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Biomaterials

Biomaterials - Poster Session D

Poster C10 - Silk Fibroin Scaffold Fabrication via More Environmentally Friendly Processing

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Silk has been used as a biomaterial in various capacities due to its biocompatibility and biodegradability.[1] Silk fibroin from the Bombyx mori species is particularly interesting in biomedical applications, as the crystalline structure of the fibroin protein, namely the β-sheet content, allows for fibroin to be used in a variety of formats with adjustable mechanical properties that contribute to stability of the biomaterial structures as well as tunable strength and elastic modulus to match the desired application.[2]
Recently, there has been a push for conservation and environmentally friendly methods for processing biomaterials, which reduce energy consumption and the use of hazardous chemicals. The typical protocol for processing cocoons utilizes sodium carbonate (Na2CO3) and lithium bromide (LiBr) for degumming (removing sericin which causes an immunogenic response) and solubilizing the silk, respectively; while this method yields isolated fibroin, sodium carbonate is produced through a process that requires ammonia sourced from processes with high levels of greenhouse gas emissions.[3,4] To form the fibroin into the desired scaffold shape, hexafluoroisopropanol (HFIP), a corrosive, volatile, and highly toxic organic solvent, is typically used to dissolve the lyophilized fibroin into a viscous solution, which is then dehydrated with NaCl crystals that simultaneously act as uniform porogens.[3] Overall, this research aims to develop a more environmentally friendly approach for producing silk fibroin scaffolds while maintaining comparable properties to those fabricated through traditional methods.

Materials and

Methods:  Control group silk fibroin samples were obtained by adding Bombyx mori cocoon fragments to boiling 0.02M Na2CO3 solution for 30 minutes while manually teasing fibers apart, following an established protocol.[3] Experimental samples were obtained by adding cocoon fragments to 0.25M NaOH solution for 90min with 100rpm stir while teasing fibers apart for 5min every 30min.[5] Remaining fibroin fibers were rinsed in double-distilled water and dried overnight. The dry silk fibroin was dissolved in 9.3M LiBr solution, heated at 60C for 4h, then dialyzed against double-distilled water for 48h. The remaining solution was centrifuged and stored at 4C.
The aqueous solutions were diluted to 6wt% silk fibroin and aliquoted into 3mL plastic molds. 7g of NaCl crystals with diameters of 500-600µm were added as porogens and containers were left sealed at room temperature for fibroin gelation. The resulting fibroin sponges were soaked in double-distilled water for 48h to leach out NaCl and subsequently cut into scaffolds with 2mm height and 4mm diameter.
Scaffolds were characterized through SEM, attenuated total reflection spectroscopy to determine the fibroin secondary structure, porosity determination via hexane displacement, and cellular response to the scaffolds, where the fibroin was seeded with human adipose-derived stem cells (hASCs) and whole human adipose tissue. The hASC-seeded scaffolds were stained with LIVE/DEAD stains and imaged via confocal microscopy for viability assessment, while tissue-seeded scaffolds were used to visualize tissue morphology and measure DNA content, triglyceride content, lipolytic function, and metabolic function of adipose tissue in scaffolds.

Results, Conclusions, and Discussions: To identify whether the experimental NaOH degumming method would isolate fibroin in a way compatible with scaffold manufacturing, it is important to recognize the removal of sericin. A similar calculated percentage of 28% of total cocoon mass loss was found through both methods of degumming, illustrating that NaOH was able to remove nearly all of the expected sericin content (25-30wt%) in Bombyx mori cocoons. Additionally, the resulting fibroin scaffolds were taken for FTIR-ATR scans, which analyzed the separation of the amide I (1620 ± 2 cm−1) and amide II (1514 ± 1 cm−1) absorbance peaks in the secondary structure of fibroin, demonstrating that the resulting scaffolds were composed of fibroin in the same β-sheet protein structure. Porosity assessment showed that scaffolds produced following the NaOH-based degumming technique had a significantly higher porosity compared to the control group scaffolds.
As for the cellular responses to the degummed scaffolds, a slight non-significant increase in viability was found on average for the NaOH-degummed scaffolds, which likely was due to the higher porosity permitting better access to cell media nutrients. The morphology of seeded hASCs was similar between scaffolds, although there visually appeared to be a greater degree of cellular attachment in the NaOH-degummed scaffolds.
Findings collectively indicate that the “green” scaffolds provided an equally biocompatible environment for soft tissue and stem cell applications in comparison to the current laboratory standard of Na2CO3-degumming methodology, with increased adherence of cells in the experimental condition scaffolds.

This study developed a more “green” method for silk fibroin scaffold fabrication through degumming Bombyx mori silk via NaOH while keeping comparable morphology, protein structure, mechanical properties, and degradation kinetics to silk scaffolds produced in non-sustainable degumming scaffold methods. In addition to being more environmentally friendly, these scaffolds also had higher porosity, with increased DNA content and better adherence of seeded tissue and cells, correlating to a non-significant trend of improved viability seen via seeded hASCs. These results suggest that the more environmentally friendly scaffolds are appropriate to use in adipose tissue applications as well as other soft tissues with similar properties.

Acknowledgements (Optional):  

References:
1. Kundu, B.; Rajkhowa, R.; Kundu, S.C.; Wang, X. Silk fibroin biomaterials for tissue regenerations. Adv. Drug Deliv. Rev. 2013, 65, 457–470.
2. Koh, L.-D.; Cheng, Y.; Teng, C.-P.; Khin, Y.-W.; Loh, X.-J.; Tee, S.-Y.; Low, M.; Ye, E.; Yu, H.-D.; Zhang, Y.-W.; et al. Structures, mechanical properties and applications of silk fibroin materials. Prog. Polym. Sci. 2015, 46, 86–110.
3. Rockwood, D.N.; Preda, R.C.; Yucel, T.; Wang, X.; Lovett, M.L.; Kaplan, D.L. Materials fabrication from Bombyx mori silk fibroin. Nat. Protoc. 2011, 6, 1612–1631.
4. Debari, M.K.; King, C.I.; Altgold, T.A.; Abbott, R.D. Silk Fibroin as a Green Material. ACS Biomater. Sci. Eng. 2021, 7, 3530–3544.
5. Roblin, N. V., DeBari, M. K., Shefter, S. L., Iizuka, E., & Abbott, R. D. Development of a More Environmentally Friendly Silk Fibroin Scaffold for Soft Tissue Applications. Journal of Functional Biomaterials. 2023, 14(4), 230.