Poster D4 - Matrix Mehanics and Composition of Entangled Collagen:Fibrin Interpenetrating Matrices Varies Temporarilly

Friday, October 25, 2024

3:30 PM - 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

ER

Eden Ramirez

Student
UTSA ESTEEMED, United States

Co-Author(s)

GC

Gennifer Chiou

Graduate research assistant
The University of Texas at San Antonio, United States
JO

Joo Ong

Senior Associate Dean of Administration and Graduate Programs
University of Texas at San Antonio, United States

Primary Investigator(s)

TG

Teja Guda, PhD

Jacobson Distinguished Professor of Innovation and Entrepreneurship
The University of Texas at San Antonio, United States

Introduction: Successful cellular and tissue regeneration requires appropriate matrix support which is both crucial for attachment and necessary for growth and development. For this reason, interpenetrating networks of collagen and fibrin offer much promise in the field of tissue regeneration. Collagen I is the primary extracellular matrix component of musculoskeletal tissues. In instances of injury, the first stabilizing transitional matrix is a temporary fibrin clot forms which serves both as an initial scaffold and provides pro-regenerative and angiogenic cues. The objective of this study was to characterize varying compositions of interpenetrating collagen and fibrin blended hydrogels and investigate the level of matrix support provided for future in vitro studies. The addition of stromal cells will introduce a cellular response to the initial matrix support, and the resulting temporal matrix changes will inform the level of matrix support available for cellular attachment, growth and differentiation and stromal induction of vascularization.

Materials and

Methods: Collagen and fibrin blend hydrogels (col:fib) were synthesized at the following concentrations: fibrin (F) at a fibrinogen density of 20 mg/mL (F20) and collagen (C) at a collagen concentration of 4 mg/mL (C4). Fibrin gels were prepared by combining fibrinogen with thrombin at a ratio of 2:5, respectively. Collagen gels were formed according to manufacturer’s protocol. Samples were allowed to gel at 37C for 30 minutes. Col:fib gels were made at the ratios of 25C75F, 50C50F, 75C25F, 100C, and 100F. Samples were characterized using an ElastoSens Bio (Rheolution, Quebec, Canada) daily over a period of 14 days or until sample degradation for storage modulus and sample height. After synthesis, samples were flash frozen and cryo-sectioned. Following sectioning, samples were stained with Masson’s Trichrome and imaged at 20x. Collagen and fibrin quantification was conducted via ImageJ (NIH) via the color deconvolution plugin. The percent of stained area was quantified for each channel, with the blue and red channel considered collagen and fibrin, respectively. Statistical Analysis: Rheology: Since the same hydrogel is measured over time, paired statistical modeling was used for improved power. A two-way ANOVA (across time and material composition) for n=3/group/time and Tukey’s post hoc test for significance determined at p< 0.05. Col:fib quantification: A one-way ANOVA on ranks for n=5/composition and Dunn’s post hoc test for significance was determined at p< 0.05 (SigmaPlot v15).

Results, Conclusions, and Discussions: Based on the characterization of these gels, we expect that matrix support provided by these col:fib hydrogels to depend on the level of fibrin. As fibrin content increased, hydrogel degradation occurred faster (Figure 1A and B), with complete degradation of 100F after day 1. The storage moduli of all groups at day 0 were significantly different compared to all other days (not shown) (Figure 1A), indicative that over time that matrices become more viscous and less elastic. Representative images of Masson’s Trichrome staining are shown in Figure 1C to visualize collagen (blue) and fibrin (red) composition. The stained micrographs demonstrate that hydrogel synthesis of blended compositions is homogenous and that with increasing amount of collagen, composition changes that occur can be visualized. Fibrin and collagen quantification was determined based on the percent area of red and blue that appeared in Masson’s Trichrome stained samples (Figure 1D). Samples were normalized by determining the ratio of percent area coverage of fibrin to collagen. There were significant differences between 100F and both 100C and 75C25F (Figure 1D). There were also differences between 100C and 25C75F (not shown) (Figure 1D). As a stromal population is added in future in vitro studies, we expect that the matrix changes that we have seen now to drastically change. We expect that in groups such as 100F or 75C25F where degradation occurs quickly, to have a slower degradation as stromal populations synthesize their own matrix. In comparison, we expect that the addition of cells to blends such as 100C to enzymatically degrade the matrix for cell proliferation and spreading.

Acknowledgements (Optional): This work is supported in part by NSF CBET#1847103, the Jacobson and USAA Foundation endowments at the University of Texas at San Antonio, and NIH 1R21DE032179-01A1. ER is supported by UTSA’s ESTEEMED Program (NIH R25EB027606).