Guide to collagen characterization for biomaterial studies.

The structure and remodeling of collagen in vivo is critical to the pathology and healing of many human diseases, as well as to normal tissue development and regeneration. In addition, collagen matrices in the form of fibers, coatings, and films are used extensively in biomaterial and biomedical applications. The specific properties of these matrices, both in terms of physical and chemical characteristics, have a direct impact on cellular adhesion, spreading, and proliferation rates, and ultimately on the rate and extent of new extracellular matrix formation in vitro or in vivo. In recent studies, it has also been shown that collagen matrix structure has a major impact on cell and tissue outcomes related to cellular aging and differentiation potential. Collagen structure is complex because of both diversity of source materials, chemistry, and structural hierarchy. With such significant impact of collagen features on biological outcomes, it becomes essential to consider an appropriate set of analytical tools, or guide, so that collagens attained from commercial vendors are characterized in a comparative manner as an integral part of studies focused on biological parameters. The analysis should include as a starting point: (a) structural detail-mainly focused on molecular mass, purity, helical content, and bulk thermal properties, (b) chemical features-mainly focused on surface elemental analysis and hydrophobicity, and (c) morphological features at different length scales. The application of these analytical techniques to the characterization of collagen biomaterial matrices is critical in order to appropriately correlate biological responses from different studies with experimental outcomes in vitro or in vivo. As a case study, the analytical tools employed for collagen biomaterial studies are reviewed in the context of collagen remodeling by fibroblasts. The goal is to highlight the necessity of understanding collagen biophysical and chemical features as a prerequisite to (a) studies with cells and tissue formation, and (b) suggest modes to establish comparative outcomes for studies conducted in different laboratories.

Phagocytosis and remodeling of collagen matrices.

The biodegradation of collagen and the deposition of new collagen-based extracellular matrices are of central importance in tissue remodeling and function. Similarly, for collagen-based biomaterials used in tissue engineering, the degradation of collagen scaffolds with accompanying cellular infiltration and generation of new extracellular matrix is critical for the integration of in vitro grown tissues in vivo. In earlier studies we observed significant impact of collagen structure on primary lung fibroblast behavior in vitro in terms of collagen uptake and matrix remodeling. Therefore, in the present work, the response of human fibroblasts (IMR-90) to the structural state of collagen was studied with respect to phagocytosis in the presence and absence of inhibitors. Protein content and transcript levels for collagen I (Col-1), matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 2 (MMP-2), tissue inhibitor of matrix metalloproteinase 1 (TIMP-1), tissue inhibitor of matrix metalloproteinase 2 (TIMP-2), and heat shock protein 70 (HSP-70) were characterized as a function of collagen matrix concentration, structure and cell culture time to assess effects on cellular collagen matrix remodeling processes. Phagocytosis of collagen was assessed quantitatively by the uptake of collagen-coated fluorescent beads incorporated into the collagen matrices. Significantly higher levels of collagen phagocytosis were observed for the cells grown on the denatured collagen versus native collagen matrices. Significant reduction in collagen phagocytosis was observed by blocking several phagocytosis pathways when the cells were grown on denatured collagen versus non-denatured collagen. Collagen phagocytosis inhibition effects were significantly greater for PDL57 IMR-90 cells versus PDL48 cells, reflecting a reduced number of collagen processing pathways available to the older cells. Transcript levels related to the deposition of new extracellular matrix proteins varied as a function of the structure of the collagen matrix presented to the cells. A four-fold increase in transcript level of Col-1 and a higher level of collagen matrix incorporation were observed for cells grown on denatured collagen versus cells grown on non-denatured collagen. The data suggest that biomaterial matrices incorporating denatured collagen may promote more active remodeling toward new extracellular matrices in comparison to cells grown on non-denatured collagen. A similar effect of cellular action toward denatured (wound-related) collagen in the remodeling of tissues in vivo may have significant impact on tissue regeneration as well as the progression of collagen-related diseases.

Extracellular matrix remodeling--methods to quantify cell-matrix interactions.

Tissue turnover during wound healing, regeneration or integration of biomedical materials depends on the rate and extent of materials trafficking into and out of cells involved in extracellular matrix (ECM) remodeling. To exploit these processes, we report the first model for matrix trafficking in which these issues are quantitatively assessed for cells grown on both native collagen (normal tissue) and denatured collagen (wound state) substrates. Human fibroblasts more rapidly remodeled denatured versus normal collagen type I to form new ECM. Fluxes to and from the cells from the collagen substrates and the formation of new ECM were quantified using radioactively labeled substrates. The model can be employed for the systematic and quantitative study of the impact of a broad range of physiological factors and disease states on tissue remodeling, integrating extracellular matrix structures and cell biology.

Impact of collagen structure on matrix trafficking by human fibroblasts.

Biodegradation of collagen biomaterial matrices and the deposition of new collagen extracellular matrix (ECM) are critical to the integration of in vitro bioengineered materials and tissues in vivo. In previous studies, we observed significant impact of collagen matrix structure on primary lung fibroblast behavior in vitro. In the present work, to begin to understand the mechanistic basis for our previous observation, the response of human fibroblasts (IMR-90) to the structural state of collagen matrices was studied with respect to cell proliferation, cell morphology, beta-galactosidase level, and transcript content for collagen (Col-1), matrix metalloproteinases (MMP-1, MMP-2), tissue inhibitors of matrix metalloproteinase (TIMP-1 and TIMP-2). Collagen digestion was assessed quantitatively by uptake of collagen-coated fluorescent beads incorporated in the preformed collagen matrix. Transcript levels related to the deposition of new ECM proteins varied as a function of the structure of the collagen matrix presented to the cells. Col-1 expression was 2-fold higher and expression for MMP-1, MMP-2, TIMP-1, and TIMP-2 increased for cells when grown on 156 microg/cm2 denatured collagen compared with cells grown on tissue culture (TC) plastic. On 156 microg/cm2 nondenatured (native) collagen, Col-1 expression was decreased by half and MMP-2 was increased by 2.5-fold compared with cells grown on TC plastic. On 78 microg/cm2 denatured collagen, Col-1 expression was 80% whereas the MMPs and TIMPs were increased by 1.25- to 2-fold compared with cells grown on TC plastic. On 78 microg/cm2 nondenatured collagen expression of all 5 transcripts was reduced 60-90% of the levels determined for the cells grown on TC plastic. Cell viability, based on cell morphology and beta-galactosidase activity, was improved on the denatured collagen. A higher level of collagen matrix incorporation was observed for cells grown on denatured collagen than on nondenatured collagen or TC plastic. These data suggest that tissue engineering matrices incorporating denatured collagen may promote more active remodeling toward new ECM in comparison to cells grown on nondenatured collagen or cells grown on TC plastic.