(396b) Digital Light Processing (DLP) Bioprinting with Methacrylated Silk Fibroin Polymers to Form Hydrogels with Tunable Controlled Release Properties

Silk fibroin-based biomaterials, derived from cocoons of Bombyx mori (BM), are investigated for use across many tissue engineering, regenerative medicine, and drug delivery applications due to properties such as its cytocompatibility, tunable degradation rates, and processibility into a wide range of materials.^1-3 Silk fibroin-based materials are typically formed by controlling the kinetics of -sheet crystillazation of the polymer chains, allowing for the formation of robust, fibrous structures as well as nanoscale structures.^1,4,5 A class of material that silk has previously been underutilized in is hydrogels. While silk fibroin can be physically crosslinked into gels through the same -sheet process as described above, these physical crosslinks are difficult to reproduce and when factors such as shear forces like those involved in extrusion 3D printing are introduced, the -sheet onset can run away and if gels are able to be formed, they have undesirable mechanical and optical properties.⁶ Thus, investigation into chemical crosslinking methods for silk fibroin hydrogels is necessary. Recent studies have explored enzymatic crosslinking mediated by horse radish peroxidase (HRP) for silk fibroin hydrogels. In this method, tyrosine residues are chemically crosslinked.^7,8 It has been shown that excess -sheeting can still occur with this crosslinking strategy, leading to poor optical and mechanical properties. The gelation kinetics are also slow compared to gelation kinetics observed when utilizing photo crosslinking methods.⁹ In this work, we seek to establish a repeatable method for the 3D printing of silk fibroin polymers into structures with desirable mechanical properties for biomedical applications. The major challenges we seek to address is limiting the onset of excess -sheet formation by utilizing a digital light processing (DLP) 3D bioprinting technique to avoid shear forces (3D Systems Volumetric LumenX+). For this method, we investigate chemical modifications to the silk polymer that allow for photo crosslinking. We also investigate the use of novel fibroin polymers from the silk source Plodia interpunctella (PI) (Figure 1 AB). PI silk fibroin is predicted to have a lower propensity to form -sheet structures compared the commonly used silk fibroin of BM (Figure 1C), which we hypothesize translates to improved processing into hydrogel formulations.

Other biopolymers such as gelatin have shown promise in the 3D printed hydrogel space when modified with a methacrylation reaction.¹⁰ In this chemistry, amine group hydrogens participate in a ring opening reaction with glycidyl methacrylate (GMA). In this work, we carry out the methacrylation of silk fibroin polymers and utilize a LAP photoinitiated polymerization to crosslink the polymer network into a hydrogel (Figure 1D). We compare the use of BM and PI fibroin polymers to determine how native fiber structure and protein sequence influence the chemical reactions and resulting materials. The material and mechanical properties of the modified silk solutions, silk films formed from these solutions, and modified silk fibroin hydrogels are assessed. We utilize the modified fibroin polymers in a digital light processing (DLP) application to 3D print hydrogel models with varying pore architecture. To further evaluate hydrogels formed from these biopolymers, we determined controlled release profiles from hydrogel formulations of varying polymer concentration and molecular weight by quantifying release of varying molecular weight (4-40 kDa) FITC-dextrans. Future work will expand on these efforts toward development and optimization of new 3D printable silk materials by exploring cytotoxicity and cell viability upon encapsulation within 3D printed constructs.

References

1 Rockwood, D. N. et al. Materials fabrication from Bombyx mori silk fibroin. Nature Protocols 6, 1612-1631, doi:10.1038/nprot.2011.379 (2011).

2 Guo, C., Li, C. & Kaplan, D. L. Enzymatic Degradation of Bombyx mori Silk Materials: A Review. Biomacromolecules 21, 1678-1686, doi:10.1021/acs.biomac.0c00090 (2020).

3 Liu, T. L. et al. Cytocompatibility of regenerated silk fibroin film: a medical biomaterial applicable to wound healing. J Zhejiang Univ Sci B 11, 10-16, doi:10.1631/jzus.B0900163 (2010).

4 Pacheco, M. O. et al. Silk Fibroin Particles as Carriers in the Development of All-Natural Hemoglobin-Based Oxygen Carriers (HBOCs). bioRxiv, doi:10.1101/2023.03.01.530637 (2023).

5 Rnjak-Kovacina, J. et al. Lyophilized Silk Sponges: A Versatile Biomaterial Platform for Soft Tissue Engineering. ACS Biomaterials Science & Engineering 1, 260-270, doi:10.1021/ab500149p (2015).

6 Farokhi, M. et al. Crosslinking strategies for silk fibroin hydrogels: promising biomedical materials. Biomed Mater 16, 022004, doi:10.1088/1748-605X/abb615 (2021).

7 Sahoo, J. K. et al. Silk degumming time controls horseradish peroxidase-catalyzed hydrogel properties. Biomater Sci 8, 4176-4185, doi:10.1039/d0bm00512f (2020).

8 Partlow, B. P. et al. Highly Tunable Elastomeric Silk Biomaterials. Advanced Functional Materials 24, 4615-4624, doi:https://doi.org/10.1002/adfm.201400526 (2014).

9 Agostinacchio, F., Mu, X., Dirè, S., Motta, A. & Kaplan, D. L. In Situ 3D Printing: Opportunities with Silk Inks. Trends Biotechnol 39, 719-730, doi:10.1016/j.tibtech.2020.11.003 (2021).

10 Song, P. et al. DLP fabricating of precision GelMA/HAp porous composite scaffold for bone tissue engineering application. Composites Part B: Engineering 244, 110163, doi:https://doi.org/10.1016/j.compositesb.2022.110163 (2022).