Cardiac valve replacement is the second most common heart operation in the Western World. Valve replacements currently available are poorly adapted for use in young patients who typically require multiple re-operations as they grow, resulting in increased morbidity and mortality. Tissue-engineering a living heart valve replacement would be an ideal substitute as it would not require drug therapies or re-operation. Bone marrow-derived mesenchymal stem cells have been shown to be capable of differentiating into a variety of lineages and may be advantageous as a cell\ud source for tissue engineering applications.\ud Attempts were made to isolate human and porcine mesenchymal stem cells (MSC) from the respective bone marrows using gradient-centrifugation to separate the cells and complete Dulbecco"s Modified Eagle's Medium (DMEM) to culture the cells. The cellular material from one bone marrow was transferred to one 25CM2 tissue Culture flask. Cell differentiation was attempted by supplementing complete culture medium with growth factors and biochemicals. The phenotype of the human MSC (hMSC) was then examined by FACS analysis. A previously developed decellularised porcine aortic valve matrix was biochemically characterised to ensure that the major matrix components had not been removed by the decellularisation procedure. Furthermore the\ud biocompatibility of the decellularised tissue compared to fresh tissue was assessed by subcutaneous implantation into mice (n--4). hMSC were then seeded onto the decellularised porcine aortic valve matrices in vitro and the ability of the cells to migrate into the tissue compared to smooth muscle cells in a static culture system was assessed.\ud Putative porcine MSC (pMSC) and hMSC were successfully isolated from their respective bone marrows. Putative pMSC were found to have a cell doubling time (CDT) of 106 hours and hMSC had a CDT of 151 hours. A failure to successfully\ud culture pMSC was found to be due to a change in the supplier of DMEM used. Attempts to differentiate porcine MSC (pMSC) produced adipogenic cells but failed to produce\ud osteogenic, chondrogenic, neurogenic, myogenic or smooth muscle lineage cells. hMSC were successfully differentiated into cells of the adipogenic, myogenic and neurogenic\ud lineage. However, attempts to clone the cells were unsuccessful. FACS analysis of hMSC indicated that the cells were CD45-,, CD13 +, D7FIB +/-, CD 105 +, CD 10 +/-, \ud LNGFR +/-, CD55 + , BNP- and AP+/-. Analysis of hydroxyproline, sulphated proteoglycan and DNA content of the decellularised porcine valve tissue indicated no change in collagen or GAG content and removal of cellular DNA. Implanted decellularised tissues were accepted by the mice in comparison to fresh tissue and appeared to be undergoing regeneration. Furthermore, the cell infiltrate into the matrices was favourable, being low in T-cells but more macrophages and endothelial cells. Seeding the decellulansed matrix with hMSC showed that the cells migrated into the tissue to up to 2% of the cell density found in native valve tissue compared to 0% of the\ud smooth muscle cells.\ud Failure to clone hMSC meant that differentiation could have been a result of multiple cell precursors being present in the bone marrow rather than a stem cell population.\ud Biochemical and biocompatibility analyses of the decellularised porcine valve matrix showed that the tissue was unaltered by the procedure and biocompatible. hMSC were\ud also observed to migrate into the tissue under static seeding conditions.\ud In conclusion,, mesenchymal stem cells represent a promising cell source for tissue engineering a living aortic heart valve. However, more research is required to further characterise the cells and optimise their growth and differentiation. The decellularised porcine valve matrix developed by shows potential as a matrix for tissue engineering an aortic heart valve.\u
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