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Abstract
Study Design / Objectives:
To investigate whether non-viral telomerase gene therapy can extend cellular lifespan while retaining functionality of cells in a safe manner. Cellular lifespan, function, karyotypic stability and transformation properties were assessed.
Furthered by the evaluation of human disc cell chemotactic potential as well as the recruitment ability of therapeutic growth factor, BMP-13, by the use of Boyden chamber assays.
Background:
Degeneration of the intervertebral disc is an age-related condition associated with the loss and deterioration of cells responsible for the maintenance and health of the disc. Telomerase can extend the cellular lifespan and function of other musculoskeletal tissues such as the heart, bones and connective tissues. Therefore extension of the cellular lifespan and matrix production of intervertebral disc cells may have the potential to delay the degeneration process. Further, the recruitment of disc cells from local non-degenerated regions may have regenerative purposes as well as enhancements for tissue/scaffold integration.
Methods:
Ovine nucleus pulposus cells were lipofectamine transfected in vitro with the hTERT-transgene. Cellular lifespan and matrix transcript levels were determined by cumulative population doublings and real time RT-PCR, respectively. G1-cell cycle checkpoint, p53 functionality, growth of transfected cells in anchorage-independent or serum starvation conditions and karyotypic analysis were performed. Human disc cells from all three regions were tested simultaneously for their chemotactic ability using the Boyden chamber assay with foetal bovine serum or BMP-13 as the chemoattractant.
Results / Conclusions:
This thesis has posed two novel therapeutic avenues for the degeneration of the intervertebral disc. By the rejuvenation of existing cells or the recruitment of local disc cells for the potential restoration of the intervertebral disc. Telomerase can extend the cellular lifespan of nucleus pulposus cells and prolong the production of extracellular matrix. Safety was unresolved, as karyotypic instability was detected but no loss of contact inhibition, mitogen dependency or G1-cell cycle checkpoint control was evident. Human endplate, annulus and nucleus pulposus cells all possess chemotactic abilities and BMP-13 showed a significant chemotactic effect for nucleus pulposus cells at levels similar to 5% foetal calf serum, a multi-component chemoattractant.