Surgeons performing revision hip joint operations are faced with a growing number of patients with extensive bone loss. This results in insufficient bone for the attachment of soft-ligamentous tissues which are important in maintaining normal joint function. The aim of this project was to develop methods for studying the attachment of soft-tissues to prosthetic implants with a view to tissue engineering the interface in vitro. Cells were isolated from the porcine medial collateral ligament (pMCL) and characterised by indirect immunofluorescent labelling using a panel of antibodies. Porcine MSCs isolated from femoral bone marrow were also characterised by fluorescent activated cell sorting and their capacity to undergo tri-lineage differentiation. The population of pMCL cells was a mixed population of fibroblasts/myofibroblasts and smooth muscle cells. The smooth muscle cells were removed from the population by magnetic activated cell sorting. The porcine anterior cruciate ligament (pACL) interface was characterised using histology and immunohistochemistry. The pACL interface was of a fibrocartilagenous nature, consisting of the ligament, uncalcified fibrocartilage, calcified fibrocartilage and bone. Segments of pMCL were cultured under a range of static conditions for 14 days and analysed for cell viability by the ATPlite-MTM assay and Live/Dead cell staining. The ligament tissue could not be viably maintained. Porcine patella tendon tissue was studied as an alternative and the results indicated that the patella tendon was 60 % viable after 14 days in organ culture. Titanium porous coated (Gription®, Porocoat® and Porocoat®/Gription®) and foam discs of different pore sizes were fabricated and characterised in terms of their ability to enable cell/tissue in-growth. To determine the pore size/volume porosity by microCT a novel method of metal suppression was developed and applied successfully. The biocompatibility of the discs was assessed using 3 types of cells and they were shown to be biocompatible. The pMCL fibroblasts and pMSCs were then seeded to the discs to determine the optimal cell/disc type in terms of in-growth and proliferation (ATPlite-M™ assay) over 21 days. There was significant ingrowth in the Porocoat® and Porocoat®/Gription® coated discs for both pMCL cells and MSCs. The methods developed and results of this study will enable future experiments to culture the ligament tissue in close contact with the optimum cell/disc scaffold type under strain in a cyclic tensile strain bioreactor (TenCell) in order to determine the effect of tensile strain on the attachment of the ligament to the scaffold and the formation of an interface like tissue.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:658608 |
| Date | January 2013 |
| Creators | Vayianos, Vassilis |
| Publisher | University of Leeds |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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