U2 - Silk Fibroin Augmentation: Modifying Silk-Based Wound Dressings for Increased Localization and Retention of Regenerative Cargo in Chronic Wounds

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Kamali Charles

Graduate Student - PhD
University of Pittsburgh, United States

Co-Author(s)

EO

Ellen L. Otto

Graduate Student - PhD
Carnegie Mellon University, United States
JW

Justin Weinbaum

Research Assistant Professor
University of Pittsburgh, Pennsylvania, United States
Charlie Ren

Associate Professor
Carnegie Mellon University, United States
PC

Phil Campbell

Research Professor in Department of Biomedical Engineering
Carnegie Mellon University
Pittsburgh, Pennsylvania, United States

Primary Investigator(s)

David A. Vorp, PhD

John A. Swanson Professor of Bioengineering and the Senior Associate Dean of Research and Facilities
University of Pittsburgh, United States

Introduction: : Silk fibroin (SF) is an ideal biomaterial for next-generation clinical wound dressings due to its biocompatibility, tunable mechanical properties, and potential to act as a scaffold for regenerative cargo1-3. Cytokines such as VEGF and TGF-β are commonly studied and utilized in regenerative medicine for chronic wound healing as they promote neovessel formation4,5 and re-epithelialization through keratinocyte migration respectively6,7. Regenerative therapies have sought to deliver cell or cell-based cargo containing these two cytokines for chronic wound treatment, however, retention of regenerative cargo is poor due to exudate “wash out”. This limitation requires repeat dosing to achieve the intended therapeutic effect leading to off-target accumulation in non-target organs8. To address concerns with delivering exogenous regenerative cargo, our lab has utilized amine reactive chemistry to conjugate our SF wound dressing with azido- (Az-)reactive dibenzocyclooctyne (DBCO) that can then attach complementary azido-tagged regenerative cargo through chemoselective immobilization.

Materials and

Methods: : SF was degummed and processed for electrospinning as previously described9. Wound dressings were electrospun using the following parameters: flow rate (100 µL/min), translational injector speed (50 mm/sec), 150 msec turn delay to improve end thickness of dressing for uniformity across the dressing , tip to mandrel distance (15 cm), rotational mandrel speed (300 rpm), mandrel diameter (25mm) applied voltage difference (15 kV), temperature (~25 °C), and humidity (~47%). A total of 3.1 mL of electrospinning solution was used and the electrospun product was removed from the mandrel and stored at room temperature (RT ~20 °C) in a sealed plastic bag. Fabricated wound dressings were then conjugated via soaking with 200μM DBCO-NHS ester with 10% DMSO in phosphate buffered saline (PBS) solution with 10% DMSO PBS solution alone acting as the vehicle control (CTRL). Dressings were then imaged using scanning electron microscopy (SEM) and processed in ImageJ for initial morphological characterization including pore size, fiber alignment, fiber distribution, and fiber diameter. Mechanical properties were analyzed using longitudinal and circumferential uniaxial tensile testing which was analyzed using MATLAB. Degradation properties were assessed using 12mm diameter circular sections excised from the elecctrospun dressings and soaked in PBS or protease XIV and weighed intermittently over 7 days. Az-Cy5 dye was utilized to assess the specific azido-alkyne binding of dye to DBCO conjugated wound dressings.

Results, Conclusions, and Discussions::

Results:
Morphological assessments of both DBCO and CTRL wound dressings are shown in Figure 1. Both wound dressings demonstrated consistent fiber characteristics from SEM images. There was no significant difference in pore size between treatments seen from ImageJ analysis. Uniaxial tensile testing demonstrated no statistical difference in ultimate strength or tangent modulus regardless of conjugation treatment. Proteolytic degradation exhibited an approximate 30% reduction in wound dressing mass after 7 days when treated with protease XIV compared to PBS, however, no statistical difference was seen when comparing DBCO to CTRL wound dressings. Successful DBCO conjugation of SF wound dressings was confirmed using Az-Cy5 dye which demonstrated fluorescence only from the DBCO conjugated wound dressings.

Discussion:
This study investigated the effects of DBCO click chemistry on a SF-based wound dressing with respect to morphology, mechanical properties, and functional capabilities. Morphological testing presented no significant differences between DBCO and CTRL in fiber characteristics indicating homogeneity in the electrospinning procedure. Consistency between both groups in fiber diameter and pore size provides evidence that amine chemistry responsible for the DBCO conjugation does not disrupt fiber formation. This result was expected as the amine linkage only occurs on amino acids with primary amines such as lysine, which constitutes only < 1% of the total amount of amino acids within the SF primary structure. Further analysis of all morphological, mechanical, and functional data demonstrated that modification of SF based wound dressings does not have a negative impact on its primary structure nor functionality. Unaltered degradation mechanics allows for predictive release of any bound cargo as this is the primary way to release chemical immobilization. These findings suggest that chemoselective immobilization is a viable option for retaining biological cargo without deteriorating the integrity of the SF wound dressing.

Conclusion:
The successful DBCO conjugation of SF had no statistically significant effect on any material properties of the wound dressing. Successful conjugation of DBCO using Az-Cy5 dye supported our hypothesis that Azido-DBCO click chemistry can improve retention of azido labeled regenerative cargo.

Acknowledgements and/or References (Optional): : References
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2. Kamalathevan, P., Ooi, P.S. & Loo, Y.L. Silk-Based Biomaterials in Cutaneous Wound Healing: A Systematic Review. Adv Skin Wound Care 31, 565-573 (2018).
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8. Xing, Y. et al. Engineering pro-angiogenic biomaterials via chemoselective extracellular vesicle immobilization. Biomaterials 281, 121357 (2022).
9. Gupta, P. et al. Bioresorbable silk grafts for small diameter vascular tissue engineering applications: In vitro and in vivo functional analysis. Acta Biomater 105, 146-158 (2020).