Poster C6 - Investigating Adult and Neonatal Fibrinogen Properties for Development of a Neonatal-Like Modified Adult Fibrinogen

Friday, October 25, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

GH

Grace Hardy

Undergraduate student
NC State University
Walkertown, North Carolina, United States

Co-Author(s)

Ana Sheridan

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

Elizabeth Byrnes

Graduate Research Assistant
NC State University, United States
SP

Sanika Pandit

Graduate research assistant
NC State University, United States
KN

Kimberly Nellenbach

Director Translational Clinical Development
SelSym Biotech, United States

Primary Investigator(s)

AB

Ashley Brown

Associate Professor
NC State University, United States

Introduction: Fibrinogen plays a major role in the wound healing process by turning into a crosslinked fibrin network that acts as a matrix to assist in hemostasis. Notably, the source of fibrinogen may lead to altered chemical and mechanical properties. For example, neonates have a molecular variant of fibrinogen with increased sialic acid content that has been shown to influence fibrin clot properties. Clots made from neonatal fibrin have been shown to be less stiff, degrade faster, enhance cellular interactions, and improve wound healing outcomes. This project explores a novel adult fibrin formulation with increased sialic acid concentration that aims to replicate the properties of neonatal fibrinogen.

Materials and

Methods: To study the role of sialic acid on wound healing properties, sialic acid was added and removed via enzyme-catalyzed reactions on both neonatal and adult fibrinogen for experimentation. Enzyme incubation times and treatments were optimized, and sialic acid concentration was quantified for each group. Fibrin-based gels were created by combining each experimental fibrinogen group with thrombin and fluorescently-labeled fibrinogen. The gels were then allowed to polymerize and were imaged using confocal microscopy. For analysis, fiber density and branching were calculated using FIJI. Mechanical properties of the fibrin-based gels were observed with atomic force microscopy. Clots were made with fibrinogen from each of the experimental groups and imaged using contact mode on an AFM to reveal the stiffness at different points of the sample within a microscopic area. These values were then averaged and compared across groups to determine the presence of any correlative relationships. To evaluate fibrin-based gels’ impact on cell attachment, fibroblasts were cultured onto the gels, labeled with fluorescent markers, and cell attachment was measured via fluorescence. Cell spreading assays were conducted by seeding fibroblasts on fibrin-based thin films, labeling the cells, and tracing the outline of the cells on FIJI.

Results, Conclusions, and Discussions: Eight preliminary treatment groups were created to analyze the impact of sialic acid on neonatal and adult fibrinogen properties. Quantification of the sialic acid content of these groups revealed that increasing incubation time and treatment number lead to increased sialic acid content on adult fibrinogen. Quantification also revealed that adding sialic acid to adult fibrinogen in 3 treatments over a 48 hour incubation (48 hr, 3x sialic acid addition group) had a final sialic acid content reflecting that found on unmodified neonatal fibrinogen. No clear quantitative trend emerged from clot fiber analysis, however clear qualitative differences could be observed between treatment groups, correlating to sialic acid content. Neonatal clots were observed to be less mechanically stiff than adult clots on AFM. Current work aims to investigate the role of increasing sialic acid on clot stiffness. Cell attachment assays revealed attachment to adult fibrin-based gels can be increased by modifying sialic acid content. Finally, thin films made using the 48 hr, 3x sialic acid addition group had significantly increased cell spreading compared to all other groups, including unmodified neonatal fibrin-based thin films. Future work for this project aims to broaden understanding of the impact of sialic acid on a number of fibrin mechanical and chemical properties by observing clot polymerization and degradation of different experimental groups, expanding cell work to include sprouting assays and a wider variety of cells, and continuing mechanical studies for all groups. The findings of this work may be used to create a more physiologically relevant neonatal blood transfusion or may be applied towards advancing dermal wound healing treatments.

Acknowledgements (Optional):