Permeation of dimethyl sulfoxide into articular cartilage at subzero temperatures

Permeation of dimethyl sulfoxide into articular cartilage at subzero temperatures first-author †E-mail:enezsz@zju.edu.cngmchen@zju.edu.cn †E-mail:enezsz@zju.edu.cngmchen@zju.edu.cn enezsz@zju.edu.cn corresp ‡Corresponding Author ‡Corresponding Author †E-mail:enezsz@zju.edu.cngmchen@zju.edu.cn †E-mail:enezsz@zju.edu.cngmchen@zju.edu.cn enezsz@zju.edu.cn Institute of Refrigeration and Cryogenics, Zhejiang University, Hangzhou 310027, China Institute of Refrigeration and Cryogenics, Zhejiang University, Hangzhou 310027, China Osteochondral allografting has been proved to be a useful method to treat diseased or damaged areas of joint surfaces. Operational long-term stocks of grafts which supply a buffer between procurement and utilization would contribute to the commercialization or industrialization of this technology. Vitrification has been thought to be a promising method for successful preservation of articular cartilage (AC), but high concentration cryoprotectants (CPAs) are used which may cause high cellular toxicity. An effective way to reduce CPA toxicity is to increase CPA concentration gradually while the temperature is lowered. Understanding the mechanism of CPA permeation at subzero temperatures is important for designing the cryopreservation protocol. In this research, the permeation of dimethyl sulfoxide (Me 2 SO) in ovine AC at subzero temperatures was studied experimentally. Pretreated AC discs were exposed in Me 2 SO solutions for different time (0, 5, 15, 30, 50, 80, and 120 min) at three temperature levels (−10, −20, and −30 °C). The Me 2 SO concentration within the tissue was determined by ultraviolet (UV) spectrophotometry. The diffusion coefficients were estimated to be 0.85×10 −6 , 0.48×10 −6 , and 0.27×10 −6 cm 2 /s at −10, −20, and −30 °C, respectively, and the corresponding activation energy was 29.23 kJ/mol. Numerical simulation was performed to compare two Me 2 SO addition protocols, and the results demonstrated that the total loading duration could be effectively reduced with the knowledge of permeation kinetics. Articular cartilage Vitrification Dimethyl sulfoxide Permeation Diffusion coefficient Subzero temperature Introduction 1 Articular cartilage (AC) is vital for normal joint function. Unlike other tissues, traumatic or degenerative cartilages do not heal well. Cartilage is described as an immunologically privileged tissue, and fresh osteochondral allografts have been proved to be effective and functional for transplantation (Chu et al., 1999 1999 ; Aubin et al., 2001 2001 ; Williams et al., 2007 2007 ; Raikin, 2009 2009 ). Furthermore, a recent study showed that fresh osteochondral autograft and fresh allograft tissues are not statistically different with respect to bony incorporation, AC composition, and biomechanical properties up to six months after implantation (Glenn et al., 2006 2006 ). However, this kind of treatment is limited by the scarcity of healthy grafts and the time difference between donation and clinical use, which necessitates the construction of an osteochondral allograft bank to preserve the AC that is from cadaver tissue donors or which might be manufactured by tissue engineering in the future. Ice-free cryopreservation, or vitrification, has been considered as a promising method for the long-term storage of biological tissues. Vitrification refers to the amorphous solidification of a liquid without crystallization. This solidification retains the normal molecular and ionic distribution of the tissue. Generally, vitrification is accomplished through the addition of a single cryoprotectant (CPA) or CPA mixtures at high concentration plus rapid cooling and thawing rates to minimize nucleation and ice-crystal growth (Rall and Fahy, 1985 1985 ; Song et al., 2000 2000 ; Wusteman et al., 2008 2008 ; Xu et al., 2009 2009 ). This type of vitrification of AC has been studied by several research groups (Song et al., 2004a 2004a ; 2004b 2004b ; Brockbank et al., 2010 2010 ). Recently, as an alternative, Pegg et al. ( 2006c 2006c ) proposed a novel approach called “liquidus-tracking” (LT) method to vitrify cartilage samples. The ability of post-thawing cartilage specimens to incorporate sulfate ( 35 S) into newly synthesized glycosaminoglycans (GAGs) was found to approach 70% of that of fresh control groups. In further work, this data reached 87% (Wang et al., 2007 2007 ). The knowledge of CPA permeation kinetics in the biological tissues is important for designing the CPA addition/removal protocols despite what kind of vitrification is used. The CPA permeation in AC at above zero temperatures has been reported in a few studies. Mukherjee et al. ( 2008 2008 ) studied the diffusions of high concentration single CPA [6.9 mol/L, dimethyl sulfoxide (Me 2 SO)] and multi-component CPA mixture (VS55, 3.1 mol/L Me 2 SO+2.2 mol/L 1,2-propanediol+3.1 mol/L formamide) in immature bovine AC. Sharma et al. ( 2007 2007 ) and Jomha et al. ( 2009 2009 ) examined the time-dependent permeations of Me 2 SO, propylene glycol (PG), ethylene glycol (EG), and glycerol into intact porcine AC at concentrations suitable for vitrification. Carsi et al. ( 2004 2004 ) determined the diffusion coefficients of Me 2 SO and glycerol in human AC at different temperatures and concentrations. The permeation of CPA in AC at subzero temperatures, however, has not been reported except when Pegg et al. ( 2006b 2006b ) measured the concentrations of Me 2 SO in AC at the end of each processing step to ensure that the AC sample was not frozen. However, the diffusion coefficients were not determined, and the CPA addition/removal protocols were more or less empirical. The successful prediction of CPA permeation into AC at subzero temperatures will aid in minimizing cell death due to CPA toxicity, osmotic damage, and ice-crystal formation when using the LT method. In this paper, the experiment was elaborately designed to obtain the apparent diffusion coefficients of Me 2 SO in isolated ovine AC at three subzero temperatures (−10, −20, and −30 °C), and the Arrhenius relation was used to express the manner in which the apparent diffusion coefficient changes with temperature. Finally, the use of these permeation kinetics in optimizing the Me 2 SO addition protocol of the LT method was discussed. Materials and methods 1 Chemicals and solutions 2 Unless otherwise specified, all chemicals used were of analytical grade and all solutions were prepared with deionized water. The main solutions were prepared as follows. A standard Me 2 SO stock solution (100 mg/L) was prepared by dissolving 100 mg of Me 2 SO (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China) in deionized water, which was then used to prepare working Me 2 SO standards (18 to 60 mg/L with an increment of 6 mg/L) through suitable dilutions. The Me 2 SO solutions of varied concentrations used in pre-equilibration and exposure (see Section 2.5) were prepared with a N -[2-hydroxyethyl] piperazine- N ′-[2-ethanesulfonic acid] (HEPES)-buffered carrier solution (145 mmol/L Na + , 3.3 mmol/L K + , and 20 mmol/L HEPES) instead of Dulbecco’s phosphate buffer solution (PBS) to avoid phosphate precipitation (Wusteman et al., 2002 2002 ). The NaCl and KCl were supplied by Sinopharm Chemical Reagent Co. Ltd., Shanghai, China, and the HEPES was from Amresco, USA. Calibration curve 2 The sulphoxide functional group present in Me 2 SO provides a potential avenue of detection of this substance by ultraviolet (UV) absorption. The absorbance of the Me 2 SO standard solutions (see Section 2.1) was measured using a TU-1810PC UV-vis spectrophotometer (Purkinje General Instrument Co. Ltd., Beijing, China) at 208 nm. Absorbance ( A ) was plotted as a function of the Me 2 SO concentration ( C , mg/L), and a linear regression equation was obtained, expressed as A =0.010 89+0.010 52 C with R 2 =0.999. Specimen preparation 2 AC samples were obtained from the back knee joints of approximately 12-month-old lambs that were killed for food in a local commercial abattoir. AC discs without bone (6 mm diameter, 0.6‒1.0 mm thickness) were taken from the weight-bearing portion using a corneal trephine and a scalpel, and weighed. They were then stored in ice-cold HEPES-buffered medium (see Section 2.1) for later use. In this research, only AC discs without bone attached were used, since studies (Xia et al., 1994 1994 ; Sharma et al., 2007 2007 ) have shown that isolated cartilage discs exhibit identical diffusion characteristics as those with bone attached. Experimental setup 2 Fig. 1 1 shows the setup for the subzero permeation experiment. The fixed temperatures needed (−10, −20, and −30 °C) were provided by a custom-built cooling device. The temperature of the Me 2 SO solution was measured by a four-wire platinum resistance thermometer and recorded using an Agilent 34970A Data Acquisition Unit (Agilent Technologies, Santa Clara, CA). The AC samples were held in 2-ml centrifuge tubes where some holes (3 mm diameter) were drilled in the walls. Around the stirring bar, which ensured good mass transfer near the surfaces of the AC samples, seven centrifuge tubes could be placed simultaneously. Experimental procedure 2 The AC discs were assigned randomly and pre-equilibrated in Me 2 SO solutions of lower concentrations at room temperature (22 °C, water bath) before they were exposed to Me 2 SO solutions of higher concentrations at subzero temperatures. Table 1 1 shows the details of the experiment. The exposure time at each subzero temperature was set as 0, 5, 15, 30, 50, 80, and 120 min. Four replications were undertaken for each exposure holding time. After exposure, the discs were removed and handled as follows: (1) The discs were lightly blotted with Kimwipes® tissues (Kimberly-Clark, Roswell, GA); (2) They were quickly rinsed with the deionized water; (3) They were lightly blotted again with Kimwipes® tissues; (4) Each was placed in a sealed centrifuge tube containing 2 ml deionized water, and held overnight at 4 °C in dark conditions to allow the Me 2 SO within the tissue to fully migrate out; (5) 0.3 ml of the resulting Me 2 SO/deionized water solution surrounding the cartilage was transferred into a flask of 50 ml for dilution; (6) the UV absorbance of the diluted solution was measured using the UV-vis spectrophotometer. As the control group, fresh AC discs without exposure to Me 2 SO solutions were directly immersed in 2 ml deionized water as in Step 4. T p : pre-equilibration temperature; c p : pre-equilibration concentration (w/v); t p : pre-equilibration time; c pc : Pre-equilibrated concentration in cartilage (w/v); T e : exposure temperature; c e : exposure concentration (w/v) Determined with an assumption that the concentration in tissue reached 90% of that of the surrounding solution at the end of the pre-equilibration The pre-equilibration time needed for each pre-equilibration concentration at 22 °C was conservatively estimated from our previous studies (Yu et al., 2010 2010 ) To avoid freezing under the corresponding exposure temperature, the equilibrium concentration in cartilage needed was determined according to the equation in (Pegg, 1986 1986 ). For computational purposes, the HEPES-buffered medium was assumed to be pure NaCl at mass fraction of 0.9% Group T p (°C) c p (%) a a t p (min) b b c pc (%) c c T e (°C) c e (%) 1 22 30.0 35 27.0 −10 47.0 2 22 41.8 48 37.6 −20 57.6 3 22 49.4 57 44.5 −30 64.5 The Me 2 SO concentration in the AC disc was expressed as weight of Me 2 SO/volume of AC interstitial fluid with the following two assumptions: (1) The volumes of the disc and fluid inside cartilage were constant; (2) The volume of the fresh AC interstitial fluid was approximated by water volume with a density of 1 g/cm 3 . The water content (mass fraction) of the fresh AC was about 77.7% (Yu et al., 2010 2010 ). Diffusion model 2 Cartilage is described as a network of pores and tortuosity. Hence, the apparent diffusion coefficient is used. The delivery of Me 2 SO in free-swelling cartilage discs is assumed to be spatially homogeneous and isotropic. The geometry of the AC disc is considered as a cylinder, and Me 2 SO can penetrate into the disc from the peripheral side as well as the top and bottom surfaces. Fick’s second law of diffusion in cylindrical coordinates can be expressed as $$\frac{{\partial c}}{{\partial t}} = - {D_{{\text{app}}}}\left[ {\frac{1}{r}\frac{\partial }{{\partial r}}\left( {r\frac{{\partial c}}{{\partial r}}} \right) + \frac{{{\partial ^2}c}}{{\partial {z^2}}}} \right]$$ , where c is the local Me 2 SO concentration in cartilage, D app is the apparent diffusion coefficient of Me 2 SO in cartilage, r is the radial position, z is the axial position, and t is the time. D app is assumed to be constant although there is a very short time (less than 20 s according to numerical estimation) for the disc temperature to drop down. The initial condition is c ( t =0)= c 0 , where c 0 is the Me 2 SO concentration in cartilage after pre-equilibration. For boundary conditions, it is assumed that the mass transfer resistance at the surfaces of the disc is negligible. Therefore, the surface concentration is equal to Kc s , where c s is the concentration of the bathing solution, and K is the partition coefficient of Me 2 SO between the tissue and the solution. In this research, K is set to 0.9 (Mukherjee et al., 2008 2008 ). The partial differential equation was solved with MATLAB software. The value of D app at each temperature was evaluated based on the least-squares best fit of the experimental data to the model prediction. Results and discussion 1 The absorbance values of the control group at 208 nm were less than 0.005 ( n =4, data not shown), which were insignificant compared to those of the treatment groups (all large than 0.2, data not shown). Consequently, the effect of possible impurity from cartilage on the measurement accuracy was not considered here. The experimental results, also the fitting curves, for the three subzero temperature levels are shown in Fig. 2 2 . The best fits for the apparent diffusion coefficients are 0.85×10 −6 cm 2 /s at −10 °C, 0.48×10 −6 cm 2 /s at −20 °C, and 0.27×10 −6 cm 2 /s at −30 °C. In no case did the final concentration in cartilage reach the concentration of the bathing solution, but stabilized at about 90% of that value (Table 2 2 ). Error bars represent the standard deviations (SDs) Data (concentration, w/v) are expressed as mean±SD Ratio of 120 min concentration to external concentration 2 Immersion time (min) 3 Me 2 SO concentration (%) # # −10 °C −20 °C −30 °C 0 27.17±0.96 36.75±1.11 44.65±1.76 5 35.23±1.74 43.84±1.89 48.05±1.72 15 39.21±1.59 46.72±0.90 51.13±1.49 30 41.27±1.10 48.90±1.20 53.90±1.63 50 42.56±1.19 50.50±1.15 56.07±0.92 80 41.83±1.20 51.31±1.07 56.72±0.71 120 43.45±1.33 51.94±1.00 57.20±1.00 0.92 * * 0.90 * * 0.89 * * The Arrhenius relation is used to express the manner in which the apparent diffusion coefficient changes with temperature: D app = A exp(− E a / RT ), where E a is the activation energy, R is the universal gas constant, T is the temperature, and A is the pre-exponential factor. Fig. 3 3 shows the natural logarithm of D app plotted against the reciprocal Kelvin temperature, with E a determined to be 29.23 kJ/mol. In Fig. 3 3 , the three data points at temperatures 1, 22, and 37 °C are obtained by fitting our previous experimental data (Yu et al., 2010 2010 ) to the current model. Due to the importance of the CPA permeation characteristics to the cryopreservation of tissues, many methods have been developed to investigate the penetration rates of CPA in tissue samples, such as using nuclear magnetic resonance (NMR) techniques (Muldrew et al., 1996 1996 ; Carsi et al., 2004 2004 ; Mukherjee et al., 2008 2008 ), high performance liquid chromatography (HPLC) (Carpenter and Dawson, 1991 1991 ; Hu and Wolfinbarger, 1994 1994 ; Pegg, 2006 2006 ), radiotracer ( 35 S) (Elford, 1970 1970 ), osmometer (Sharma et al., 2007 2007 ; Jomha et al., 2009 2009 ; Yu et al., 2010 2010 ), conductivity meter (Chen et al., 2009 2009 ), or electronic microbalance (Pegg, 2006 2006 ). In this study, the quantity of Me 2 SO in ovine AC was measured using a spectrophotometer. The range of 0–60 mg/L was chosen for measurement calibration. In fact, the Beer’s law can also be used well in a larger range of Me 2 SO concentrations according to our trials (data not shown). The application of spectrophotometry is feasible in determining the quantity of Me 2 SO, which is a common CPA in the cryopreservation field. Investigation of Me 2 SO permeation into AC has been studied with ovine (Pegg et al., 2006b 2006b ), porcine (Muldrew et al., 1996 1996 ; Sharma et al., 2007 2007 ; Jomha et al., 2009 2009 ), bovine (Mukherjee et al., 2008 2008 ), and human AC (Carsi et al., 2004 2004 ). In these studies, the temperature levels were between approximately 0 and 37 °C. The reported apparent diffusion coefficients ranged from 1.5×10 −6 to 17.9×10 −6 cm 2 /s depending on the temperature and tissue species. The current study obtained apparent diffusion coefficients of Me 2 SO in ovine AC at subzero temperatures −10, −20, and −30 °C. Arrhenius behavior was followed with R 2 =0.998 (Fig. 3 3 ). The activation energy E a presented here was higher than those reported [17.99 kJ/mol (Jomha et al., 2009 2009 ), 6.35 kJ/mol (Sharma et al., 2007 2007 )], but those two values were obtained with only three suprazero apparent diffusion coefficients for porcine AC. From Table 2 2 , it can be seen that the final concentration of Me 2 SO in cartilage samples after 120 min immersion did not reach the concentration value of the bathing solution. This phenomenon has been observed at above-zero temperatures (Pegg et al., 2006b 2006b ; Sharma et al., 2007 2007 ; Yu et al., 2010 2010 ), which might be due to multiple factors. Briefly, (1) there exists an equilibrium pressure difference between the cartilage and the surrounding bathing solution (Elmoazzen et al., 2005 2005 ); (2) “Bound” water exists in tissues, i.e., not all of the tissue water is available to dissolve Me 2 SO (Pegg et al., 1987 1987 ; Zhang and Pegg, 2007 2007 ). With the Me 2 SO diffusion kinetics known, it is possible to do some work in the optimization of the loading protocol. Here, as an example, two protocols named as Protocol I (Table 3 3 ) and Protocol II (Table 4 4 ) are compared. The temperatures and concentrations of the bathing solution for both protocols follow Pegg et al. ( 2006c 2006c )’s values. In Protocol I, as did Pegg et al. ( 2006c 2006c ), the duration for each step is set to be 30 min except for two steps at 22 °C, while in Protocol II, the duration for each subzero step is varied so that the T m,c [melting point of tissue calculated by the final center concentration of current step using the published equation (Pegg, 1986 1986 )] at the end of current step is 1 °C lower than the exposure temperature of the next step. The initial concentration in AC is zero and the objective is to increase the center concentration (mass fraction) to 47.6%, the minimal concentration for vitrification (Hua and Ren, 1994 1994 ). It can be noted that for Protocol II, although there was one more step than Protocol I, the total loading time (104 min) is nearly one third less than that of Protocol I (140 min). The time cutoff is due to the relatively steep concentration change at the initial stage of each step as shown in Fig. 2 2 . The exposure reduction would decrease the cellular toxicity of the CPA, but it should be noted that the final bathing solution concentration of Protocol II is higher than that of Protocol I despite a lower temperature. For further optimization of the Me 2 SO addition protocol, the CPA toxicity kinetics (cell viability loss from CPA exposure as a function of time, concentration, and temperature) are critical, which can be used to improve the coupling of the bathing solution temperature, concentration, and the duration for each step. The toxicity of Me 2 SO to chondrocytes [isolated (Tomford et al., 1984 1984 ; Pegg et al., 2006a 2006a ) or in situ (Elmoazzen et al., 2007 2007 ; Jomha et al., 2008 2008 )] at above-zero temperatures has been investigated, but no systematic data for subzero temperatures are available. BST: bathing solution temperature; BSC: bathing solution concentration (mass fraction); ICC: initial center concentration (mass fraction) in AC; IMC: initial mean concentration (mass fraction) in AC; FCC: final center concentration (mass fraction) in AC; FMC: final mean concentration (mass fraction) in AC Step BST (°C) BSC (%) Duration (min) ICC (%) IMC (%) FCC (%) FMC (%) T m,c (°C) 1 22.0 10 10 0.0 0.0 8.8 8.9 2 22.0 20 10 8.8 8.9 17.8 17.9 3 −5.0 29 30 17.8 17.9 25.8 25.9 −5.8 4 −8.5 38 30 25.8 25.9 33.6 33.9 −10.2 5 −16.0 47 30 33.6 33.9 40.9 41.5 −17.6 6 −23.0 56 30 40.9 41.5 47.6 48.8 −28.5 The abbreviations are the same as those shown in Table 3 3 Step BST (°C) BSC (%) Duration (min) ICC (%) IMC (%) FCC (%) FMC (%) T m,c (°C) 1 22.0 10 10.0 0.0 0.0 8.8 8.9 2 22.0 20 10.0 8.8 8.9 17.8 17.9 3 −5.0 29 17.4 17.8 17.9 24.8 25.4 −5.8 4 −8.5 38 23.1 24.8 25.4 33.1 33.6 −9.5 5 −16.0 47 14.8 33.1 33.6 38.2 39.9 −17.0 6 −23.0 56 18.3 38.2 39.9 44.7 47.1 −24.0 7 −35.0 63 10.7 44.7 47.1 47.6 50.8 −36.0 Conclusions 1 Vitrification has been proved to be promising for the long-term preservation of AC and other tissues. This paper presented the measurements of Me 2 SO permeation into ovine AC at three subzero temperatures using the method of UV spectrophotometry. The equilibrium concentration of Me 2 SO in AC was approximately 90% of that of the bathing solution. The fitted apparent diffusion coefficients followed the Arrhenius relation well. Using the obtained diffusivities, two addition protocols of Me 2 SO were simulated and compared, which demonstrated that the total loading duration could be effectively reduced with the knowledge of permeation kinetics. Otherwise, the LT method could be further optimized when coupling permeation kinetics with toxicity kinetics. Investigations of Me 2 SO toxicity kinetics to AC at subzero temperatures are now under way in our laboratory. References PP Aubin , HK Cheah , AM Davis , AE Gross . Long-term followup of fresh femoral osteochondral allografts for posttraumatic knee defects . Clin Orthop Relat R , 2001 . 391 ( Suppl. ): 318 - 327 . DOI: 10.1097/00003086-200110001-00029 http://doi.org/10.1097/00003086-200110001-00029 . Long-term followup of fresh femoral osteochondral allografts for posttraumatic knee defects KGM Brockbank , ZZ Chen , YC Song . Vitrification of porcine articular cartilage . Cryobiology , 2010 . 60 ( 2 ): 217 - 221 . 20026102 DOI: 10.1016/j.cryobiol.2009.12.003 http://doi.org/10.1016/j.cryobiol.2009.12.003 . Vitrification of porcine articular cartilage JF Carpenter , PE Dawson . Quantitation of dimethyl sulfoxide in solutions and tissues by high performance liquid chromatography . Cryobiology , 1991 . 28 ( 3 ): 210 - 215 . 1864079 DOI: 10.1016/0011-2240(91)90025-J http://doi.org/10.1016/0011-2240(91)90025-J . Quantitation of dimethyl sulfoxide in solutions and tissues by high performance liquid chromatography B Carsi , JL Lopez-Lacomba , J Sanz , F Marco , L Lopez-Duran . Cryoprotectant permeation through human articular cartilage . Osteoarthr Cartilage , 2004 . 12 ( 10 ): 787 - 792 . DOI: 10.1016/j.joca.2004.06.013 http://doi.org/10.1016/j.joca.2004.06.013 . Cryoprotectant permeation through human articular cartilage HH Chen , XM Zhou , ZQ Shu , EJ Woods , DY Gao . Electrical conductivity measurements for the ternary systems of glycerol/sodium chloride/water and ethylene glycol/sodium chloride/water and their applications in cryopreservation . Biopreserv Biobank , 2009 . 7 ( 1 ): 13 - 17 . DOI: 10.1089/bio.2009.0001 http://doi.org/10.1089/bio.2009.0001 . Electrical conductivity measurements for the ternary systems of glycerol/sodium chloride/water and ethylene glycol/sodium chloride/water and their applications in cryopreservation CR Chu , FR Convery , WH Akeson , M Meyers , D Amiel . Articular cartilage transplantation—clinical results in the knee . Clin Orthop Relat R , 1999 . 360 159 - 168 . DOI: 10.1097/00003086-199903000-00019 http://doi.org/10.1097/00003086-199903000-00019 . Articular cartilage transplantation—clinical results in the knee BC Elford . Diffusion and distribution of dimethyl sulphoxide in the isolated guinea-pig taenia coli . J Physiol , 1970 . 209 ( 1 ): 187 - 208 . 5499041 . Diffusion and distribution of dimethyl sulphoxide in the isolated guinea-pig taenia coli HY Elmoazzen , JAW Elliott , LE McGann . Cryoprotectant equilibration in tissues . Cryobiology , 2005 . 51 ( 1 ): 85 - 91 . 15979064 DOI: 10.1016/j.cryobiol.2005.05.003 http://doi.org/10.1016/j.cryobiol.2005.05.003 . Cryoprotectant equilibration in tissues HY Elmoazzen , A Poovadan , GK Law , JAW Elliott , LE McGann , NM Jomha . Dimethyl sulfoxide toxicity kinetics in intact articular cartilage . Cell Tissue Bank , 2007 . 8 ( 2 ): 125 - 133 . 17063260 DOI: 10.1007/s10561-006-9023-y http://doi.org/10.1007/s10561-006-9023-y . Dimethyl sulfoxide toxicity kinetics in intact articular cartilage RE Glenn , Jr EC McCarty , HG Potter , SF Juliao , JF Gordon , KP Spindler . Comparison of fresh osteochondral autografts and allografts: a canine model . Am J Sports Med , 2006 . 34 ( 7 ): 1084 - 1093 . 16452265 DOI: 10.1177/0363546505284846 http://doi.org/10.1177/0363546505284846 . Comparison of fresh osteochondral autografts and allografts: a canine model JF Hu , Jr L Wolfinbarger . Dimethyl sulfoxide concentration in fresh and cryopreserved porcine valved conduit tissues . Cryobiology , 1994 . 31 ( 5 ): 461 - 467 . 7988155 DOI: 10.1006/cryo.1994.1056 http://doi.org/10.1006/cryo.1994.1056 . Dimethyl sulfoxide concentration in fresh and cryopreserved porcine valved conduit tissues TC Hua , HS Ren . Cryobiomedical Technologies , 1994 . : Beijing, China Science Press , 120 (in Chinese) . Cryobiomedical Technologies NM Jomha , LE McGann , HY Elmoazzen . Modeling cryoprotectant toxicity in articular cartilage . J Bone Joint Surg Br , 2008 . 90B ( Suppl. ): 105 . Modeling cryoprotectant toxicity in articular cartilage NM Jomha , GK Law , A Abazari , K Rekieh , JA W Elliott , LE McGann . Permeation of several cryoprotectant agents into porcine articular cartilage . Cryobiology , 2009 . 58 ( 1 ): 110 - 114 . 19041639 DOI: 10.1016/j.cryobiol.2008.11.004 http://doi.org/10.1016/j.cryobiol.2008.11.004 . Permeation of several cryoprotectant agents into porcine articular cartilage IN Mukherjee , Y Li , YC Song , Jr RC Long , A Sambanis . Cryoprotectant transport through articular cartilage for long-term storage: experimental and modeling studies . Osteoarthr Cartilage , 2008 . 16 ( 11 ): 1379 - 1386 . DOI: 10.1016/j.joca.2008.03.027 http://doi.org/10.1016/j.joca.2008.03.027 . Cryoprotectant transport through articular cartilage for long-term storage: experimental and modeling studies K Muldrew , B Sykes , N Schachar , LE McGann . Permeation kinetics of dimethyl sulfoxide in articular cartilage . Cryoletters , 1996 . 17 331 - 340 . . Permeation kinetics of dimethyl sulfoxide in articular cartilage DE Pegg . Equations for obtaining melting points and eutectic temperatures for the ternary system dimethyl sulphoxide/sodium chloride/water . Cryoletters , 1986 . 7 387 - 394 . . Equations for obtaining melting points and eutectic temperatures for the ternary system dimethyl sulphoxide/sodium chloride/water DE Pegg . Immersion weighing as a method for monitoring the permeation of tissues by cryoprotectants . Cryobiology , 2006 . 53 ( 3 ): 383 DOI: 10.1016/j.cryobiol.2006.10.038 http://doi.org/10.1016/j.cryobiol.2006.10.038 . Immersion weighing as a method for monitoring the permeation of tissues by cryoprotectants DE Pegg , CJ Hunt , LP Fong . Osmotic properties of the rabbit corneal endothelium and their relevance to cryopreservation . Cell Biochem Biophys , 1987 . 10 ( 2 ): 169 - 189 . . Osmotic properties of the rabbit corneal endothelium and their relevance to cryopreservation DE Pegg , MC Wusteman , LH Wang . Cryopreservation of articular cartilage. Part 1: conventional cryopreservation methods . Cryobiology , 2006 . 52 ( 3 ): 335 - 346 . 16524570 DOI: 10.1016/j.cryobiol.2006.01.005 http://doi.org/10.1016/j.cryobiol.2006.01.005 . Cryopreservation of articular cartilage. Part 1: conventional cryopreservation methods DE Pegg , LH Wang , D Vaughan , CJ Hunt . Cryopreservation of articular cartilage. Part 2: mechanisms of cryoinjury . Cryobiology , 2006 . 52 ( 3 ): 347 - 359 . 16527262 DOI: 10.1016/j.cryobiol.2006.01.007 http://doi.org/10.1016/j.cryobiol.2006.01.007 . Cryopreservation of articular cartilage. Part 2: mechanisms of cryoinjury DE Pegg , LH Wang , D Vaughan . Cryopreservation of articular cartilage. Part 3: the liquidus-tracking method . Cryobiology , 2006 . 52 ( 3 ): 360 - 368 . 16527263 DOI: 10.1016/j.cryobiol.2006.01.004 http://doi.org/10.1016/j.cryobiol.2006.01.004 . Cryopreservation of articular cartilage. Part 3: the liquidus-tracking method SM Raikin . Fresh osteochondral allografts for large-volume cystic osteochondral defects of the talus . J Bone Joint Surg Am , 2009 . 91 ( 12 ): 2818 - 2826 . 19952243 DOI: 10.2106/JBJS.I.00398 http://doi.org/10.2106/JBJS.I.00398 . Fresh osteochondral allografts for large-volume cystic osteochondral defects of the talus WF Rall , GM Fahy . Ice-free cryopreservation of mouse embryos at −196 °C by vitrification . Nature , 1985 . 313 ( 6003 ): 573 - 575 . 3969158 DOI: 10.1038/313573a0 http://doi.org/10.1038/313573a0 . Ice-free cryopreservation of mouse embryos at −196 °C by vitrification R Sharma , GK Law , K Rehieh , A Abazari , JA W Elliott , LE McGann , NM Jomha . A novel method to measure cryoprotectant permeation into intact articular cartilage . Cryobiology , 2007 . 54 ( 2 ): 196 - 203 . 17379205 DOI: 10.1016/j.cryobiol.2007.01.006 http://doi.org/10.1016/j.cryobiol.2007.01.006 . A novel method to measure cryoprotectant permeation into intact articular cartilage YC Song , BS Khirabadi , F Lightfoot , KG M Brockbank , MJ Taylor . Vitreous cryopreservation maintains the function of vascular grafts . Nat Biotechnol , 2000 . 18 ( 3 ): 296 - 299 . 10700144 DOI: 10.1038/73737 http://doi.org/10.1038/73737 . Vitreous cryopreservation maintains the function of vascular grafts YC Song , YH An , QK Kang , CY Li , JM Boggs , ZZ Chen , MJ Taylor , KGM Brockbank . Vitreous preservation of articular cartilage grafts . J Invest Surg , 2004 . 17 ( 2 ): 65 - 70 . 15204712 DOI: 10.1080/08941930490422438 http://doi.org/10.1080/08941930490422438 . Vitreous preservation of articular cartilage grafts YC Song , FG Lightfoot , ZZ Chen , MJ Taylor , KGM Brockbank . Vitreous preservation of rabbit articular cartilage . Cell Preserv Technol , 2004 . 2 ( 1 ): 67 - 74 . DOI: 10.1089/153834404322708772 http://doi.org/10.1089/153834404322708772 . Vitreous preservation of rabbit articular cartilage WW Tomford , GR Fredericks , HJ Mankin . Studies on cryopreservation of articular cartilage chondrocytes . J Bone Joint Surg Am , 1984 . 66 ( 2 ): 253 - 259 . 6693452 . Studies on cryopreservation of articular cartilage chondrocytes LH Wang , DE Pegg , J Lorrison , D Vaughan , P Rooney . Further work on the cryopreservation of articular cartilage with particular reference to the liquidus tracking (LT) method . Cryobiology , 2007 . 55 ( 2 ): 138 - 147 . 17678641 DOI: 10.1016/j.cryobiol.2007.06.005 http://doi.org/10.1016/j.cryobiol.2007.06.005 . Further work on the cryopreservation of articular cartilage with particular reference to the liquidus tracking (LT) method SK Williams , D Amiel , ST Ball , RT Allen , WL Tontz , Jr BC Emmerson , NM Badlani , SC Emery , P Haghighi , WD Bugbee . Analysis of cartilage tissue on a cellular level in fresh osteochondral allograft retrievals . Am J Sports Med , 2007 . 35 ( 12 ): 2022 - 2032 . 17724095 DOI: 10.1177/0363546507305017 http://doi.org/10.1177/0363546507305017 . Analysis of cartilage tissue on a cellular level in fresh osteochondral allograft retrievals MC Wusteman , DE Pegg , MP Robinson , LH Wang , P Fitch . Vitrification media: toxicity, permeability, and dielectric properties . Cryobiology , 2002 . 44 ( 1 ): 24 - 37 . 12061845 DOI: 10.1016/S0011-2240(02)00002-0 http://doi.org/10.1016/S0011-2240(02)00002-0 . Vitrification media: toxicity, permeability, and dielectric properties MC Wusteman , J Simmonds , D Vaughan , DE Pegg . Vitrification of rabbit tissues with propylene glycol and trehalose . Cryobiology , 2008 . 56 ( 1 ): 62 - 71 . 18093578 DOI: 10.1016/j.cryobiol.2007.10.177 http://doi.org/10.1016/j.cryobiol.2007.10.177 . Vitrification of rabbit tissues with propylene glycol and trehalose Y Xia , T Farquhar , N Burton-Wurster , E Ray , LW Jelinski . Diffusion and relaxation mapping of cartilage-bone plugs and excised disks using microscopic magnetic resonance imaging . Magn Reson Med , 1994 . 31 ( 3 ): 273 - 282 . 8057798 DOI: 10.1002/mrm.1910310306 http://doi.org/10.1002/mrm.1910310306 . Diffusion and relaxation mapping of cartilage-bone plugs and excised disks using microscopic magnetic resonance imaging H Xu , HC Shi , WF Zang , D Lu . An experimental research on cryopreserving rabbit trachea by vitrification . Cryobiology , 2009 . 58 ( 2 ): 225 - 231 . 19162000 DOI: 10.1016/j.cryobiol.2008.12.009 http://doi.org/10.1016/j.cryobiol.2008.12.009 . An experimental research on cryopreserving rabbit trachea by vitrification XY Yu , SZ Zhang , MJ Xu , GM Chen . Study on the permeation of dimethyl sulfoxide into articular cartilage . J Eng Thermophys , 2010 . 31 ( 8 ): 1363 - 1366 . (in Chinese) . Study on the permeation of dimethyl sulfoxide into articular cartilage SZ Zhang , DE Pegg . Analysis of the permeation of cryoprotectants in cartilage . Cryobiology , 2007 . 54 ( 2 ): 146 - 153 . 17300774 DOI: 10.1016/j.cryobiol.2006.12.001 http://doi.org/10.1016/j.cryobiol.2006.12.001 . Analysis of the permeation of cryoprotectants in cartilage We would like to thank Mrs. Debra KWITEROVICH-HOOVER (Villanova University, USA) for revising the grammar of the paper. 10.1631/jzus.B11a0041 Project supported by the National Natural Science Foundation of China (No. 50606032) and the Graduate Innovation Research Program of Zhejiang Province (No. YK2008020), China 2011-02-20 2012-02-11 2011-07-26 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2012 2012 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 03 13