Pre-Staining Silk Fibroin with Sudan Black B Attenuates Autofluorescence and Maintains Hydrogel Cytocompatibility and Physiologically-Relevant Mechanical Properties

Introduction: Biomaterial-based tissue engineering approaches have utilized silk fibroin (SF) from the Bombyx mori silkworm because of its biocompatibility and tunable mechanical properties1. SF biomaterials have been particularly promising in their utilization for tissue regeneration applications as they stiffen over time due to increased beta-sheet formation, mimicking extracellular stiffening during embryonic development. Despite their excellent versatility, a known issue with silk materials is persistent autofluorescence, which only gets worse over in culture. The autofluorescence makes it nearly impossible to assess embedded cells at longer culture time-points (2 - 6weeks) using any light microscopy modality. Previous literature has utilized Sudan Black B (SBB) treatment to quench SF hydrogel autofluorescence at the end of cell culture experiments, once the gels and encapsulated cells are fixed. However, this removes the ability for live cell imaging in otherwise opaque and highly autofluorescent constructs2. The goal of this study was to test the efficacy of treating SF with SBB before cell seeding and crosslinking the SF hydrogels, while silk is still raw. Our overall goal was to use SBB pre-culture treatment to maintain the favorable biocompatible and mechanical properties of SF hydrogels to allow 1) real-time analysis of cellular behavior in mechanically tunable SF biomaterials and 2) decrease total experiment time by avoiding cumbersome post-staining protocols. We hypothesized that pre-culture treatment of SF hydrogels with SBB would significantly attenuate the autofluorescent signal of SF while not significantly altering cell viability or hydrogel mechanical properties.

Materials and

Methods: SF was prepared from Bombyx mori cocoons using an established and widely standardized protocol3. For pre-culture SBB treated SF hydrogels (SBB-SF), SBB was added to dissolved silk in LiBr at 0.3% w/v; utilizing the DI water mediated dialysis step to encourage hydrophobic interactions between the SBB and SF. SF and SBB-SF hydrogels were synthesized at 3% w/v as previously described3. Cellular constructs were seeded with 1.5x106 red fluorescent protein (RPF) C2C12 mouse myoblasts. All constructs were incubated at 5% CO2, 37°C, in 10% fetal bovine serum (FBS), 1% Antibiotic/Antimycotic (AA) Dulbecco’s Modified Eagle’s Medium (DMEM). Mechanical testing was performed after 24 hrs of incubation using compression testing and atomic force microscopy (AFM). Compression testing was performed using an unconfined custom loading frame4 equipped with a 1N load cell where all samples were preloaded to 0.02N and brought to 30% strain at 1% strain•sec-1. AFM imaging was done in ambient fluid conditions using Bruker’s Dimension FastScan Bio SPM with a 64-bit Nanoscope V Controller utilized in combination with PeakForce-HiRs-F-B probes. Cell viability in hydrogels was assessed after 72 hours by a live/dead assay and phalloidin and Dapi fluorescent staining. The attenuation of the autofluorescence was assessed by deriving a signal (cell) to background (autofluorescence) ratio (SBR) for both SF and SBB-SF constructs from live and fixed cell confocal imaging after 48 hrs of incubation, respectively. Comparisons between SF and SBB-SF for derived Young’s modulus (YM), percent live cells, and SBRs were made using Paired Student’s T-tests in R.

Results, Conclusions, and Discussions: No significant differences were found between YM of SF and SBB-SF derived from compression testing (p = 0.2157). SBB-SF had a significant decrease in YM derived from AFM imaging compared to SF gels (p < 2.2e-16) (Figure 1). There was no significant difference in the percentage of live cells between conditions (p = 0.4062) (Figure 2). The SBB-SF condition had a significant increase in SBR compared to the SF condition (p = 1.03e-9) (Figure 3). Fluorescent imaging of the cells between the constructs showed a much higher proliferation rate of the C2C12 cells in the SBB-SF condition when compared to the SF (Figure 4).
Pre-culture treatment of SF with SBB does not significantly affect the macroscale (compression testing) mechanical properties of SF hydrogels, however it significantly decreased the YM at the nanoscale (AFM) level. This change is likely explained by SBB being a large ink molecule that potentially decreases crosslink density through steric hindrance. This significant difference likely is also responsible for the increased proliferation rate observed in the SBB-SF condition through fluorescence imaging as all other conditions were identical. Pre-culture treatment of SF with SBB additionally had no significant effect on cell viability however it did significantly quench the autofluorescence from the SF when compared to the non-treated group.
The findings of this study suggest that pre-culture SBB treatment of SF is an efficacious protocol in attenuating SF autofluorescence without compromising the favorable mechanical properties and cell viability. Applications of this protocol will allow for real-time analysis of cellular behavior through live cell imaging in these highly tunable biomaterials. This will allow for a greater understanding of cellular behavior in these constructs which will inform novel approaches to tissue engineering based regenerative medicine utilizing these materials, and potentially other hydrophobic biomaterials commonly used in tissue engineering applications as well.

Acknowledgements (Optional): Acknowledgements: We would like to thank the Surface Science Lab for assistance with obtaining and interpreting the nanomechanical measurements, and the Northwest Tissue Mechanics Lab for the use of their custom load frame. 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:
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2. Foster, O. et al. Sudan Black B Pretreatment to Suppress Autofluorescence in Silk Fibroin Scaffolds. ACS Biomater. Sci. Eng. 9, 3193–3205 (2023).
3. Rockwood, D. N. et al. Materials fabrication from Bombyx mori silk fibroin. Nat. Protoc. 6, 1612–1631 (2011).
4. Semodji, A. M. et al. A novel strain energy-based method to dynamically stimulate three-dimensional cellular constructs. in (Vail, Colorado, 2023).