Evaluation of the permeation kinetics of formamide in porcine articular cartilage

Cryopreservation of articular cartilage will increase tissue availability for osteochondral allografting and improve clinical outcomes. However, successful cryopreservation of articular cartilage requires the precise determination of cryoprotectant permeation kinetics to develop effective vitrification protocols. To date, permeation kinetics of the cryoprotectant formamide in articular cartilage have not been sufficiently explored. The objective of this study was to determine the permeation kinetics of formamide into porcine articular cartilage for application in vitrification. The permeation of dimethyl sulfoxide was first measured to validate existing methods from our previously published literature. Osteochondral dowels from dissected porcine femoral condyles were incubated in 6.5 M dimethyl sulfoxide for a designated treatment time (1 s, 1 min, 2 min, 5 min, 10 min, 15 min, 30 min, 60 min, 120 min, 180 min, 24 h) at 22 degrees C (N = 3). Methods were then repeated with 6.5 M formamide at one of three temperatures: 4 degrees C, 22 degrees C, 37 degrees C (N = 3). Following incubation, cryoprotectant efflux into a wash solution occurred, and osmolality was measured from each equilibrated wash solution. Concentrations of effluxed cryoprotectant were calculated and diffusion coefficients were determined using an analytical solution to Fick's law for axial and radial diffusion in combination with a least squares approach. The activation energy of formamide was determined from the Arrhenius equation. The diffusion coefficient (2.7-3.3 x 10-10 m2/s depending on temperature) and activation energy (0.9 +/- 0.6 kcal/mol) for formamide permeation in porcine articular cartilage were established. The determined permeation kinetics of formamide will facilitate its precise use in future articular cartilage vitrification protocols.