Biomechanics and remodeling of free-floating tissue equivalents

Since its introduction in 1979, the free-floating fibroblast-populated collagen lattice (FF-FPCL) has been employed in hundreds of studies examining cell-matrix interactions and cell contractility mechanisms. While it has been shown in constrained tissue equivalents that fibroblasts seek to develop and maintain a preferred mechanical environment, little work has been done to mechanically characterize the free-floating construct as it was assumed, since the lattices are unconstrained, cells cannot develop tension in the matrix. Similarly, the FF-FPCL is often used to examine remodeling during the wound healing process, yet very few studies have been carried out to determine how cells "lock-in" or entrench the matrix after actively remodeling it. The overall goal of this dissertation is to shed light on the mechanical environment of the FF-FPCL and to determine the mechanisms by which cells remodel and entrench the extant matrix to increase its utility as a model system for mechanobiological studies.;First, initiation of collagen lattice compaction was examined mechanically using an incompressibility assumption and traction-free radial and axial boundary conditions. It was found that radially varying material properties and active cell stresses were required to allow for nontrivial solutions and that a residual-type stress field would develop with equibiaxial compression in the central region transitioning to circumferential tensile stress in the periphery of the lattice. Next, the mechanical framework was adjusted to allow for compressibility as the FF-FPCL undergoes a significant change in volume during culture. Again, it was shown mathematically that the experimentally observed deformations are possible when considering radially varying material properties and active cell stresses and this results in the development of a residual-type stress field. The presence of residual stresses was verified experimentally by applying a radial cut from the center of the gel to the outer edge the relieve the circumferential stresses, which resulted in the development of an opening angle, and the application of a circular punch in the central region, which resulted in an equibiaxial contraction of the applied hole, indicative of the release of an equibiaxial compressive stress.;To examine the remodeling and entrenchment of the FF-FPCL, the role of covalent crosslinkers, namely tissue transglutaminase (tTG) and lysyl oxidase (LOX), was examined, as well as the contractile mechanisms (Ca2+-dependent vs. -independent) by which the cells actively deform the matrix. It was found that tTG was necessary for normal FF-FPCL remodeling at early times (through 2 days) and the preferred contractile mechanism up to day 1 appeared to be Ca2+-dependent. While LOX was not necessary in early remodeling, its inhibition reduced further compaction at later times (3 to 4 days in culture). Similarly, there was a shift towards Ca2+-independent contractility dominating at later times.;Finally, a growth & remodeling (G&R) theory-based constrained mixture model was developed to computationally examine the evolution of the first 2 days of culture of the FF-FPCL when little to no new matrix deposition occurs. This approach employs the idea that the cells seek to develop a preferred level of stress and remodel the matrix in an effort to achieve this homeostatic value and that remodeling of the extant matrix results in a composite of original and remodeled matrix that are constrained to deform together. With this approach, the gross geometric evolution of the FF-FPCL can be modeled including the development of opening angles in the reference configuration. This model also provides the first approximation of how the stresses within the FF-FPCL can evolve over time as the resident cells seek to establish their preferred mechanical environment.;In conclusion, this work provides a mechanical framework for the FF-FPCL and its variants that accounts for prior experimental observations related to cell and matrix orientations and provides experimental verification of the presence of a residual-type stress field. This mechanical framework allows for radially varying cellular responses to be correlated to the mechanical environment and improves the utility of the FF-FPCL in mechanobiological investigations. The results related to tTG and LOX inhibition provide a potential timeline for how cells initially remodel extant matrix in an attempt to quickly develop or restore a preferred mechanical environment. Finally, the computational model provides the first model of FF-FPCL evolution to account for the changing mechanical environment and the entrenchment of the matrix and provides a hypothesis testing framework to guide future cell-matrix interaction studies utilizing the FF-FPCL. Future investigation related to this work includes the refinement of the computational model as new experimental data become available and implementation of this framework in biaxially constrained tissue equivalents to allow for precise control of the mechanical environment.