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BACKGROUND: Contemporary dermal substitutes are avascular and prone to high failure rates secondary to failure of incorporation or infection, especially when applied in complex wound beds, such as those previously irradiated or those with exposed hardware, bone or tendon. To overcome these shortcomings, we fabricated a novel collagen hydrogel scaffold with regularly spaced interfaces of differential collagen density to guide and optimize cellular invasion and neovascularization.
METHODS: Utilizing Kepler's conjecture of sphere packing, which states that the arrangement of spheres in a 3 dimensional space has a density of 74%, we fabricated 7 mm diameter microsphere scaffolds (MSS) with a regular arrangement of density gradients. 1%, type I collagen microspheres were manufactured via a water-in-oil emulsion technique and their morphology was confirmed via scanning electron microscopy (SEM). MSS were fabricated by encasing higher density, 1% collagen microspheres into lower density, 0.3% collagen bulk so that 3/4 of the scaffold's volume was comprised of microspheres and 1/4 of bulk collagen. MSS underwent thermal gelation at 37°C for 1 hour. Non-microsphere-containing 1% or 0.3% collagen scaffolds were fabricated as controls. Additionally, 7mm diameter Integra™ disks served as controls. All scaffolds were implanted subcutaneously in the dorsa of 8 week old WT C57bl/6 mice and harvested for histological and immunohistochemical analysis after 7 or 14 days of implantation.
RESULTS: SEM revealed the fabrication of smoothly contoured collagen microspheres, ranging 50 to 150 µm in diameter. After 7, 14 and 28 days, fluorescent microscopy revealed MSS with robust cellular invasion spanning the scaffold depth. Comparatively, cells sporadically invaded 0.3% collagen scaffolds and failed to invade 1% collagen scaffolds altogether, remaining confined to the scaffold periphery. The area fraction of cells was significantly higher in MSS samples (7d: 6.767±3.032;14d: 10.367±3.964) compared to that of 1% collagen scaffold samples (7d: 2.829±0.934, p<0.01; 14d: 5.52.938, p<0.01) and 0.3% collagen scaffold samples (7d: 2.4±0.92, p<0.01; 14d: 6.675±2.118, p<0.05). The area fraction of cells in Integra was not statistically different from than that of MSS. Immunohistochemical analysis identified CD31 expressing endothelial cells within MSS even after 7 days of implantation, with continued proliferation after 14 days of implantation, indicative of invading endothelial precursors and angiogenesis.
CONCLUSIONS: We have successfully accelerated cellular invasion and neovascularization into tissue engineered hydrogel constructs by enhancing mechanical and spatial cues within MSS through the regular arrangement of differential collagen density gradients. Although we have shown here that manipulation of the microstructure of the collagen has a powerful influence on cellular invasion, the microspheres utilized herein can also serve as drug delivery systems, making their therapeutic potential even greater. Our innovative, tissue engineered scaffolds hold tremendous promise for the development of the next generation dermal replacement product.