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Optimizing Cellular Invasion Into Hydrogel Scaffolds Using Microspheres To Create Interfaces Of Differential Densities
Opeyemi A. Asanbe, M.D., Rachel C. Hooper, M.D., John Morgan, Ph.D. Candidate, Jeremiah Joyce, B.A., Ross H. Weinreb, B.A. candidate, Adam Jacoby, B.A., Kadria N. Derrick, M.D, Abraham D. Stroock, Ph.D, Jason A. Spector, M.D., FACS.
Weill Cornell Medical College, New York, NY, USA.
PURPOSE- Although several acellular engineered tissue templates are available for clinical use, their success is limited to application within well-vascularized wound beds. In poorly vascularized wounds, such as those that have been irradiated or those with exposed hardware, bone or tendon, cellular and vascular invasion into tissue-engineered templates remains largely insufficient, leading to failure of incorporation or infection. Previous work in our lab demonstrated that cells preferentially invade scaffolds at the interface of differential densities, in some cases even more robustly than in scaffolds with well-defined microfeatures such as pores. As such, we fabricated a novel scaffold containing closely packed higher density collagen microspheres encased in a lower density collagen bulk, which created regularly spaced interfaces of differential densities so as to optimize cellular invasion and neovascularization.
METHODS- Using an oil emulsion technique, 1% collagen microspheres, ranging 50 to 150 µm in diameter, were created using neutralized type 1 collagen. 7 mm diameter microsphere scaffolds were fabricated by embedding 1% collagen microspheres in 0.3% type 1 collagen bulk. According to Kepler’s conjecture of close-packed spheres, approximately 74% of the density of the scaffold was comprised of higher density microspheres, and the remaining density was taken up by the 0.3% collagen. Microsphere scaffolds underwent thermal gelation at 37° C for 1 hour. Non-microsphere-containing 1% collagen scaffolds and non-microsphere-containing 0.3% collagen scaffolds were also fabricated for comparison. Microsphere and non-microsphere-containing scaffolds were implanted subcutaneously in dorsa of WT C57bl/6 mice. Following 7 or 14 days of implantation, scaffolds were procured and subsequently processed for histological analysis.
RESULTS- Histological analysis following procurement of microsphere scaffolds from mice dorsa after 7 days of implantation revealed substantial and uniform cellular invasion spanning the entire depth of the scaffold. After 14 days of implantation, immunohistochemical analysis identified CD31+ endothelial precursors within microsphere scaffolds, indicative of a progression in cellular invasion with the formation of neovasculature. Comparatively, even after 14 days, cells sporadically and only partially invaded the 0.3% collagen scaffolds and failed to invade the 1% collagen scaffolds, instead proliferating along the periphery of the scaffold.
CONCLUSIONS- We have demonstrated that altering the mechanical and spatial cues within hydrogel scaffolds by creating interfaces of differential collagen densities significantly improves cellular invasion. In addition to optimizing the architectural and structural cues sensed by cells, microspheres may also be impregnated with chemical moieties to further promote cellular invasion. We believe this approach holds tremendous promise for creating the optimal wound scaffold.
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