Poster N17 - Silk microfiber-reinforced bioink for muscle tissue repair and in situ printing by a handheld 3D printer

Introduction: Extremity muscle injuries exceeding 20% muscle loss, termed volumetric muscle loss (VML), surpass the body's innate capacity for self-repair and become irreparable. As a result, there is a pressing need for effective regenerative therapies to address these debilitating injuries. In this study, we propose a novel approach for VML treatment using a custom-made handheld 3D printer to fabricate bio-constructs composed of silk microfiber-reinforced silk fibroin hydrogel. The handheld 3D printer enables the precise and on-demand fabrication of scaffolds directly at the defect site, for personalized and minimally invasive treatment.

Materials and

Methods: Our goal is to develop a working prototype of a device that can be used to print freeform biomaterial patterns on a portable scale, to deposit live cells with good viability, and to be easily integrated into a robotic arm in the future. Firstly, custom-made handheld 3D printer was developed consisting of two units such as base platform and printing module. Secondly, different concentrations of silk fibroin gel, gelatin and silk-microfiber bioink were formulated and assessed their printability. Formulated gels were crosslinked by sonication method and incubating at 37 °C humidified incubator. Further, biocompatibility and in situ 3D bioprinting was evaluated with C2C12 cells.

Results, Conclusions, and Discussions: Silk fibroin gel (SF) and Silk microfibers (MF) were extracted from silk cocoons and mixed with gelatin (G) at varied concentrations. The MF inclusion to the SF and G gel increased the overall viscosity and storage modulus (G’). Additionally, MF reinforcement enhanced the mechanical strength of the scaffold. Time-dependent complex viscosity plot illustrates an increase in viscosity over time following sonication, characterized by an initial logarithmic phase and reaching a plateau after 1000 s, indicative of fully crosslinked gels. Handheld 3D printer performance ascertained that increasing printing speed resulted in a larger line width. Live/dead and cytoskeleton staining images of C2C12-encapsulated scaffolds revealed that optimal concentration of MF (1% w/v) incorporation positively impacted the cell-biomaterial interaction. Ex vivo printing demonstrates printed structure retained its shape post-printing and cells exhibited a well-spread cytoskeletal structure and were attached around the MF.
In this research, we presented a silk microfiber reinforced silk fibroin bioink and custom-made handheld 3D printer for potential VML tissue repair and regeneration. Formulated silk microfiber reinforced silk fibroin bioink exhibited enhanced mechanical properties and cell-biomaterial interaction. Fiber-incorporated hydrogel would possibly improve the tissue repair process. Overall, these results highlight the promising prospects of in situ 3D bioprinting using the developed handheld printer, paving the way for developing advanced therapies that leverage the precision and flexibility of additive manufacturing techniques in regenerative medicine.

Acknowledgements (Optional): The authors would like to acknowledge the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (Award Numbers DP2AR082471).