Biophysical and biochemical microenvironmental cues for tenogenic phenotype maintenance, differentiation and trans-differentiation in vitro
Ryan, Christina
Ryan, Christina
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Publication Date
2019-07-19
Type
Thesis
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Abstract
Tendon injuries affect tens of millions of people worldwide each year and as the average lifespan increases their occurrence also increases, Achilles rupture rates alone have increased by ≈ 1000% in the last 40 years. Current treatments most often fail to restore tendon function to pre-injury levels, thus cell-based strategies aim to engineer functional tendon tissue in vitro to replace damaged suboptimal tendon tissue. However, in standard in vitro conditions the tenogenic phenotype is notoriously unstable, therefore it is critical to engineer the optimal in vitro niche for the maintenance of the tenogenic phenotype or the differentiation of stem cells and trans-differentiation dermal fibroblasts towards the tenogenic lineage. In this work, biophysical cues (electric field stimulation, substrate stiffness, surface topography) were engineered and investigated for their suitability for use in a multifactorial in vitro culture system. Subsequently, the effect of combined biophysical (substrate stiffness, surface topography, macromolecular crowding) and biochemical (collagen type I coating) cues on the behaviour and phenotype of permanently differentiated cells (human tenocytes and human dermal fibroblasts) and stem cells (human mesenchymal stem cells) was assessed. Two different electric field stimulation apparatuses (two-point electrode system, parallel wire electrode system) were fabricated and their suitability for the induction of cellular alignment was assessed. However, both apparatuses developed were unsuccessful in inducing the cellular alignment of dermal fibroblasts in vitro. Therefore, as an alternative strategy, PDMS substrates at a range of stiffnesses (1000 kPa, 130 kPa, 50 kPa, 30 kPa, 15 kPa) with a planar and grooved surface topography and collagen type I coating (0 mg/ml, 0.1 mg/ml, 0.5 mg/ml, 1 mg/ml) were fabricated and characterised. PDMS substrates of 30 kPa and 15 kPa failed to retain the topographical pattern of the silicon template and were not used for further investigation. 1,000 kPa, 130 kPa and 50 kPa PDMS substrates with uniform grooved and planar surface topographies and a 0.5 mg/ml collagen type I coating were found to be suitable for cell culture for up to 14 days. Finally, the influence of 1000 kPa, 130 kPa and 50 kPa PDMS substrates, with and without collagen coating, with and without anisotropic topography and with and without a macromolecular crowding agent (carrageenan) on human tenocytes, human bone marrow stem cells and human dermal fibroblast function was assessed. The substrate stiffnesses investigated did not show any clear effect on cellular morphology, matrix deposition or gene expression. The grooved surface topography induced the alignment and elongation of all cell types parallel to the groove direction, leading to the alignment of fibronectin. Collagen type I coating promoted overall cell adhesion and the deposition of various extracellular matrices dependent on cell type. Carrageenan accelerated the deposition of collagen I, III, IV, V and VI, however it reduced fibronectin deposition by tenocytes and dermal fibroblasts. Carrageenan also reduced cell number and FAK concentration and altered the aligned, fibrillar structure of the deposited matrix. Nonetheless, the macromolecular crowding agent had a robust effect on gene expression, particularly in hBMSCs by accelerating differentiation. Collectively, this work provides further knowledge on the combined use of biophysical and biochemical microenvironmental cues to engineer the tenogenic niche in vitro.
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NUI Galway
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Attribution-NonCommercial-NoDerivs 3.0 Ireland