Modeling Neovascularization of Tissues through the Encapsulation of Equine Endothelial Progenitor Cells in PEG-Fibrinogen Hydrogels Shasta NK Rizzi1,3, Wen J Seeto1,3, Petra Kerscher1, Alexander J Hodge1, Margaret M Salter2, Anne A Wooldridge2, Elizabeth A Lipke1,3

1. Cardiac Regeneration and Tissue Engineering Lab, Department of Chemical Engineering, Samuel Ginn College of Engineering2. Equine Research Lab, Department of Clinical Sciences, College of Veterinary Medicine

3. NSF REU Site in Micro/Nano-Structured Materials, Therapeutics, and DevicesAuburn University, Auburn, AL 36849, USA

Objective

AcknowledgmentsFunding was awarded by the NSF EEC Award #1063107, NSF CBET Award #1150854, and the Auburn University College of Veterinary Medicine Animal Health and Disease Research Program. The authors would like to thank all members of the Lipke and Wooldridge Labs for their support and advice during this study.

Cell Culture

Equine Regenerative Medicine

Fig. 3. Representation of a blood vessel. Endothelial Cells comprise the innermost lining of a blood vessel. They are supported by smooth muscle cells.

Background

Equine EPCs (Fig. 4) were isolated from peripheral horse blood and cultured in Lonza EGM-2 BulletKit media with 10% horse serum. Equine MSCs (Fig. 4) were isolated from horse bone marrow and cultured in DME/F-12 media with 10% fetal bovine serum and antibiotics.

Cell Encapsulation and Hydrogel Crosslinking

Fig. 5. Hydrogel Crosslinking. Hydrogels are crosslinked with visual light.

Prior to cell encapsulation, cells were fluorescently labeled with CellTracker Red (EPCs) and CellTracker Green (MSCs). EPCs, MSCs, and a co-culture of EPCs and MSCs were encapsulated in PEG-fibrinogen and pipetted into a PDMS mold on acrylated glass slides (Fig. 5). Hydrogels were then crosslinked (Fig. 6).

Conclusions and Future ResearchThis study demonstrated that equine EPCs could produce vasculature when cultured both alone and with MSCs in 3D PEG-fibrinogen scaffolds. It was unclear if the MSCs had a significant effect on vessel formation. Ensuing research will concentrate on quantifying tubule formation through lumen density, branching points, and average vessel length. This quantitative data will help to determine the effect of hydrogel thickness as well as the influence of MSCs on tubule formation and stability. Eventually this technology could advance to clinical trials for ultimate use as therapy for wound healing in horses.

Cell ViabilityFig. 7. EPCs remain viable in PEG-Fibrinogen Hydrogels after Crosslinking and Vascularization. Fluorescent images of EPCs encapsulated in PEG-fibrinogen taken (A) 24 hours and (B) 96 hours after crosslinking. Green represents live cells and red represents dead cells.

Four days after crosslinking, encapsulated cells were fixed with 5% paraformaldehyde and stained F-actin and nuclei for with phalloidin and DAPI. Stained hydrogels were imaged with confocal microscopy (Fig. 11).

Fig. 11. Morphology of Encapsulated Cell Types 4 Days Post Encapsulation. (A) Equine EPCs. (B) Equine MSCs. (C) Co-cultured equine EPCs and MSCs.

Fig. 4. Equine EPCs and MSCs. Phase contrast images of equine (A) EPCs and (B) MSCs.

Fig. 6. Cell Encapsulation and Hydrogel Crosslinking Process. (A) Hydrogel precursor and cells in media suspension. (B) Cells are encapsulated in hydrogel precursor and dispensed onto acrylated slide. (C) Hydrogel precursor with encapsulated cells is exposed to visual light. (D) Crosslinked hydrogels are suspended in media. (E) Encapsulated cells are viewed using a microscope.

Tubule Formation

References[1] J. J. Moon, J. E. Saik, R. A. Poche, J. E. Leslie-barbick, A. A. Smith, M. E. Dickinson, and J. L. West, “Biomimetic

hydrogels with pro-angiogenic properties,” Biomaterials, vol. 31, no. 14, pp. 3840–3847, 2010.[2] L. Almany and D. Seliktar, “Biosynthetic hydrogel scaffolds made from fibrinogen and polyethylene glycol for 3D cell

cultures.,” Biomaterials, vol. 26, no. 15, pp. 2467–77, May 2005. [3] K. K. Hirschi, D. a Ingram, and M. C. Yoder, “Assessing identity, phenotype, and fate of endothelial progenitor

cells.,” Arteriosclerosis, thrombosis, and vascular biology, vol. 28, no. 9, pp. 1584–95, Sep. 2008.

Endothelial Progenitor Cells (EPCs)

3D Cell Culture3D cell culture provides an alternative to 2D cell culture that more accurately predicts in vivo conditions. The scaffold used in this study was a hydrogel—a polymeric biomaterial with high water retention—composed of polyethylene glycol (PEG) and fibrinogen (Fig. 2). The hydrophilicity of PEG, a synthetic polymer, gives the scaffold the high water retention it needs to mimic tissues. Fibrinogen, a naturally occurring protein, is responsible for cell attachment within the scaffold [2].

NeovascularizationNeovascularization represents a significant area of research due to its applications in tissue engineering and regenerative medicine. In tissue engineered constructs, vascularization is essential to the survival of encapsulated cells [1].

Fig. 2. PEG-Fibrinogen Structure. Crosslinked PEG-fibrinogen network [2].

Fig. 9. Vasculature Increase. Phase contrast images of encapsulated EPCs and MSCs in co-culture taken (A) 1 day after crosslinking, (B) 2 days after crosslinking, (C) 3 days after crosslinking and (D) 4 days after crosslinking.

Fig. 8. EPCs Form Tubules One Day After Crosslinking. Phase contrast image of tubule formation in PEG-fibrinogen hydrogel containing equine EPCs 1 day after crosslinking.

Fig. 10. Vasculature Formation in PEG-Fibrinogen Hydrogels. Phase contrast (A,D,G) and fluorescent (B, E, H) images of encapsulated EPCs (A,B,C), encapsulated MSCs (D,E,F), and encapsulated EPCs and MSCs (G,H,I). Overlay (C,F,I).

(D) t=4 days

Mesenchymal Stem Cells (MSCs)Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into fat, bone, muscle, cartilage, tendon, and skin cells. When differentiated into smooth muscle cells, MSCs can be utilized to improve stability of blood vessels by providing mechanical support to endothelial cells comprising the formed vasculature (Fig. 3).

This study assessed neovascularization of 3D engineered constructs by equine endothelial progenitor cells (EPCs) and the ability of co-cultured equine mesenchymal stem cells (MSCs) to influence vessel formation and stability.

Fig. 1. Neovascularization. The formation of new blood vessels in the absence of preexisting vessels.

Equine research is at the forefront of regenerative veterinary medicine. Correlating with a decrease in muscle tissue, the amount of blood vessels decreases in proximity to a horse’s hooves. This decrease in vasculature, combined with a lack of rest, manifests in prolonged healing time for leg wounds. Neovascularization at sites of injury could shorten healing times as well as improve function of healed tissues.

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EPCs: 12E6 cells/mLMSCs: 3E6 cells/mL

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Tubule formation was tracked daily through phase contrast and fluorescent imaging. Tubules were detected one day after crosslinking in hydrogels containing EPCs only as well as hydrogels containing EPCs and MSCs in co-culture (Fig. 8). Vasculature increased significantly over a four day period (Fig. 9). Tubules did not appear in hydrogels containing only MSCs (Fig. 10).

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(A) t=1 day (B) t=2 days (C) t=3 days (D) t=4 days

(A) t=24 hours (B) t=96 hours

Vasculature in PEG-Fibrinogen HydrogelsMSCsEPCs EPCs + MSCs

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Endothelial progenitor cells (EPCs)—cells that have the ability to differentiate into mature, functioning endothelial cells [3]—are important to neovascularization because they constitute the innermost layer of blood vessels (Fig. 3). This was the first study to attempt vascularization using equine EPCs.