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Evaluating the Effectiveness of Vascularized Skin Substitutes in Repairing Full-thickness Skin Defects in Rodent Models: A Time-Course Study
Gurtej Singh
1, Shi Fu
2, Huiting Luo
2, Kuan-Che Feng
2, Shiffoni Sukhlal
1, Brooklyn Ratel
2, Divleen Singh
1, Ethan Lester
1, Marcia Simon
3, Miriam Rafailovich
2, Sami U. Khan
1, Duc Bui
1, Alexander B. Dagum
1
1Surgery, Stony Brook University, USA; 2Materials Science and Chemical Engineering, Stony Brook University, USA; 3Department of Oral Biology and Pathology, Stony Brook University, USA
Background: Advances in tissue engineering have led to the creation of skin constructs that, despite their potential, frequently fail due to poor vascularization. We address this by testing pre-vascularized constructs, hypothesizing their quick integration and improved survival in murine models. This study evaluates the temporal efficacy of these constructs, examining their performance over critical healing phases.
Methods: Our team developed vascularized skin constructs by adding a collagen-fibrin layer with Green Fluorescent Protein-labeled Human Umbilical Vein Endothelial Cells (GFP-HUVECs) and mural cells such Dental Pulp Stem Cells (DPSCs) to our previous non-vascularized constructs made of human keratinocytes, dermal fibroblasts, and rat tail collagen type I. To evaluate these, we induced full-thickness wounds on athymic mice, treating them with the constructs and observing healing over 1, 4, 7, and 14 days. Wounds were dressed to promote healing, and at each time point, mice were analyzed for construct integration and vascular network formation through Hematoxylin & Eosin and immunohistochemical staining using anti-GFP antibodies.
Results: Histological analysis showed successful skin construct integration at all assessed time points. Anastomosis began on Day 4, with substantial healing by Day 7, including wound contraction and absence of scar tissue. Anti-GFP antibodies revealed anastomoses with host vasculature, showing clear capillary networks by Day 7. By Day 14, however, a decrease in blood vessel density was observed, suggesting dynamic changes in vascularization post-grafting.
Conclusion: We've optimized culture conditions for endothelial and mural cells, producing pre-vascularized skin constructs that integrate and form anastomoses with host blood vessels. Our current research focuses on the roles of pre-vascularization and mural cells in enhancing anastomosis. We also aim to study these aspects in decellularized constructs to further understand their functionality and applications.
Application and Histological Analysis of Skin Constructs Over Time
(scale bar - 275 microns).
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