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Tissue Engineering

Enhancing the Vascularization of Collagen-GAG Scaffolds through improved cell distribution and fibrin incorporation

(I-337) Enhancing the Vascularization of Collagen-GAG Scaffolds through improved cell distribution and fibrin incorporation

Thursday, October 12, 2023

2:00 PM – 3:00 PM PDT

Location: Exhibit Hall - Row I - Poster # 337

Presenting Author(s)

BT

Bridget Twombly (she/her/hers)

undergrad research assistant
Kearney Lab, University of Massachusetts Amherst
Amherst, Massachusetts, United States

Co-Author(s)

John J. Amante (he/him/his)

PhD Candidate
University of Massachusetts Amherst
Amherst, Massachusetts, United States

Primary Investigator(s)

CK

Cathal Kearney

Assistant Professor
University of Massachusetts Amherst, United States

Introduction:: Chronic wounds are a significant problem across the world, affecting 6.5 million patients per year in the United States alone. A key reason that these wounds are unable to heal is poor revascularization. A current clinical approach for healing these wounds is collagen-glycosaminoglycan (collagen-GAG) scaffolds, which help promote tissue growth at a wound site. While collagen-GAG scaffolds support tissue formation, they do not currently help drive revascularization. Prior research shows that the inclusion of fibrin in polyethylene glycol hydrogels increases mechanical strength and promotes highly mature vessel-like structures. We propose that adding fibrin to the collagen-GAG scaffold will promote vascularization throughout the whole scaffold. As a second goal, seeding and centrifuging steps will be used to achieve enhanced cell distribution, which is a persistent challenge for these scaffolds in vitro. The combination of these improvements is expected to yield a collagen-GAG scaffold with enhanced vascularization.

Materials and Methods:: Collagen-GAG scaffolds were made using established freeze-drying techniques, then dehydrothermally crosslinked, cut with 8mm biopsy punches, and chemically crosslinked using 1-Ethyl-3-(3-Dimethlamniopropyl)-carbodimide and N-Hydroxysuccinimide. For seeding and centrifugation steps, each scaffold was seeded with 300,000 HEK293t cells. There were three groups (slow-spin, fast-spin, and no-spin; n = 3). Scaffolds were each placed in a well of a 24-well plate, 150,000 cells added to each scaffold, and centrifuged for five minutes three times at: 0 rcf (no spin), 28 rcf (slow-spin), or 300 rcf (fast-spin). Between each spin, runoff cell suspension was added back onto the scaffold. The scaffolds were flipped, 150,000 cells were added, and the scaffolds were spun three more times. Scaffolds were moved to 6-well plates, and 5 ml of media was added to each well and incubated. The scaffolds were fixed in PFA after 24 hours, then frozen in OCT. The samples were sectioned and stained for H&E to evaluate cell distribution. To further understand cell distribution, fluorescent tracking dye was used on some groups: one side of the scaffolds were seeded with GFP tagged cells and the other was seeded with Cy5 tagged cells. To incorporate fibrin into the scaffold, 50µl of thrombin was added with the cells to both sides of the scaffold. No-spin, slow-spin, and fast-spin steps were performed as described above. After cell seeding, 125 µl of fibrinogen was added to each side of the scaffolds to form fibrin within the pores. Fibrin distribution was evaluated using scanning electron microscopy.

Results, Conclusions, and Discussions:: Cell dispersion throughout the whole scaffold was improved with both a slow spin and a fast spin. In contrast to the no-spin group, for the spin groups, cells were distributed throughout the scaffold as identified on H&E (see figure 1A). Similar results were observed with the cell fluorescence (see figure 1B): the no-spin group had groups of cells clumped together with little distribution throughout the scaffold. The slow-spin group had the most even distribution throughout the scaffold with both GFP and Cy5 tagged cells throughout the scaffold. The cells in the fast-spin group were more evenly dispersed than the control, but there were fewer total cells. When fibrin was added to the seeded scaffold, the number of cells spread throughout the scaffold improved compared to a seeded collagen-GAG scaffold without fibrin (see figure 1C). While some cells were able to spread throughout the scaffold in the no-spin group, the majority of them stayed grouped together around the periphery of the scaffold. The slow-spin group had cells evenly dispersed throughout the entirety of the scaffold. Again, the fast-spin group had cells spread throughout the scaffold but also had the least amount of cells. This research shows promise in improving cell distribution within collagen-GAG scaffolds to ultimately enhance the vascularization of these scaffolds and aid in wound healing. In ongoing work, we will add endothelial cells and look at the vascularization potential of these scaffolds.

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