Summary

Vitrification and nanowarming outperformed conventional methods for rescuing cartilage cells and maintaining viability.

Abstract

Introduction

Due to the limited resources of fresh cartilage allograft and the extended time needed for serologic and microbiologic testing and donor-recipient matching, an improved cartilage preservation technique is in a great need to further extend the implementation of the osteochondral allograft transplantation. Vitrification, a process that transfers a liquid solution into a glassy solid without ice formation, is a promising technique for tissue cryopreservation. The authors hypothesize that the combination of nanowarming with ice-free cryopreservation by vitrification will help scale up articular cartilage cryopreservation to larger specimen sizes while improving cell viability and maintaining cartilage matrix properties compared with conventional vitrified tissue warming using large articular cartilage specimens.

Methods

Femoral weight bearing condyles of sexually mature domestic Yorkshire cross pigs were obtained and no animals were sacrificed specifically for this study. Osteochondral pieces of the femoral condyle were harvested and separated into three groups: fresh, convection and nanowarming. The osteochondral pieces in the fresh group were kept in a Dulbecco’s minimum essential media while convection and nanowarming specimens were both vitrified ± 2 mg/mL Fe nanoparticles. The vitrified tissues were then cooled and rewarmed using either a two stage convective warming process while the nanowarming group was rewarmed in 80 seconds using a solid state induction power supply. Cell viability (using fluorescence live/dead staining with calcein AM and ethidium homodimer) and metabolic activity (using alamarBlue assay) were then assessed among the three groups.

Results

Fluorescence live/dead staining images revealed that an increased number of living cells with nanowarming cartilage compared to the convection warmed group. Cell metabolic activity increased in both the convection group and nanowarming groups; however significantly higher cell metabolic activity was found in the nanowarming group when compared to the convection group (p < 0.0001). No significant difference in conductivity was found between the fresh cartilage group, convection or the nanowarming group.

Discussion And Conclusion

The live/dead staining and cell activity results indicated that nanowarming outperformed the conventional convection warming method by rescuing more cells and maintaining cell activity for large cartilage cryopreservation. Nanowarming caused very limited alterations on the cartilage permeability with conductivity results as well. This study pioneers the application of nanowarming for large sized articular cartilage cryopreserved specimens. This biobanking method for cartilage preservation has the chance to improve the utilization of the limited supply of allografts, increase long term allograft survival in vivo, and extend the indications for osteochondral allograft transplantation.