Optimization of Vitrification for Human Articular Cartilage

• Author / Creator

  Hahn, Joshua N

• Osteoarthritis, a disease resulting in the breakdown of cartilage and bone within joints, is a global burden that is growing in scope. There is no cure for osteoarthritis, and the current treatments are all lacking in some form or another. One treatment which attempts to prevent degeneration of cartilage injury into osteoarthritis is osteochondral allografting. This surgery involves the transplantation of healthy bone and cartilage to replace damaged and diseased areas. Osteochondral allografting application is limited primarily by the supply of fresh, healthy tissue and the lack of a long-term storage method that maintains cell viability within cartilage. Vitrification is a method of cryopreservation that preserves cells and tissues at temperatures low enough to halt all biological activity while maintaining cell health when applied properly. Previous work within this lab resulted in successful vitrification of human articular cartilage, but there is room for improvement. The current research was performed to explore the use of additive compounds as well as the use of a vitrification protocol with altered cryoprotectant exposure criteria in an attempt to improve the post-warmed health of the cryopreserved cartilage tissue. The use of chondroitin sulphate, tetramethylpyrazine, a combination of these two, ascorbic acid, and glucosamine was investigated in a set of cryoprotectant toxicity mitigation experiments. We found that when evaluating the effect of exposure to these compounds in a toxic cryoprotectant solution coupled with a two-day incubation, that all but the chondroitin sulphate alone were capable of improving tissue health, while there were no benefits seen when evaluated before the incubation period. The use of additive compounds has been shown to reduce long-term deleterious effects of CPA exposure, indicating that their use may be beneficial to a vitrification application due to the high CPA concentrations involved. This thesis also experimentally explored an altered cryoprotectant protocol proposed by another student, Nadia Shardt, who used Fick’s 1-D law of diffusion to determine the minimum time required for the diffusion of cryoprotectants into articular cartilage in concentrations that were adequate for vitrification. These modifications reduced the protocol length by one and a half hours, but did not result in viability results that were significantly improved over the standard protocol. As the experimental trials in this thesis work all produced a recovery cell viability that is much lower than the previously published results for the standard vitrification protocol, no conclusions can be made regarding which protocol to use based on these data. The experimental groups did not have an obvious deleterious effect on cell viability and, therefore, the reduction in protocol time may be beneficial. The standard vitrification protocol for articular cartilage has shown good results. The experiments performed here demonstrate that there are two potential avenues that may be exploited to enhance cell recovery and streamline the technical aspects of the protocol. Further research should lead to improvements in the results of this protocol.

• Subjects / Keywords

  • Articular Cartilage
  • Vitrification
  • Cryopreservation

• Graduation date

  Fall 2015

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3901ZQ1H

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.