Collagen threads with mechanical properties and fibrillar substructure similar to native tissue have been synthesized for the repair of injured tendon and ligament. While these scaffolding materials have demonstrated the potential for inducing tissue regeneration, one limitation has been an insufficient rate of tissue ingrowth for complete regeneration. We hypothesize that the structural hierarchy and biochemical cues on the surfaces of these threads will enhance the rate of cell migration and ultimately the rate of new tissue ingrowth. We developed an in vitro assay to measure the effects of various collagen sources and crosslinking on the rate of fibroblast migration on the surfaces of collagen threads. Threads were suspended from elevated platforms and seeded with fibroblast-populated collagen lattices. Cell migration rates ranging from 0.75 to 1.25 mm/day were measured as the fibroblasts left the lattices and migrated onto various thread types. Threads self-assembled from type I collagen were found to have migration rates similar to native tendon threads while crosslinking by severe dehydration decreased the rate. This novel in vitro model system allows examination of cell migration from a wound margin onto biomaterials to determine the effects of various cell types, matrix materials, and surface biochemistries on cell-matrix interactions. Ultimately, this assay will allow us to identify design parameters that will be most effective for enhancing the rate of tissue ingrowth on fiber-based collagen scaffolds for soft tissue regeneration.

The rational design of future bioengineered skin substitutes requires an understanding of the mechanisms by which the three-dimensional microarchitecture of tissue scaffolds modulates keratinocyte function. Microtextured basal lamina analogs were developed to investigate the relationship between the characteristic topography at the dermal-epidermal interface of native skin and keratinocyte function. Microfabrication techniques were used to create master patterns, negative replicates, and collagen membranes with ridges and channels of length scales (e.g., grooves of 50-200 microm in depth and width) similar to the invaginations found in basal lamina at the dermal-epidermal junction of native skin. Keratinocytes were seeded on the surfaces of basal lamina analogs, and histological analyses were performed after 7 days of tissue culture at the air-liquid interface. The keratinocytes formed a differentiated and stratified epidermis that conformed to the features of the microtextured membranes. Morphometric analyses of immunostained skin equivalents suggest that keratinocyte stratification and differentiation increases as channel depth increases and channel width decreases. This trend was most pronounced in channels with the highest depth-to-width ratios (i.e., 200 microm deep, 50 microm wide). It is anticipated that the findings from these studies will elucidate design parameters to enhance the performance of future bioengineered skin substitutes.

The dermal-epidermal junction of skin contains extracellular matrix proteins that are involved in initiating and controlling keratinocyte signaling events such as attachment, proliferation, and terminal differentiation. To characterize the relationship between extracellular matrix proteins and keratinocyte attachment, a biomimetic design approach was used to precisely tailor the surface of basal lamina analogs with biochemistries that emulate the native biochemical composition found at the dermal-epidermal junction. A high-throughput screening device was developed by our laboratory that allows for the simultaneous investigation of the conjugation of individual extracellular matrix proteins (e.g. collagen type I, collagen type IV, laminin, or fibronectin) as well as their effect on keratinocyte attachment, on the surface of an implantable collagen membrane. Fluorescence microscopy coupled with quantitative digital image analyses indicated that the extracellular matrix proteins adsorbed to the collagen-GAG membranes in a dose-dependent manner. To determine the relationship between extracellular matrix protein signaling cues and keratinocyte attachment, cells were seeded on protein-conjugated collagen-GAG membranes and a tetrazolium-based colorimetric assay was used to quantify viable keratinocyte attachment. Our results indicate that keratinocyte attachment was significantly enhanced on the surfaces of collagen membranes that were conjugated with fibronectin and type IV collagen. These findings define a set of design parameters that will enhance keratinocyte binding efficiency on the surface of collagen membranes and ultimately improve the rate of epithelialization for dermal equivalents.