Design and realisation of 3D collagen gel models for the study of connective tissue remodelling and integration in vitro

Cell spatial remodelling in tensile connective tissue is at the base of fundamental biological processes including tissue morphology, growth adaptation and repair. It is increasingly clear that cell-force generation plays a central role in the remodelling process, however the understanding of how cell generated forces lead to stable 3-dimensional collagenous structures formation is poorly understood. In order to investigate two different processes occurring during cellular connective tissue remodelling, two models were developed using 3D fibroblast populated collagen lattices (FPCLs): 1) The process of stable structural remodelling of the matrix, was investigated in uniaxially tethered gels in the tensioning Culture Force Monitor (tCFM). The tension retained in the matrix after blocking cell force, by cytoskeletal disrupting agent cytochalasin D, was a measure of the degree of stable structural remodelling. 2) The processes of adhesion formation and integration between interfaces was investigated using an interface model made of a cell-free and a cell-seeded collagen lattice. The mechanical adhesion strength was measured to quantify the cellular remodelling process. Results showed that the residual matrix tension (RMT) retained in the collagen matrix, after cytochalasin D treatment, increases with culture time. Treatment with TGF-β1 and external cychcal loading seemed to increase RMT by different mechanisms. TGF-β1 by upregulating cell force generation, hence contraction; cychcal loading by increasing the alignment of collagen fibrils, hence accelerating the remodelling process. Adhesion strength in the interface model increased with culture time and was correlated with cellular migration across the interface zone. The distribution of failure stresses in the interface construct was determined by a computer model using Finite Element method. Results are discussed in terms of a mechanism of cell-matrix interaction in collagen lattices. This suggests that cell-matrix interactions are modulated by a constant iterative feed back relation between cell force generation and mechanical properties of the matrix both at microscopic (i.e. fibril packing and alignment) and macroscopic level (i.e. contraction force generation and uniform alignment of cells and matrix throughout the lattices). A computer model using Finite Element (FE) analysis, was developed in order to simulate the process of cell-matrix interaction, following the iterative concept to model the remodelling process. The results suggested a possible mechanism by which randomly orientated tractional forces in the matrix lead to highly aligned structures. Identifying the mechanisms which regulate the cell-matrix spatial remodelling process can provide strategies to tackle important pathologies such as tissue contractures and tissue adhesions or poor-integration. The final part of this study presents the development of an optical method, based on elastic scattering spectroscopy (ESS) with the potential of minimally invasive monitoring of tissue formation. Changes in ESS spectra obtained from fibroblast-populated lattices were correlated to cellular remodelling (i.e. contraction) measured as lattice compaction or development of tension. Additionally distinct spectra were obtained for different areas of the interface constructs, indicating the ability of the ESS method to discriminate different structure within the same construct. This study has presented an interdisciplinary model platform which can be useful for the development of a new generation of complex bioreactors in which tissue formation can be analysed under different simultaneous parameters (e.g. biochemical, mechanical and structural)