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Regeneration of decellularised tendon by human mesenchymal stem cells in response to uniaxial tensile strain

Sandora, Normalina (2016) Regeneration of decellularised tendon by human mesenchymal stem cells in response to uniaxial tensile strain. PhD thesis, University of Leeds.

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Abstract

Injury of ligaments is very common, and a total tear of the anterior cruciate ligament (ACL) demands replacement. Ideally, a graft for ligament replacement should be able to regenerate into a native like ligament, and restore physiological and anatomical function immediately after transplantation. The University of Leeds has developed a protocol to generate an acellular tendon scaffold derived from allograft/ xenograft using decellularisation technology. The aims of the study were to study the differentiation of human bone marrow derived multipotential stromal cells (BM-MSC) seeded onto the decellularised porcine patella tendon scaffold in response to cyclic tensile strain. Porcine patellar tendons were harvested and decellularised using the Leeds protocol. The decellularised tendon scaffolds were then characterised to determine their: (i) acellularity, (ii) histoarchitecture, (iii) extracellular matrix components (iv) levels of DNA (v) sterility, and (vi) biocompatibility. The decellularisation protocol was found to have minimal effect on the tissue histoarchitecture, and consistently generated sterile, non-toxic acellular scaffolds among different batches, with 98 – 99 % DNA removal compared to native tissue. Both porcine and human BM-MSCs were characterised using a range of antibodies to CD markers and trilineage differentiation and shown to have properties consistent with multipotential stromal cells. Human BM-MSCs were seeded directly onto appropriately sized samples of porcine patella tendon scaffolds at 1 x 105 cells.cm-2 for 12 hours, and then transferred to culture wells of TenCell-1 (a physically interactive rig for delivery of cyclic tensile strain). The cell-seeded tendon scaffolds were cultured either statically or with 4, 6 or 8 % cyclic tensile strain for 4/24 hours at 1 Hz, for 7 days. The response of the cells to cyclic tensile strain was investigated using viability assays (Live/Dead assay and ATPlite™ assay), histology, immunohistochemistry and gene expression analysis (RT-qPCR and gel electrophoresis). The mechanical properties of the scaffolds before and after incubation with cells were determined using uniaxial tensile testing. Under 4, 6 and 8 % cyclic tensile strain, the cell seeded scaffolds had a histological appearance of tendon-like tissue. When cultured under 6 and 8 % cyclic tensile strain, there was evidence that the MSCs were differentiating into tenogenic cells by expressing scleraxis, tenascin C, collagen I and collagen III genes. Cell-seeded scaffolds cultured under 6 % cyclic tensile strain had the highest viability and the matrix stiffness, was significantly increased compared to cell-seeded scaffolds cultured at 4 or 8 % cyclic tensile strain. The cell-seeded scaffolds incubated statically for 7 days showed matrix disorganization, had lower cell viability and less cell infiltration compared to samples incubated with cyclic strain.

Item Type: Thesis (PhD)
Keywords: ACL, decellularisation, ligament tissue engineering, mesenchymal stem cells, mechanotransduction,tenogenesis
Academic Units: The University of Leeds > Faculty of Engineering (Leeds) > School of Mechanical Engineering (Leeds)
The University of Leeds > Faculty of Engineering (Leeds) > School of Mechanical Engineering (Leeds) > Institute of Medical and Biological Engineering (iMBE)(Leeds)
Depositing User: Dr Normalina Sandora
Date Deposited: 09 Mar 2017 12:09
Last Modified: 09 Mar 2017 12:09
URI: http://etheses.whiterose.ac.uk/id/eprint/16429

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