Micropatterned Microsphere Scaffolds: Optimizing the Performance of Engineered Dermal Substitutes
Karel-Bart Celie, B.A.1, Yoshiko Toyoda, B.A.2, Justin S. Buro, B.A.3, Alexandra J. Lin, B.A.1, Jonathan Xu, B.A.1, Andrew Miller, B.A.2, John Morgan, Ph.D.2, Jason A. Spector, M.D. F.A.C.S.2.
1Columbia University College of Physicians and Surgeons, New York, NY, USA, 2Weill Cornell Medicine, New York, NY, USA, 3George Washington University School of Medicine & Health Sciences, Washington, D.C., DC, USA.
PURPOSE: Current dermal replacement products perform sub-optimally in complex wound beds, such as those that have been irradiated or those with exposed hardware, mostly as a result of insufficient cell invasion and vascularization. We have previously observed more robust endothelial cell invasion of scaffolds containing a micropatterned matrix of differential collagen stiffness in a murine model when compared to collagen controls. Herein we compare the performance of our micropatterned microsphere hydrogels (MSS) to a widely utilized commercially available dermal replacement product in vitro and in vivo.
METHODS: Microspheres composed of 1% type I collagen 50-150um in diameter were created and encased in a 0.3% type I collagen bulk. For our in vitro study, polydimethylsiloxane (PDMS) wells of 4mm diameter and 2mm height were filled with the microsphere scaffolds. 3x2mm IntegraŽ disks were placed inside PDMS wells. Non-microsphere containing 1% and 0.3% collagen scaffolds served as controls. A monolayer of endothelial cells was seeded onto this three-dimensional platform, activated for invasion with 1uM sphingosine-1-phosphate, and cultured for 3 days. The collagen hydrogels were then analyzed using confocal microscopy to quantify cell invasion. For our in vivo study, 8x2mm MSS disks were created, along with 1% and 0.3% collagen controls. 8mm IntegraŽ disks were created, and the unilateral silicone layer was removed. A disk of each type was then implanted subcutaneously in the dorsum of 8-week old wild-type mice. The scaffolds were removed at 7 and 14 days, imaged, and analyzed with ImageJ.
RESULTS: Cells formed a confluent monolayer on the surface of the collagen disks, and migrated at a significantly higher rate into the MSS scaffold during 3 days of culture compared to control cultures as well as IntegraŽ (142um MSS vs 45um IntegraŽ, p<0.0001) (Figure 1). Furthermore, in our in vivo study, MSS and IntegraŽ both demonstrated robust cellular invasion spanning the depth of the scaffold at 7 and 14 days. IntegraŽ had a lower cell density within the bulk at all depths with 705 cells per mm2 compared to 1,454 cells per mm2 in MSS at 14 days. Control collagen disks demonstrated minimal cell invasion and notable volumetric contraction.
CONCLUSION: Micropatterned differential stiffness microsphere hydrogels (MSS) promote significantly more cellular invasion both in vivo and in vitro when compared to a commercially available dermal substitute. This enhanced cellular invasion and accelerated neovascularization, which results solely from the unique architecture of the scaffolds, indicates superior efficacy in the rate of integration into the host wound bed and may result in decreased length of time between its application and definitive wound closure. Further, the significantly more robust cellular and vascular invasion and integration of these scaffolds indicates their applicability in the treatment of suboptimal wound beds, which is beyond the capability of currently available dermal substitutes.
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