title: Growth by stretch: an interdisciplinary approach
Creators: name: Unadkat, Ricky Ashok
Publisher: name: University of Glasgow
Year: 2020
Subjects: subject: mechanotransduction, plastic flow, soft tissue, fibroblasts, cell sheets.

description: Tissue expansion is a technique used by plastic and restorative surgeons to cause the body to grow additional skin, bone or other tissues. Distraction osteogenesis (DO) is an example of tissue expansion which has been widely applied in lower limb surgery (trauma/congenital), and congenital upper limb reconstruction (e.g. radial dysplasia). This complex and tightly regulated expansion process has resulted in adverse effects such as severe soft-tissue contractures and loss of nerve function as well as microtrauma and micro-haematoma formation (Natu et al., 2014). Thus far, the procedure can only be optimised by long-term animal or human experimentation. This thesis explains the development of an in vitro model that will allow extension regimes (µm/h, continuous/ intermittent) and molecular pathways involved in soft tissue damage related to DO to be explored. Cells cultured onto polycaprolactone (PCL) polymer films can be stretched at very low, adjustable speeds, using a stepper motor and various 3D printed and laser cut designs. The idea here is that plastic flow of PCL can be utilised to enable the material to stay extended upon strain being released, to represent permanent stretching of soft tissue. PCL film for the purposes of this project was made using a solvent in conjunction with a spin-coating process; A semi-crystalline and amorphous derivative of the polymer was made (C-PCL and A-PCL respectively). Testing the two polymer sheets indicated that C-PCL is a more rigid material and that strain occurs in more localised regions when it is stretched in comparison to A-PCL. The profile of the stress-strain curve for both C-PCL and A-PCL closely resemble that of a typical soft tissue after it has passed its yield point (33% strain). Due to the known involvement of fibroblasts in mechanical loading of tissue (B. Hinz, 2004), they were used as an initial cell line to develop an in vitro model for growth by stretch. Both A-PCL and C-PCL were used as substrates and were stretched passed their yield point (33% strain) before cells were cultured on. Following fibroblast proliferation to confluency substrates were further stretched by 1mm (2.5% strain) over 24 hours (stepped stretching at 0.04mm per hour). Orientation analysis indicated that cells grown on C-PCL initially elongate and orient to the direction of pre-stretch (when substrates are initially stretched passed their yield point), then contract upon being further stretched by 1mm over 24 hours. Cells cultured on A-PCL, under the same stretching regime initially align to the direction of pre-stretch; after being further stretched by 1mm the majority of cells remain aligned, but also elongate in the direction of stretch. Initial alignment on both materials was deemed a result of tension in the material and/or or topographical features which formed during stretching of substrates before cells were cultured on. The alignment was more pronounced on the C-PCL substrate and cell nuclei were analysed to be more elongated indicating the topography caused the fibroblasts to reside in a stressed state. This aligned cell effect was lost on C-PCL during further 1mm stretching due to; stress relaxation after each step of stretching; and/or localised strain regions causing cells to round during the stepped 1mm stretch. A-PCL was further investigated as a substrate to model soft tissue expansion in relation to DO where MRTF-A nuclear translocation was shown to increase in response to stretch (by 3-fold). F-actin texture analysis further implied cytoskeletal involvement in the stretching regime utilised for this project. Based on the results obtained, it was concluded that A-PCL with the stretching regime detailed (where plastic flow is utilised), provides the basis for a representative in vitro model of stretching soft tissue in relation to DO. Future work outlined to build on this model would be to: further investigate the relation between strain and cell response at the cell level for both materials using live imaging (in conjunction with fiducial markers in the substrate) and atomic force microscopy methods; and to develop understanding of extracellular matrix (ECM) interactions with cells in response to the stretching in the plastic flow region by again using live imaging methods (fluorescently tagging ECM components).