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Biomaterials

Biomaterials - Poster Session E

Poster O18 - Predictable Stiffness and Rapid Crosslinking: 3D Printing with Novel SF-Alginate Bioink

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Introduction
Silk stiffens over time at a predictable rate, however, there has been limited success in 3D inks due to long crosslinking times (Agostinacchio et al., 2024; Vepari & Kaplan, 2007). Alginate has also been used but fails to provide cells with attachment sites necessary and lacks the biologically relevant dynamic mechanical characteristic of SF (Lee & Mooney, 2012). To realize this potential, we propose modifying silk and alginate composition to formulate a bioink that is readily printable with rapid crosslinking. With a carefully tailored g-code protocol optimized for SF-Alginate-ECM bioink extrusion, this setup should ensure precise deposition of bioink layers, vital for crosslinking with CaCl2 leading to the formation of stable, biocompatible scaffolds. Our goal was to synthesize a new SF-based bioink with optimized cross-linking time, through the introduction of alginate, without compromising the dynamic mechanical landscape of SF hydrogels. We hypothesized that our new hydrogels would exhibit predictable rates of stiffness increase associated with SF gels (Kuzucu et al., 2021). By integrating SF's mechanical strength, alginate's biocompatibility, this novel bioink formulation and printing technique holds great promise for advancing tissue engineering and regenerative medicine applications.

Materials and

Methods:  Materials and Methods
10% silk fibroin (SF) solution was prepared by boiling raw SF for 60 minutes with calcium carbonate. SF was then washed in deionized water (H2O), dissolved in 9.3 M LiBr at 60 ˚C, and dialyzed against dH2O using 3 kDa molecular weight cutoff dialysis tubing, as previously described (Rockwood et al., 2011). 11.9% Alginate was prepared from bulk alginate powder. A Geeetech A20T-based 3D bioprinter served as a precise mold injection device for SF-Alginate-ECM bioink (Schneider et al., 2023). G-code was written and modified using Repetier Host after slicing through PrusaSlicer (Agostinacchio et al., 2024). The final gcode enabled extrusion into a PDMS (Polydimethysiloxane) mold comprised of 9 wells of 10mm diameter, 8mm depth in a 3x3 array and fixated into a petri dish, all sterilized with 70% Ethanol bath prior to the experiment. The bioink was extruded at an optimal feed rate of 50 mm/min and extruded volume in each sink was approximately 550 cubic mm from a 5mL syringe with a 20gauge 0.51” needle tip, 0.6 to be the optimal extrusion factor. To counter continuous print in-between sinks, retraction was added after completion of each sink. The bio-ink consisted of 4% w/V SF, 4% w/V Alginate, 4% HRP, 1% H2O2 and finally 2 mM 4% CaCl2.H2O2 for crosslinking after printing. All constructs were incubated at 5% CO2, 37°C. Mechanical testing was performed after 24, 96 and 168 hrs of incubation using unconfined compression testing. All samples were brought to failure at 1% strain.sec-1.

Results, Conclusions, and Discussions: Results
The mechanical properties of 4% Alginate / 4% SF hydrogels were assessed by measuring Young's Modulus at three time points: Day 1 (D1), Day 4 (D4), and Day 7 (D7). A significant increase in stiffness was observed from D1 to D4, with the Young's Modulus rising from approximately 10 kPa to 17 kPa (p = 0.04463). From D4 to D7, no significant change in stiffness was detected (p = 0.3899). Additionally, no significant difference in stiffness was observed between D1 and D7 (p = 0.2187) [Figure 3].

Discussion
The results indicate a marked increase in the stiffness of SF-Alginate hydrogels within the first four days. This initial stiffening phase is likely due to cross-linking reactions facilitated using CaCl₂.H₂O₂, which promotes ionic cross-linking of alginate and possibly enhances the interactions between alginate and silk fibroin components. The subsequent stabilization of stiffness from Day 4 to Day 7 suggests that these interactions reach equilibrium, and the mechanical properties of the hydrogel remain consistent thereafter. Interestingly enough, the hydrogels morphed into irregular shape and swelled up. This could be due to non-uniform crosslinking, imbalanced composition, or suboptimal 3D printing parameters [Figure 1(b)]. Optimization of these factors is necessary to achieve consistent hydrogel structures and further investigation.

Conclusion
The study demonstrates that 4% Alginate / 4% SF hydrogels experience a significant increase in stiffness within the first four days, followed by a stabilization period where no further significant changes in mechanical properties occur. This behavior indicates that after an initial period of conditioning, these hydrogels can provide consistent mechanical support, making them suitable for various biomedical applications. Moreover, our preliminary results showed that SF stiffening still occurs in this new biomaterial, allowing us to reach ranges of Young's Modulus not achievable with SF alone. In future studies, the integration of decellularized extracellular matrix (dECM) into these hydrogels will be explored with live/dead cell assay and imaging. The biological activity of dECM, along with its potential to enhance cell viability, holds promise for further improving the performance and application range of these hydrogels in tissue engineering and biomaterials.

Acknowledgements (Optional):  Acknowledgements
We acknowledge support from the Institutional Development Awards (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grants #P20GM103408, P20GM109095, and 1C06RR020533. We also acknowledge support from The Biomolecular Research Center at Boise State, BSU-Biomolecular Research Center, RRID:SCR_019174, with funding from the National Science Foundation, Grants #0619793 and #0923535; the M. J. Murdock Charitable Trust; Lori and Duane Stueckle, and the Idaho State Board of Education.

References
1. Agostinacchio, F., Fitzpatrick, V., Dire, S., Kaplan, D. L., & Motta, A. (2024). Silk fibroin-based inks for in situ 3D printing using a double crosslinking process. Bioactive Materials, 35, 122–134.
2. Kuzucu, M., Vera, G., Beaumont, M., Fischer, S., Wei, P., Shastri, V. P., & Forget, A. (2021). Extrusion-based 3D bioprinting of gradients of stiffness, cell density, and immobilized peptide using thermogelling hydrogels. ACS Biomaterials Science & Engineering, 7(6), 2192–2197.
3. Lee, K. Y., & Mooney, D. J. (2012). Alginate: Properties and biomedical applications. Progress in Polymer Science, 37(1), 106–126.
4. Rockwood, D. N., Preda, R. C., Yücel, T., Wang, X., Lovett, M. L., & Kaplan, D. L. (2011). Materials fabrication from Bombyx mori silk fibroin. Nature Protocols, 6(10), 1612–1631.
5. Schneider, K. H., Goldberg, B. J., Hasturk, O., Mu, X., Dötzlhofer, M., Eder, G., Theodossiou, S., Pichelkastner, L., Riess, P., & Rohringer, S. (2023). Silk fibroin, gelatin, and human placenta extracellular matrix-based composite hydrogels for 3D bioprinting and soft tissue engineering. Biomaterials Research, 27(1), 117.
6. Vepari, C., & Kaplan, D. L. (2007). Silk as a biomaterial. Progress in Polymer Science, 32(8–9), 991–1007.