Integrated biophysical characterization of fibrillar collagen-based hydrogels

Abstract

This paper describes an experimental characterization scheme of the biophysical properties of reconstituted hydrogel matrices based on indentation testing, quantification of transport via microfluidics, and confocal reflectance microscopy analysis. While methods for characterizing hydrogels exist and are widely used, they often do not measure diffusive and convective transport concurrently, determine the relationship between microstructure and transport efficiency, and decouple matrix mechanics and transport properties. Our integrated approach enabled independent and quantitative measurements of the structural, mechanical, and transport properties of hydrogels in a single study. We used fibrillar type I collagen as the base matrix and investigated the effects of two different matrix modifications: 1) cross-linking with human recombinant tissue transglutaminase II (hrTGII) and 2) supplementation with the non-fibrillar matrix constituent hyaluronic acid (HA). hrTGII modified matrix structure and transport but not mechanical parameters. Furthermore, changes in matrix structure due to hrTGII were seen to be dependent on the concentration of collagen. In contrast, supplementation of HA at different collagen concentrations altered matrix microstructure and mechanical indentation behavior but not transport parameters. These experimental observations reveal the important relationship between ECM composition and biophysical properties. The integrated techniques are versatile, robust, and accessible; and as matrix-cell interactions are instrumental for many biological processes, the methods and findings described here should be broadly applicable for characterizing hydrogel materials used for 3-D tissue engineered culture models.

ECM-derived biophysical cues mediate interstitial flow-induced sprouting angiogenesis

“…It was also described that increasing fibrin ECM density limits vessel morphogenesis by hindering molecular diffusion from a proangiogenic source (stromal fibroblasts) [38c] . Yet, unlike with standalone collagen or fibrin ECM where there is an inverse relationship between ECM concentration and matrix pore size, we previously reported [34] , and confirmed in the present study, that addition of non-fibrillar HA increases fibrillar collagen matrix pore size due to swelling of this GAG constituent [39] .…”

“…These stiffness measurements were in line with our previous reports for the same ECM compositions. [21,34] Moreover, addition of HA to the collagen-based ECM significantly increased the mean pore size by 13% from 1.70 µm to 1.92 µm compared to collagen-only ECM (Figure 8). We previously reported a similar outcome on matrix pore size when comparing the same ECM compositions [34] .…”

“…To characterize the stiffness profiles of the acellular ECM hydrogel mixtures, we used indentation testing, as previously described by our group. [21,34] To characterize the mean matrix pore size, we employed confocal reflectance microscopy and image analysis techniques described previously by our group [34] .…”

“…The mechanical stiffness of ECM hydrogels was measured using the protocols previously reported by our group [21,34] . Briefly, 300 µL of 3 mg/mL concentration of collagen-only and collagen/HA were casted on the 10.4 mm diameter custom made polystyrene housing holder.…”

Architected Hydrogels for Functional Tissue Engineering Applications

“…46 Further, fiber alignment reduces the flow resistance parallel to the fibers. 48 Increased transport of nutrients and waste inside fibrillar hydrogels support cell survival and growth, and larger pore sizes allow for cell migration. 49 In fibrillar hydrogels, clusters of cell adhesion ligands are present on the surface of the fibers similar to in vivo conditions.…”