 1E.
Conclusion: In this study, a series of 4D dynamic cell culture substrates with different micropatterns, including micro-columns, triangles, anti-triangles, squares, and anti-squares, were successfully fabricated. Moreover, the microwells imprinted on SMP substrates exhibited the ability to return to a flat configuration within 7 days at 37°C. Notably, compared to the static aligned substrate control, the dynamic SMP substrates imprinted with micropatterns demonstrated a higher level of proliferation. This phenomenon is thought to be linked to capacity of the 4D micropatterns to facilitate the early cell aggregation behaviors of NSCs.


Acknowledgements (Optional): : The authors would like to thank the NSF EBMS program grant # 2110842 for financial support.


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