Long-Term Banking of Vascularized Composite Grafts Using Ice-Free Cryopreservation by Vitrification and Nanowarming Technologies
Abstract
This proposal targets the development of ice-free cryopreservation and rapid rewarming methods for the preservation of vascularized composite tissues, such as muscle flaps, digits, faces, and limbs, to radically improve restorative and reconstructive medicine. In the United States there are approximately 150,000 civilian limb amputations performed annually in addition to high profile military cases. Over 4 years ago, more than 1,000 military men and women had lost an arm or leg in the conflicts in Iraq and Afghanistan and 20 percent had lost two or more limbs. Hand surgeons are often called upon to reattach severed parts such as fingers, thumbs, hands, forearms, and more recently faces. These complex tissues have recently been included as organs by the Department of Health and Human Services in the definition of organs governing the operation of the US Organ Procurement and Transplantation Network. It is well established in the scientific literature that cryopreservation methods that permit ice to form in organ tissues result in cell death and extracellular matrix damage if warming is too slow. Ice formation can be prevented by the use of very high concentrations of chemicals called cryoprotectants in ice-free cryopreservation methods; however, again if the warming rate is too slow, the cells will die due to cryoprotectant toxicity. Use of ice-free cryopreservation methods has been limited to small structures, 3 milliliters or less in volume, because we were not able to warm large samples fast enough to prevent either ice formation or cryoprotectant toxicity. Our preservation and warming technology may be used to establish banks of organs from donors for best match transplantation. Until recently, this proposal would not have been feasible; however, the convergence of new analytical techniques that permit visualization of events inside organs during cryopreservation procedures and innovative new warming methods enable the development of radical new organ cryopreservation techniques employing ice-free preservation methods. Dr. Brockbank has previously demonstrated that his ice-free cryopreservation method can preserve 80%-100% of cell viability and functions in a variety of individual tissues. Dr. Bischof has developed a novel, innovative, tissue warming method using low radiofrequency wave lengths combined with coated iron nanoparticles that are distributed around and within tissues to focus rapid controlled heat generation. Dr. Brandacher is part of one of the most experienced clinical and research teams for human extremity and animal limb model research. The ability of radiofrequency rewarming of nanoparticles to effectively rewarm ice-free cryopreserved rat limbs will be studied in three specific aims. In Aim 1, the lead ice-free cryopreservation method from the Brockbank group will be scaled up to large volumes for rat hind limb preservation in combination with different concentrations of iron (magnetic) nanoparticles produced by the Bischof group. A range of low radiofrequencies will be evaluated for rewarming and the best combinations of radiofrequency and nanoparticle concentration compared with state-of-the-art cryopreservation by freezing and untreated controls. In Aim 2, the Brandacher group will implant radiofrequency rewarmed limbs in genetically identical rats to permit investigation without the complications of immunological rejection. In Aim 3, the Brandacher group will perform transplant studies in which the rats are from genetically different strains simulating the reality of human organ transplantation. In these aims, rat limbs will be procured from animals under the supervision of Dr. Helke at the Medical University of South Carolina after the procurement team has been trained by the Brandacher group. The limbs will be cryopreserved by the Brockbank group in Charleston using nanoparticles produced by the Bischof group. Rewarming will be supervised remotely by the Bischof group from Minn
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jan 31, 2017
- Source ID
- W81XWH1610508
Entities
People
- Kelvin Brockbank
Organizations
- United States Army