A "Rational" Dermal Replacement: A Tissue Engineered Microsphere Hydrogel Scaffold
Yoshiko Toyoda, B.A., John P. Morgan, Ph.D., Xue Dong, M.D. Ph.D., Julia Jin, B.S., Jason A. Spector, M.D. F.A.C.S..
Weill Cornell Medical College, New York, NY, USA.
Background: Tissue-engineered dermal replacements require host vascular invasion to achieve permanent integration, a process that can span several weeks and as a result places the patient at risk for infection, multiple dressing changes, and the potential for poor wound healing. Insufficient vascularization of commercially available products has largely limited their clinical utility to only the most optimal wound beds. We hypothesize that a "rationally" designed dermal replacement scaffold with specific pore size, distribution, and material chemistry would become vascularized more rapidly than currently available commercial products. We have previously demonstrated accelerated cellular invasion and neovascularization into a novel hydrogel microsphere scaffold containing differential collagen density in a mouse model. Herein, we extend this analysis using a three-dimensional in vitro model of the microsphere scaffold and further characterize and quantify endothelial cell migration in scaffolds of varying density. This platform provides enhanced, quantitative invasion metrics and objective comparison with commercially available products.
Methods: The microsphere scaffolds were fabricated by encasing 1% type 1 collagen microspheres 50-150um in diameter into 0.3% collagen bulk. Polydimethylsiloxane wells of 4mm diameter and 2mm height were coated with polyethylenimine and glutaraldehyde, then subsequently filled with the microsphere scaffolds. Non-microsphere containing 1% and 0.3% collagen scaffolds served as controls. A monolayer of green fluorescent (GFP)-labeled human umbilical vein endothelial cells were seeded on this three dimensional platform, activated for invasion and cultured for 3 days. The scaffolds were subsequently analyzed via immunohistochemical staining for DAPI (nuclei) and CD31 (endothelial phenotype) and confocal microscopy. Endothelial cell migration into the collagen wells was quantified using image analysis with ImageJ/FIJI and MATLAB.
Results: Activated GFP labeled cells comprising an initial confluent monolayer migrated into the bulk collagen scaffold during 3 days of culture, compared to control cultures. The microsphere scaffolds demonstrated robust endothelial cell migration along the interfaces of differential density and exhibited greater cellular invasion compared to 1% and 0.3% native collagen scaffolds, as quantified by the average invasion depth. Three dimensional vertical plane, z-stack imaging revealed the presence of sprouting structures with evidence of lumen formation.
Conclusion: We have demonstrated that custom-made, type I collagen hydrogel microsphere tissue scaffolds of interconnected differential densities result in more rapid and extended cellular infiltration and vascularization compared with similar commercially available products on the market today. This in vitro model of endothelial cell migration within our microsphere scaffold supports our previous observation of significantly accelerated cellular invasion and neovascularization in vivo, via enhanced mechanical and spatial cues, and through the engineering of a regular arrangement of differential density collagen interfaces. Activation of the endothelia predictably resulted in disruption of the endothelial monolayer, resulting in increased permeability and cellular migration throughout the scaffolds. The development of more rapidly vascularized dermal replacements, including those that may be placed overlying avascular surfaces, will have wide application to oncologic, traumatic, and burn reconstruction. Furthermore, these studies provide crucial information regarding the geometric and chemical cues that drive angiogenesis within hydrogel scaffolds.
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