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 °C (N = 3). Methods were then repeated with 6.5 M formamide at one of three temperatures: 4 °C, 22 °C, 37 °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 × 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.

Methodology for clinical genotyping of CYP2D6 and CYP2C19.

Many antidepressants, atomoxetine, and several antipsychotics are metabolized by the cytochrome P450 enzymes CYP2D6 and CYP2C19, and guidelines for prescribers based on genetic variants exist. Although some laboratories offer such testing, there is no consensus regarding validated methodology for clinical genotyping of CYP2D6 and CYP2C19. The aim of this paper was to cross-validate multiple technologies for genotyping CYP2D6 and CYP2C19 against each other, and to contribute to feasibility for clinical implementation by providing an enhanced range of assay options, customizable automated translation of data into haplotypes, and a workflow algorithm. AmpliChip CYP450 and some TaqMan single nucleotide variant (SNV) and copy number variant (CNV) data in the Genome-based therapeutic drugs for depression (GENDEP) study were used to select 95 samples (out of 853) to represent as broad a range of CYP2D6 and CYP2C19 genotypes as possible. These 95 included a larger range of CYP2D6 hybrid configurations than have previously been reported using inter-technology data. Genotyping techniques employed were: further TaqMan CNV and SNV assays, xTAGv3 Luminex CYP2D6 and CYP2C19, PharmacoScan, the Ion AmpliSeq Pharmacogenomics Panel, and, for samples with CYP2D6 hybrid configurations, long-range polymerase chain reactions (L-PCRs) with Sanger sequencing and Luminex. Agena MassARRAY was also used for CYP2C19. This study has led to the development of a broader range of TaqMan SNV assays, haplotype phasing methodology with TaqMan adaptable for other technologies, a multiplex genotyping method for efficient identification of some hybrid haplotypes, a customizable automated translation of SNV and CNV data into haplotypes, and a clinical workflow algorithm.

Evaluation of five additives to mitigate toxicity of cryoprotective agents on porcine chondrocytes.

Cryoprotective agents (CPAs) are used in cryopreservation protocols to achieve vitrification. However, the high CPA concentrations required to vitrify a tissue such as articular cartilage are a major drawback due to their cellular toxicity. Oxidation is one factor related to CPA toxicity to cells and tissues. Addition of antioxidants has proven to be beneficial to cell survival and cellular functions after cryopreservation. Investigation of additives for mitigating cellular CPA toxicity will aid in developing successful cryopreservation protocols. The current work shows that antioxidant additives can reduce the toxic effect of CPAs on porcine chondrocytes. Our findings showed that chondroitin sulphate, glucosamine, 2,3,5,6-tetramethylpyrazine and ascorbic acid improved chondrocyte cell survival after exposure to high concentrations of CPAs according to a live-dead cell viability assay. In addition, similar results were seen when additives were added during CPA removal and articular cartilage sample incubation post CPA exposure. Furthermore, we found that incubation of articular cartilage in the presence of additives for 2 days improved chondrocyte recovery compared with those incubated for 4 days. The current results indicated that the inclusion of antioxidant additives during exposure to high concentrations of CPAs is beneficial to chondrocyte survival and recovery in porcine articular cartilage and provided knowledge to improve vitrification protocols for tissue banking of articular cartilage.