Novel osteochondral repair tissue engineering strategies are investigating the use of a single scaffold, with a portion for osteogenic and chondrogenic differentiation, and a single cell source, most notably the mesenchymal stem cell, to facilitate osteochondral differentiation and repair in a single construct. However, this approach requires robust differentiation protocols to ensure that the correct balance of each cell type is produced and maintained. Techniques used to analyse osteogenic and chondrogenic differentiation are well established, but many of the current methods described are qualitative, based on imaging stained cells or sections under a microscope. To facilitate higher throughput screening of chondrogenic differentiation in human MSCs, a novel culture technique using V shaped 96 well plates has been developed combining three robust and quantitative assays. Additionally, two reporter cell lines have been developed that express luciferase under the control of an osteogenic (osteocalcin) or chondrogenic (col2a1) promoter in order to streamline differentiation assays. The use of growth factors to elicit differentiation is well established; with BMP-2 used to enhance osteogenic differentiation and TGF-61 used to enhance chondrogenic differentiation. However, there are several limitations of using growth factors in regenerative medicine and consequently, the use of growth factor mimics was investigated. Two promising growth factor mimics were identified that could support both osteogenic and chondrogenic differentiation; LE135 and imperatorin. LE135, a retinoic acid receptor antagonist, significantly enhanced chondrogenesis with increased GAG production, col2a1 promoter activity and versican mRNA expression and had no significant effect on osteogenic differentiation. Imperatorin, a coumarin derivative, significantly enhanced early stage osteogenesis (alkaline phosphatase activity) and had no significant effect on late stage osteogenesis (mineralisation). Furthermore, chondrogenic differentiation was enhanced by imperatorin with significantly increased GAG production.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:727949 |
| Date | January 2015 |
| Creators | Prosser, Amy |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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