Research Summary

Designing highly functional scaffolds for tissue engineering requires a fundamental understanding of the mechanisms by which the three-dimensional architecture and the biochemical composition of the tissue scaffolds modulate cellular adhesion, proliferation, and differentiation, as well as the regeneration of native tissue functions. These scaffolds must replicate both native tissue microstructures with cellular microenvironments (1-10 µm) and organ-scale structures with cellular resolution (10-1000 µm) to better maintain in vivo-like cellular function. Additionally, it has been shown that the control of cellular functions is largely modulated by cells interacting with extracellular matrix (ECM) proteins, which have extremely complex structures in the submicron or nanoscale range (100 – 300 nm). The research projects in my laboratory investigate the interactions between cells and precisely bioengineered scaffolds with micron and submicron scale topographic features and ECM cues that mimic native tissue structures. We anticipate that the results of these studies will be used to develop precisely tailored biomaterials that direct cell-matrix interactions and enhance the rate of tissue regeneration; these will improve the design of tissue engineered analogs for the repair of soft tissue injuries. Presently, three projects in my laboratory are focused on this initiative.

The Influence of Microtextured Basal Lamina Analog Topography on Keratinocyte Function and Epidermal Organization

The design of future bioengineered skin substitutes requires an understanding of the mechanisms by which the three-dimensional microarchitecture and the biochemical composition of tissue scaffolds modulate keratinocyte adhesion, proliferation and differentiation, as well as the morphogenesis of cells into analogs of highly functional skin. It is our hypothesis that microfabricated basal lamina analogs with three-dimensional features and ECM cues which mimic the cellular microenvironments of the dermal-epidermal junction will increase epidermal stem cell clustering on the surface of bioengineered skin substitutes and will promote the rapid regeneration of a robust epidermal layer. Specifically, we seek to characterize the microstructural and biochemical cues that regulate keratinocyte function on the surface of basal lamina analogs with topographic features and ECM compositions that mimic the native dermal-epidermal junction (DEJ).

Recently, we developed methods for fabricating basal lamina analogs with topographic features comparable to native tissue structures. Microfabrication techniques were used to create master patterns, negative replicates, and collagen membranes with ridges and channels of length scales similar to the invaginations found in native basal lamina at the dermal-epidermal junction of native skin (e.g., grooves 50-200 um in depth and width). Keratinocytes were seeded on the surfaces of the basal lamina analogs, and histological analyses were performed after 7 days of tissue culture. The keratinocytes formed a differentiated and stratified epidermis that conformed to the features of the microtextured membrane. Morphometric analyses of these immunostained skin equivalents indicated that the rates of keratinocyte stratification and differentiation increased as channel depth increased and channel width decreased. This trend was most pronounced in channels with the highest depth-to-width aspect ratios (i.e., 200 um deep, 50 um wide).

To characterize the relationships between the ECM compositions of basal lamina analogs and keratinocyte functions on the surfaces of bioengineered skin substitutes, we devised a novel high-throughput screening device. This system allows for several cellular adhesion assays with varying protein concentrations to be performed in parallel under normal culturing conditions. Customized Delrin™ templates were designed to hold collagen-GAG membranes and to create a multiwell assay system. To modify the surfaces of the membranes, serial dilutions of fluorescently labeled ECM proteins known to enhance keratinocyte adhesion, including type I collagen, fibronectin, laminin, were absorbed onto the surfaces. Quantitative fluorescence image analyses of the individual wells confirmed that the ECM molecules were bound to the membranes in a dose-dependent manner. Keratinocytes were seeded onto the modified surfaces of the membranes in the high-throughput screening device and a tetrazolium-based (MTT) colorimetric assay was used to show which concentration of proteins provided optimal cellular attachment. To date, findings from this study show that increasing concentrations of fibronectin or type I collagen adsorbed to the surfaces of collagen membranes enhances keratinocyte adhesion to the membranes.

By testing this hypothesis, we will gain a better understanding of the cell-matrix interactions that mediate keratinocyte function on the surface of microfabricated basal lamina analogs. These findings will also provide us with design parameters to fabricate basal lamina analogs that will enhance the performance of tissue engineered skin substitutes by increasing their structural and mechanical stability.

Multiphoton Excited (MPE) Nano- and Microfabrication of Scaffolds for Tissue Engineering

In tissues like cornea and skin, epithelial cells, such as keratinocytes, attach to basement membranes that present a complex milieu of topographic and ECM cues to cells through cell-surface receptors. Studies of basement membrane composition indicate that these thin structures consist of filaments containing collagen, laminin, fibronectin and proteoglycans as well as other fibrils which form complex nanometer sized topographies (22 nm to 191 nm) of fibers and ridges. In addition to providing structural support, these membranes provide biophysical signals that mediate cell proliferation and differentiation as well as new tissue synthesis. Thus, if we wish to influence and direct cell function, we must precisely tailor the nanometer scale architecture on the surfaces of biomaterial scaffolds.

In collaboration with Dr. Paul Campagnola, a biophotonics expert in the Department of Cell Biology, at the University of Connecticut Health Center, we have begun to characterize cellular responses to MPE crosslinked three-dimensional ECM proteins on the micron and submicron size scales. Dermal fibroblasts were seeded on MPE fabricated grid patterns composed of bovine serum albumin (BSA), fibronectin (FN), fibrinogen (FG) or type I collagen (CI). Each of these linear structures had widths in the range of 600-1000 nm. Qualitative analyses of fibroblast morphologies on MPE fabricated ECM patterns, 3 hours after seeding, suggest that fibronectin and collagen structures mediate matrix directed adhesion, spreading and lamellapodia alignment. In contrast, cells cultured on BSA and fibrinogen do not appear to be influenced by the underlying scaffold.

To make quantitative assessments of cell functions on MPE fabricated patterns, we analyzed cellular circularity, elongation and alignment as a function of ECM protein and pitch width (spacing between features). Cellular circularity is a parameter that is used to characterize the degree of cell spreading. A value of one indicates that a cell is circular and a value approaching zero indicates that a cell is an increasingly elongated or has a linear shape. For cells seeded on MPE fabricated patterns, circularity values ranged from 0.27 +/- 0.05 to 0.37 +/- 0.12, depending on the pitch width. In contrast, cells seeded on BSA control surfaces without patterns had circularity values of 0.44 +/- 0.19. These findings indicate that fibroblasts seeded on MPE fabricated patterns exhibit a more spread morphology than fibroblasts cultured on BSA control surfaces without patterns. Preliminary measurements of cell orientation and elongation were also compared for fibroblasts seeded on fibronectin, fibrinogen, collagen type I, and bovine serum albumin with various pitch widths. At 20 um pitch widths, cells on type collagen type I had an average angle of orientation of 4 degrees and cells on fibrinogen at 40 um pitch widths had an average angle of orientation of 4.2 degrees with respect to the MPE patterned lines, indicating that the cells are highly aligned. MPE fabricated patterns of BSA showed no difference in elongation from that of the flat surfaces for any geometry studied. Together, these preliminary finding suggests that the ECM composition and the pitch width of MPE patterns play significant roles in mediating contact guidance and cell function on the surfaces of precisely tailored biomaterials. Ultimately, we anticipate that the findings from these studies will provide a better understanding of how specific cell/scaffold interactions contribute to tissue development, disease pathologies and tissue regeneration.

Characterizing fibroblast migration on discrete collagen threads for applications in tissue regeneration

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. It is our hypothesis 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. To predict the rate of new tissue ingrowth onto these aligned fibrous scaffolds for soft tissue regeneration, several studies have focused on developing in vitro assays to measure contact-guided cell migration. While each of these techniques provides some measure of tissue responses to implantable threads, there is presently no quantitative and definitive in vitro method for characterizing cellular functions such as fibroblast migration on the surfaces of collagen threads with precisely engineered surface topographies and extracellular matrix compositions.

Recently, our laboratory 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. In our initial experiments, we compared the effects of two different collagen sources, as well as the effect of crosslinking, on the rate of fibroblast migration across the surfaces of collagen threads. Threads were suspended from elevated platforms and seeded with fibroblast-populated collagen lattices. Fibroblast migration rates were determined by measuring the distances that cells traveled along the lengths of the various thread types as a function of time. 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 of cell migration.

Ultimately, this novel in vitro model system may be a valuable tool for measuring contact-guided cell migration from a wound margin onto biomaterials with precisely engineered surface topographies and extracellular matrix compositions. Furthermore, 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.