Effective cryopreservation of articular cartilage (AC) could improve clinical results of osteochondral allografting and provide a useful treatment alternative for large cartilage defects. Vitrification (a form of cryopreservation) incorporates high concentrations of cryoprotectant agents (CPAs) and rapid cooling rates to preserve cells in suspended animation without detrimental ice formation. Effective vitrification requires high concentrations of CPAs within the cartilage matrix but the time-dependent toxicity of CPAs hinders their usefulness. The objective of this experiment was determine the CPA permeation parameters for four commonly used CPAs. This data will enable the use of mathematical models to develop novel vitrification procedures to preserve AC. We hypothesised that the time dependency of CPA permeation into intact AC can be determined by exposing AC to CPAs for specific times and then allowing the CPA to be removed into a known volume of PBS, the osmolarity of which is then measured. Full thickness 10mm diameter osteochondral dowels were harvested from the medial femoral condyles of sexually mature pigs. The dowels were randomly immersed in one of four CPAs (DMSO, propylene glycol, ethylene glycol, and glycerol) for various lengths of time (1–15 min). The cartilage was then immersed in 4ml of 1X PBS in a sealed container for twenty-four hours. The equilibrated solution was measured for osmolarity. The cartilage was weighed before and after treatment and this data was used to calculate the CPA concentration within the AC. This will be repeated in triplicate. Preliminary results (minimum n=2) indicated a marked difference in permeation for the four CPAs. Ethylene glycol had the most rapid permeation with almost complete permeation (84%) within 15 min. Conversely, glycerol had the least permeation (29%) after 15 min most of which occurred within the first minute. DMSO (63%) and propylene glycol (40%) had intermediate rates of permeation that gradually increased over time. Cryoprotectant agent permeation into intact porcine AC can be calculated using the method described in this study. This will allow us to successfully document the permeation kinetics of four commonly used CPAs within intact AC. This valuable data will markedly improve our ability to create novel vitrification solutions using mathematical models to add and remove CPAs to limit their toxic effects at high concentrations.
Osteochondral allografts (frozen uncontrolled, or cryo-protected with dimethyl sulfoxide) were transplanted into medial femoral condyles of eighteen sheep. Cores from the ipsilateral graft site served as autografts for the contralateral limb. Analysis of graft and host cancellous bone microarchitecture by μCT at three months post transplant demonstrated no significant differences among the treatment groups. Dramatic bone resorption at the graft–host interface, however, occurred in up to 1/3 of condyles from all treatment groups, including fresh autografts suggesting that factors other than donor source or tissue storage played an important role in the bone incorporation of osteochondral grafts. The purpose of this study was to study the effect of different freezing protocols on periarticular cancellous bone architecture after osteochondral allograft transplantation. There were no significant differences in graft or host cancellous bone architecture among the groups (autografts, frozen allografts, cryopreserved allografts). Dramatic resorption of graft bone in condyles from all treatment groups suggested that factors other than donor source or tissue storage played important roles during incorporation of osteochondral grafts. Graft positioning, graft orientation, and recipient bed necrosis may play significant roles during incorporation of osteochondral graft bone. Osteochondral allografts (10 mm diameter) were transplanted into medial femoral condyles of eighteen skeletally mature Suffolk ewes. Allografts were frozen (–80°C) without cryoprotectant (FROZ) or treated with dimethyl sulfoxide (cryoprotectant) and frozen (–80°C at 1°C · min−1) (CRYO). Osteochondral cores removed from ipsilateral graft sites served as fresh autografts (AUTO) for the contralateral medial femoral condyles. Condyles were harvested at three months and scanned (micro computed tomography –μCT). Three dimensional μCT data of graft and host cancellous bone regions were analyzed for bone volume fraction, trabecular thickness, bone surface–volume ratio, and trabecular anisotropy. No morphological differences were found among treatment groups. Excessive bone resorption of graft and interface precluded analysis of some samples from each group (ALLO — 2/9, CRYO — 3/9, AUTO — 6/18). Dramatic bone loss did not correlate with poor graft orientation, placement, infection, or recipient–bed necrosis, but a combination of these factors may contribute to excessive cancellous bone resorption in osteochondral grafts.
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Cryopreserving agents (CPAs) can cryopreserve articular cartilage (AC) but their use is limited due to cellular toxicity. This study examined the time-dependent penetration of multiple CPAs into intact porcine AC. Porcine AC was immersed in CPAs for various amounts of time at three temperatures (4°C, 22°C, and 37°C). The results demonstrated an initial sharp rise in CPA concentration within the matrix for dimethyl sulfoxide and propylene glycol with maximum concentration after three to six hours. The trehalose and glucose concentration increased minimally even after twenty-four hours of exposure. The information from this study provides insight into the penetration kinetics of cryoprotectant agents into AC. This study examined the time-dependent penetration of cryoprotectant agents (CPAs) [dimethyl sulfoxide (DMSO), propylene glycol (PG), trehalose and glucose] into intact porcine articular cartilage (AC). Penetration of DMSO and PG into AC was rapid but time and temperature dependent while trehalose and glucose had poor penetration. The information gathered from this study can determine concentrations of CPAs within the cartilage matrix to create cryopreservation/vitrification solutions while minimizing toxicity. The results demonstrated there was a sharp rise in the CPA concentration within 15–30min exposure to DMSO and PG and the concentration peaked after three to six hours exposure at a concentration approximately 90% of the original concentration (6.5 molar). This was temperature dependent with slower penetration at lower temperatures. The trehalose and glucose had very poor penetration into the matrix at all temperatures, with a maximum penetration of 2% of the original concentration. Dowels of porcine AC (10mm diameter) were immersed in high concentration of each CPA for various amounts of time (0min, 15min, 30min, 60min, 3hr, 6hr, and 24hr) at three temperatures (4°C, 22°C, and 37°C). The cartilage was excised and the amount of cryoprotectant within the matrix determined. Successful cryopreservation of AC could improve clinical results of osteochondral allografting and provide a useful treatment alternative for large cartilage defects. However, successful cartilage cryopreservation is limited by chondrocyte death and matrix disruption due to inadequate CPA penetration.
Cryoprotectant toxicity has become more relevant because of increased use of high concentrations of cryoprotectants for vitrification of biologic tissues. A single toxicity model that integrates cryoprotectant concentration, time and temperature is essential to optimize the cryopreservation of tissues. The Weibull probabilistic distribution has been used in environmental toxicology research. This objective of this study was to fit the Weibull model to experimental data for chondrocyte recovery from articular cartilage exposed to various concentrations of dimethyl sulfoxide at different temperatures as a function of time. This study indicated that the Weibull model is an appropriate model to describe cryoprotectant toxicity to chondrocytes in articular cartilage. This study was designed to examine the toxicity of dimethyl sulfoxide (DMSO) on chondrocytes in porcine articular cartilage (AC) as a function of time, temperature and concentration. The Weibull model is suitable for modeling cryoprotectant toxicity in cartilage and can be further extended to other cellular and tissue systems. The model provides a simple method to predict toxicity and to assess the feasibility of cryopreservation protocols. The model proved to be a good fit for the entire data set of concentration, temperature and time, yielding an R2 value of 0.87 and a maximum discrepancy of 20% between the experimental data and the model. Estimates of the model’s parameters within a confidence interval of 95% were found to be: _=30±2, _=0.67±0.05, _C=0.38±0.03, _T=−2300±300 and _CT=700±100. Sliced porcine AC was exposed to DMSO (1, 3, 5, 6M) at different temperatures (0, 22, 37°C) for various durations. Cellular viability was determined by membrane integrity stains. Experimental data for chondrocyte recovery was fit to the global Weibull probabilistic distribution model using SPSS SigmaPlot 2000 to estimate the five parameters. A model integrating concentration, time, and temperature of exposure is required to optimize addition and removal protocols of high concentrations of cryoprotectant for cryopreservation. The Weibull distribution is a simple and flexible model used to describe similar processes. In the current study, chondrocyte viability decreased with increased concentration, temperature and time of exposure. The model indicated a significant interaction between the toxic effects of concentration and temperature.
