Thesis

Studies on collagen stability and mechanical properties in tissue engineering applications

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Awarding institution
  • University of Strathclyde
Date of award
  • 2014
Thesis identifier
  • T13905
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Abstract
  • Objective: This project aims to measure the stiffness of collagen based gel matrix. This work provides mechanical study of 0.3 % collagen hydrogel monolayer properties under compression. In order to understand how the collagen gel properties change in relation with time and cell concentrations, the mechanical analysis was performed with different cell densities and time rates. They have been analysed before the culture (to report the stiffness characteristics of non-altered collagen gel) and during the culture, with the influence of the cells. Background: Cells are profoundly affected by the physical properties of the environment, including the stiffness of the matrix. The stiffness of cell adhesion substrates is increasingly appreciated as an important mediator of cell behaviour; it can regulate cell signalling broadly, with effects on growth, survival, differentiation and motility. The stiffness of ECM-based collagen gels can be manipulated, and so varied to be suitable for growing cells derived from soft tissues. However, the cells are capable of remodelling the gel, altering its stiffness during culture; this could damage the final culture characteristics, leading to altered cell properties. Methodology: The 0.3 % collagen hydrogel was prepared from collagen type I, obtained from rat tail tendons; it was then placed in the wells of a standard 24-well plate. The collagen gel monolayer was seeded with human hepatoma cells (HepG2) at different densities: 105, 5*104, 2.5*104, 104 and 0.5*104. Before testing the wells, the viability of the cells was ensured with the MTT assay. The normal sample was represented by a single well of collagen hydrogel which was not seeded with cells, but left for the same period of the culture filled with medium in the same incubator as the samples. The confined compression strength was measured through BOSE ElectroForce machine together with the WinTest software. The data acquired were analysed with Matlab software in order to evaluate the Aggregate Modulus (HA), Hydraulic Permeability (k), Coefficient of Permeability and Correlation Coefficient (r). Results and Conclusions: The data showed that the Aggregate Modulus has a general trend of increasing with culture time, except for the collagen gel without cells; the differences between culture durations are statistically significant just for the cases of 0.5*104 and 105 cells per cm2 cultures. Cells grow and increase in number during culture time, indeed cells in gels cultured for one day present a smaller cell concentration than the ones cultured for 5 days. The increase in number can contribute to increase in the Aggregate Modulus, therefore in the stiffness, since, as showed in the literature, the stiffness tends to increase with cells because they add resistance to the gel (Saddiq et al., 2008). It is generally true that cells act to weaken the collagen gel, but the rate in which they make it soft (by causing the secretion of degradative enzymes and by exerting mechanical forces) could be probably lower than the rate of cells growth, in the specific case of HepG2 cells. For this reason, the cell growth effect outweighs the effect of weakness. Seeding cells on collagen gels will usually lead to one of two possible effects on the Aggregate Modulus: the Aggregate Modulus can reduce which will be due to cells weakening the gels or the Aggregate Modulus can increase which can be due to physical presence of cells contributing to higher stiffness measurements. However, for HepG2 cell line, the increase in stiffness seems to be the predominant action. On the other hand, regarding the changes in stiffness during the different cell densities, the data acquired do not show a trend which can be interpreted with logical deductions.
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DOI
Date Created
  • 2014
Former identifier
  • 1041975

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