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Title: Development and Characterisation of an Acellular Porcine Medial Meniscus for use in Tissue Engineering.
Author: Stapleton, Thomas William
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2007
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The meniscus is a fibrocartilage structure found within the knee joint whose functions. include; stress distribution, load transmission, stability and lubrication ofthe joint. Demand for a suitable, 'off the shelf meniscal replacementhas increased considerably due to the rising number of injuries to the knee. This predisposition to meniscal injury relates to the limited vascularisation of the tissue resulting in restricted access of necessary repair factors. Damage to the avascular areas of the meniscus may lead to loss of function, compromising the tribological function of the knee joint. Allografts represent the gold standard in meniscal replacement, but problems related to foreign body reaction, sizing/shrinkage of the implant and lack ofchondroprotection, have yet to be solved. Synthetic products such as the Collagen Meniscal Implant™ are available which allow repair of large defects, however, this only functions when the exterior meniscal rim is intact and has been associated with poor biomechanical functioning. The overall aim of this project was to develop and evaluate the potential ofan acellular porcine medial meniscus for use as a biological matrix for a tissue engineered meniscal substitute. Following several iterations, whole porcine medial menisci were decellularised by subjecting the tissue to freeze thaw cycles, incubation in hypotonic tris buffer, 0.15% (w/v) sodium dodecyl sulfate in hypotonic buffer plus protease inhibitors, nucleases, hypertonic buffer followed by disinfection using 0.1% (v/v) peracetic acid and final washing in , phosphate buffered saline. The presence or absence ofcells was confirmed histologically, whilst DNA levels were qualitatively and quantitatively assessed using a range of molecular techniques including agarose gel electrophoresis and nano-spectrophotometry (amount ofDNA). Histological analysis demonstrated an absence of cells within the decellularised tissue whilst DNA levels were less than 2 . Histologica.l, immunohistochemical and biochemical analysis (collagen and glycosaminoglycans) of the decellularised tissue in comparison to the fresh porcine medial meniscal tissue confirmed the retention of the major structural proteins (collagen: fresh, 83.4% (± 13.9,95% confidence limits [CLl) (w/w); decellularised, 74.3% (± 21.7,95% iii CL). There was however a 59.4% loss ofglycosaminoglycans as determined by the sulfated/carboxylated sugar assay. In addition, electron microscopy allowed visualisation of areas ofsynovial-like membrane damage following decellularisation. Biomechanical properties were determined by indentation and uni-axial tensile tests which confirmed the' retention of biomechanical function following decellularisation. Biocompatibility of the acellular scaffold was determined using in vitro contactand extract cytotoxicity tests. Unfortunately, deep meniscal tissue was found to be cytotoxic. Modifications to the original decellularisation protocol were incorporated to overcome this problem. These included, reduced sodium dodecyl sulfate concentration (0.1% (w/v)), reduced detergent wash temperature (45°C) and extended terminal buffer wash. Consequently, decellularised tissue and extracts were found not to be cytotoxic to cells. Residual sodium dodecyl sulfate present within the acellular scaffold was determined using radio labelled 14C sodium dodecyl sulphate (0.022% (± 0.08, 95% CL) (w/w) SDS), whilst differential scanning calorimetry demonstrated retention of the thermal stability of the decelhilarised meniscus (denaturation enthalpy: fresh outer meniscus, 510.4 J.g-1 (± 153.19, 95% CL); fresh inner meniscus 399.23 Ig-I (± 99.4, 95% CL);--deceIlularised outer meniscus, 492 J.g-1 (± 73.99, 95% CL), decellularised outer meniscus, 283.86 J.g-1 (± 115.75,95% CL). Immunohistochemistry and antibody adsorption assays showed no evidence ofthe expression of the major antigenic epitope, galactose-a-I,3-galactose in the decellularised porcine medial meniscus. The biocompatibility of the acellular scaffold was assessed in vivo by subcutaneous implantation into galactosyl-transferase knockout mice. The explanted tissue was assessed for foreign body response (capsule thickness), cellular _infiltr~te phenotype (F4/80, CD3, CD4, CD34 and C3c), as well as the presence of antigalactose- a-I,3-galactose antibodies in the mouse serum. A non-specific foreign-body response was observed following implantation of the decellularised porcine medial meniscal tissue. Preliminary investigations into in vitro recellularisation of the acellular medial meniscal scaffold demonstrated the successful attachment ofprimary human dermal fibroblasts and primary porcine meniscal cells. In addition, primary human dermal fibroblasts were able to iv infiltrate the scaffold following seven days in culture. Studies into the potential incorporation of bone blocks demonstrated almost complete cell removal following application of the established decellularisation treatment, whilst the galactose-a-l ,3galactose epitope was not detected by immunohistochemistry and antibody adsorption assay. In conclusion, this study has generated preliminary results into the production of a biocompatible, biomechanically functional scaffold which has excellent potential for development ofa tissue engineered solution to meniscal repair.
Supervisor: Not available Sponsor: Not available
Qualification Name: University of Leeds, 2007 Qualification Level: Doctoral
EThOS ID:  DOI: Not available