(K-405) In Vitro Study of DC Fibroblast-Mediated Collagen Matrix Remodeling

Fibroblasts play a key role in the onset and progression of fibrosis by generating excessive mechanical tension on the extracellular collagen matrix coupled with specific intracellular signaling pathways. The fibroblast-populated collagen matrix (FPCM) anchored on the substrate is a commonly used tissue model for studying cell-dependent remodeling in wound healing and fibrosis. The goal of this project is to characterize the cellular and biomechanical properties of the FPCM as a model for Dupuytren’s contracture (DC). DC is the most common connective tissue disorder caused by fibroblastic proliferation and remodeling in the hand that leads to finger contracture. In this study, we compared the biomechanics of the collagen matrices that are populated with either normal human dermal fibroblasts or fibroblasts from DC patients. We measured over time the signaling onset of the myofibroblast phenotype correlated to the structural and mechanical changes in the matrix. The structural remodeling of the FPCM was measured by the optical coherence tomography (OCT) imaging, and the associated cell migrations were measured by the immunohistochemical staining. Our results showed that fibroblasts progressively compact and remodel the collagen matrix structure during development and at the same time pull on the matrix to generate tension. The results shed new light on the biomechanical mechanisms of normal and fibrotic tissue development.

Anchored FPCM were set up as previously described, using 125,000 cells/ml of 1-mg/ml collagen solution. Initial mixing conditions (duration and temperature etc.) were optimized to achieve approximately homogeneous collagen matrices with evenly-distributed fibroblasts. In addition, normal and DC fibroblast are set up to provide differentiation and confirmation.  The FPCM were cultured continuously at 37°C with 5% CO2 for up to 8 days, with half volume of media replaced daily. General cellular characterizations were performed accordingly. The cross-sectional morphology of FPCM during development was visualized and measured by OCT imaging. Generated mechanical tensions in these FPCM were probed by detaching the lattices from the substrate and recording the subsequent contraction over time with the OCT and microscopy. Furthermore, immunohistochemical staining is performed after measuring each FPCM every two days and is then quantified and categorized.

The cell-dependent compaction and remodeling of FPCM, as indicated by continuous reduction of their thickness and volume, increased considerably during the development while maintaining about same attachment area to the substrate. The DC fibroblasts remodeled the collagen matrix more quickly and further compared with normal fibroblasts. The top surface of the FPCM would frequently develop buckles or folds during earlier developmental days, which is a distinctive mechanical signature associated with heterogeneous tension distribution during compaction. In addition, myofibroblasts did not appear until after 3 days of collagen remodeling and tension generation, suggesting corresponding mechanical trigger to the upregulation of signaling factors. Furthermore, DC fibroblasts differentiated into myofibroblasts more rapidly than normal fibroblasts, in association with faster cell proliferation rates and more prominent stress fibers, thereby corresponding well to the more tension generation in the FPCM.

In conclusion, anchored FPCM were developed as a tissue model for DC progression. Our preliminary results showed that the cellular and biomechanical changes of developing tissue depend strongly on the type and initial distribution of cells, and that biomechanical and biochemical signaling factors are closely coupled in the tissue remodeling. This study would lead to better understanding of the biophysical mechanisms behind cancer progression, wound healing, and tissue engineering.

This work was supported by the CURE-STEM Scholarship and a student RCSA grant from the Office of High Impact Practices at the University of Central Oklahoma.