T32 - Integration of Decellularized Extracellular Matrix and Platelet Like Particles for Enhanced Chronic Wound Healing

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

SG

Sofia Galletti

Undergraduate Researcher in the Young Scholars Program
North Carolina State University, United States

Co-Author(s)

Krista Hilmas

Graduate Research Assistant
NC State University
Raleigh, North Carolina, United States
Chloe Hincher

Graduate Student
North Carolina State University
Raleigh, North Carolina, United States
Ashley C. Brown

Lampe Distinguished Professor
North Carolina State University and University of North Carolina at Chapel Hill
Raleigh, North Carolina, United States
Donald Freytes

Associate Professor
NCSU/UNC Lampe Joint Department of Biomedical Engineering
Raleigh, North Carolina, United States

Last Author(s)

Donald Freytes

Associate Professor
NCSU/UNC Lampe Joint Department of Biomedical Engineering
Raleigh, North Carolina, United States

Introduction: : Conditions such as obesity and diabetes impair the natural healing process, resulting in chronic wounds. Existing solutions, including skin grafts and wound dressings, have poor biocompatibility and high infection risk, resulting in unregulated collagen production and fibrosis. This prolongs the healing process and increases the likelihood of scarring, pain, and long-term impairment. This project proposes an alternative approach: combining a biocompatible decellularized extracellular matrix (dECM) with synthetic platelet-like particles (PLPs), a deformable microgel with fibrin-binding antibodies to enhance provisional matrix stiffness, to create a topical hydrogel wound dressing. These components are designed to support healing post-hemostasis, minimize fibrosis by deriving a stable dECM hydrogel containing a fibrous matrix that is enhanced via PLP-mediated crosslinking. This matrix supports cell proliferation and migration to reduce fibrosis and improve the healing process of chronic wounds. Confocal fluorescence microscopy confirmed uniform PLP integration within the dECM-fibrinogen matrix, indicating crosslinking between fibrin and PLPs within the matrix, showing scaffold reinforcement and stability. Mechanical testing is performed to evaluate differences in polymerization and measure the strength of the dECM-fibrinogen matrix, with and without PLP crosslinking. To support imaging and validate a more effective approach, PLPs were integrated directly during the gelation process. Current work includes cytocompatibility testing with neonatal human dermal fibroblasts (HDFns) via live/dead assays, collagen contraction and migration assays, confirming the hydrogel’s potential in cell proliferation and collagen migration.

Materials and

Methods: : Porcine tissue was chopped into approximately 1 mm3 uniform tissue pieces. Tissue was manually decellularized using alternating hypertonic and hypotonic washes, along with chemical and enzymatic reagents, including trypsin-EDTA, sodium deoxycholate, DNase, and peracetic acid, applied for 120-minute intervals. The decellularized tissue was cryogenically powdered and then lyophilized for at least 24 hours. Residual DNA was quantified using Promega QuantiFluor® dsDNA fluorescent dye assay. To prepare the dECM hydrogel, lyophilized powder was enzymatically digested with pepsin for 24 hours.

The hydrogel was formulated by neutralizing the dECM digest to pH 7.2–7.4, followed by the addition of 1X HEPES, 1X PBS, and 7.5 µL of fibrinogen and thrombin. The mixture was cast in a low-attachment 24-well plate and incubated at 37°C for 60 minutes, then stored in 1X PBS overnight. For imaging, 488-labeled fibrinogen (1:15 ratio to unlabeled fibrinogen) was added prior to gelation. After polymerization, hydrogels were transferred to Ibidi µ-slide 8-well (Ibidi GmbH, Germany) plates, and AlexaFluor 647-labeled PLPs, diluted in 1X PBS, were added. Z-stack images in 5 μm slices were captured via confocal microscopy. FIJI software was used to calculate the fibrin and PLP density using pixel intensity ratios.

Rheological testing included 1% strain at 1 Hz for 1080 seconds during gelation, followed by a sweep from 0.1% to 1000% strain across 41 points for a total of a 3-hour gelation period with the gel mixture being plated on the rheometer to compare polymerization of gels with and without 1 µL of 10 mg/mL PLPs.

Results, Conclusions, and Discussions:: Mechanical analysis revealed substantial differences in gelation kinetics and mechanical stability between control and PLP-containing hydrogels. The negative control hydrogel, without PLP’s, demonstrated a rapid gelation profile, with the crossover point occurring at 0.833 minutes, indicating an increasingly stable dECM-fibrous matrix formation. In contrast, preliminary data showed the hydrogel incorporating 1 µL of PLPs exhibited a delayed crossover, suggesting the presence of PLPs disrupts the gelation process. This disruption was further supported by a reduction in the maximum storage modulus (G'), indicating compromised mechanical properties in the PLP-containing samples.

Confocal imaging revealed that PLPs are primarily localized to fibrinogen-rich regions, particularly along the gel periphery, when the hydrogels are immersed in a PLP suspension. This suggests that PLPs may selectively bind to areas with high fibrinogen levels, potentially influencing local gel architecture and contributing to inhomogeneous matrix formation. Notably, signs of fibrinolysis were observed after overnight gelation and additional PLP soaking solution. The gels were incubated in a non-humidity-controlled incubator. Mobile fibrin fragments within the hydrogel were also observed, impairing imaging clarity and indicating incomplete or unstable fibrin network formation.

Additionally, not all fibrinogen appeared to be successfully crosslinked within the matrix, implying insufficient enzymatic conversion or structural interference during gelation. Overall, these findings suggest that while PLPs interact strongly with fibrin-rich environments, their inclusion in the hydrogel formulation compromises both gelation efficiency and structural integrity at this high concentration of added thrombin and fibrinogen.

Further research is being conducted to include cytocompatibility through Live/Dead imaging with HDFns, cellular migration assays, and collagen contraction assays. These studies aim to determine whether this hydrogel formulation supports cell viability, migration, and extracellular matrix remodeling, which are critical for promoting healing in chronic wound environments. Additional mechanical testing and confocal fluorescence imaging will be conducted at increasing amounts of PLPs and alternative combinations of fibrinogen and thrombin, to better analyze and understand their influence on gelation kinetics, mechanical integrity, and crosslinking effects when incorporated before gelation and polymerization.

Acknowledgements and/or References (Optional): : I would like to thank my amazing mentors for their support, time, teaching me, and encouraging me to continue my research. As well as the Brown and Freytes Laboratories for their guidance and professional support, and the Comparative Medicine Institute for providing funding and support for my first undergraduate research experience.