Radiofrequency heating preserved cells in vitrified porcine cartilage during rewarming
Radiofrequency heating preserved cells in vitrified porcine cartilage during rewarming
On July 16, Biofabrication published an accepted manuscript describing an experiment with porcine articular cartilage. A piece measuring about 15 × 10 × 2,5 mm was vitrified in 10 ml of cryoprotectant solution. After rewarming with an electric field, about 90% of the cells remained viable throughout the full thickness of the cartilage.
Vitrification converts water in tissue into a glass-like state and prevents ice crystals from forming. The greatest risk comes during rewarming. A water bath heats the outer regions of the cartilage quickly, while the center remains colder. This temperature difference creates mechanical stress and gives ice time to grow.
Articular cartilage presents a second challenge because it has no blood vessels. The cryoprotectant solution VS55 must diffuse from the surface to the center to protect the cells during cooling and rewarming. A higher concentration reduces the risk of ice formation but damages cells. After a three-hour stepwise loading procedure, the authors achieved more than 85% of the target concentration at the center of the cartilage sample.
A controlled cryoprotectant concentration gradient has already helped cells, oocytes, and skin samples about 2 mm thick survive thawing. In the new experiment, the solution had to diffuse through 2,5 mm of avascular cartilage.
The sample and the surrounding solution were then placed between electrodes. A 40 MHz field heated the cryoprotectant solution throughout the system, including the solution inside the cartilage. Polar molecules in the solution moved with the oscillating field and converted its energy into heat. The equipment accounted for changes in the electrical properties of the solution and adjusted the power accordingly. Within the critical temperature range, the difference between the edge and the center remained below 10 °C. The water bath produced a difference of about 76 °C.
The water bath rewarmed the system containing the cartilage in 10 ml of solution at about 31 °C per minute. The radiofrequency system achieved about 110 °C per minute. After water-bath rewarming, cell viability at the center of the cartilage samples fell to about 60%. After radiofrequency rewarming, about 90% of the cells remained viable throughout the full thickness. By the third day in culture, metabolic activity exceeded 70% of the level measured in fresh control cartilage.
In experiments with smaller cartilage discs, radiofrequency rewarming preserved more than 90% of the glycosaminoglycans, which are matrix components that retain water and help cartilage withstand mechanical loads. After the procedure, elasticity and permeability were close to those of fresh cartilage.
Earlier work from the same research program used magnetic nanoparticles to heat cartilage. The molecules in the cryoprotectant solution are about an order of magnitude smaller than the particles and can penetrate the dense matrix more easily. In the new approach, the field heats the solution that has already entered the tissue.
The authors tested porcine cartilage samples and observed them in culture for five days. Heating a system with a volume of up to 65 ml has so far been assessed only through calculations. The authors did not transplant the treated material. Several coauthors are affiliated with TDA Research, and patent applications have been filed for the system and the loading protocol.
Vitrification of an entire rabbit brain using the M22 solution also preserved fine structural detail after rewarming. The cartilage experiment adds measurements that are necessary for dense tissue: whether the cryoprotectant reached the center and how far the center lagged behind the surface in temperature.