Vitrification of intact human articular cartilage.

Articular cartilage injuries do not heal and large defects result in osteoarthritis with major personal and socioeconomic costs. Osteochondral transplantation is an effective treatment for large joint defects but its use is limited by the inability to store cartilage for long periods of time. Cryopreservation/vitrification is one method to enable banking of this tissue but decades of research have been unable to successfully preserve the tissue while maintaining cartilage on its bone base - a requirement for transplantation. To address this limitation, human knee articular cartilage from total knee arthroplasty patients and deceased donors was exposed to specified concentrations of 4 different cryoprotective agents for mathematically determined periods of time at lowering temperatures. After complete exposure, the cartilage was immersed in liquid nitrogen for up to 3 months. Cell viability was 75.4 ± 12.1% determined by membrane integrity stains and confirmed with a mitochondrial assay and pellet culture documented production of sulfated glycosaminoglycans and collagen II similar to controls. This report documents successful vitrification of intact human articular cartilage on its bone base making it possible to bank this tissue indefinitely.

Cryoprotectant agent toxicity in porcine articular chondrocytes.

Large articular cartilage defects have proven difficult to treat and often result in osteoarthritis of the affected joint. Cryopreservation of articular cartilage can provide an increased supply of tissues for osteochondral allograft but cryoprotective agents are required; however, few studies have been performed on the toxicity of these agents. This study was designed to determine the order of toxicity of five commonly used cryoprotectant agents as well as interactions that occur between them. Isolated porcine articular chondrocytes were exposed to individual cryoprotectant agents and combinations of these agents at 1M and 3M concentrations for 5 min and 120 min. Cell viability was determined using membrane integrity dyes and a metabolic activity assay. Subsequently, a regression analysis based study was undertaken to extract the maximum amount of information from this data. Results of this study demonstrated that all 1M solutions were minimally toxic. The 3M solutions demonstrated varying toxicity after 120 min. Ethylene glycol and glycerol were less toxic than propylene glycol, dimethyl sulfoxide, and formamide. Combinations of cryoprotectant agents were less toxic than single cryoprotectant agents at the same concentration. This is the most comprehensive study investigating cryoprotectant agent toxicity in articular chondrocytes and has resulted in important information regarding the order of toxicity and interactions that occur between these agents.

Statistical prediction of the vitrifiability and glass stability of multi-component cryoprotective agent solutions.

Long-term biologic storage of articular cartilage has proven elusive due to cellular degradation over time or acute damage during attempts at cryopreservation. Vitrification is one option that may result in successful cryopreservation but difficulty with cryoprotective agent (CPA) toxicity at high concentrations of a single cryoprotectant has hindered development of successful protocols. This study was designed to determine the vitrifiability and glass stability of solutions containing combinations of commonly used CPAs and to document CPA interactions that occur. One hundred and sixty-four multi-CPA combination solutions of 6-9 M were evaluated for vitrifiability and glass stability using direct visualization after immersion in liquid nitrogen for 30 min and upon warming. Binary and ordinal logistic regression analysis was used to statistically analyze each CPA for its ability to vitrify and its effect on glass stability in multi-component CPA solutions. Propylene glycol had the greatest incremental contribution to vitrification while formamide had the least contribution. A threshold was established whereby the ability of a solution to vitrify could be determined by calculation. Glass stability was not as clearly defined due to variability in the results; however, contributions of interactions between CPAs to the glass stability of solutions were determined. This study provided values that predict if a solution will vitrify. Furthermore, the glass stability of solutions containing multiple CPAs do not behave as linear additions of binary solutions and interactions between CPAs have a significant effect on the glass stability of these solutions. These variables should be considered when designing vitrification solutions.

A biomechanical triphasic approach to the transport of nondilute solutions in articular cartilage.

Biomechanical models for biological tissues such as articular cartilage generally contain an ideal, dilute solution assumption. In this article, a biomechanical triphasic model of cartilage is described that includes nondilute treatment of concentrated solutions such as those applied in vitrification of biological tissues. The chemical potential equations of the triphasic model are modified and the transport equations are adjusted for the volume fraction and frictional coefficients of the solutes that are not negligible in such solutions. Four transport parameters, i.e., water permeability, solute permeability, diffusion coefficient of solute in solvent within the cartilage, and the cartilage stiffness modulus, are defined as four degrees of freedom for the model. Water and solute transport in cartilage were simulated using the model and predictions of average concentration increase and cartilage weight were fit to experimental data to obtain the values of the four transport parameters. As far as we know, this is the first study to formulate the solvent and solute transport equations of nondilute solutions in the cartilage matrix. It is shown that the values obtained for the transport parameters are within the ranges reported in the available literature, which confirms the proposed model approach.

Permeation of several cryoprotectant agents into porcine articular cartilage.

OBJECTIVE: Osteochondral allografting is an effective method to treat large osteochondral defects but difficulties in tissue preservation have significantly limited the application of this technique. Successful cryopreservation of articular cartilage (AC) could improve the clinical availability of osteochondral tissue and enhance clinical outcomes but cryopreservation of large tissues is hampered by a lack of knowledge of permeation kinetics within these tissues. This study describes the refinement and extension of a recently published technique to measure the permeation kinetics of cryoprotectant agents (CPAs) within porcine AC. DESIGN: Dowels of porcine AC (10mm diameter) were immersed in solutions containing 6.5 M concentrations of four commonly used CPAs [dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG) and glycerol] for different times (1s, 1, 2, 5, 10, 15, 30, 60, 120, 180 min , 24h) at three different temperatures (4, 22, and 37 degrees C). The cartilage was isolated and the amount of CPA within the matrix was determined. RESULTS: Diffusion coefficients (DMSO=2.4-6.2x10(-6)cm2/s; PG=0.8-2.7x10(-6)cm2/s; EG=1.7-4.2x10(-6)cm2/s; and glycerol=0.8-2.4x10(-6)cm2/s) and activation energies (DMSO=4.33 kcal/mol, PG=6.29 kcal/mol, EG=3.77 kcal/mol, and glycerol=5.56 kcal/mol) were determined for each CPA. CONCLUSION: The results of this experiment provide accurate permeation kinetics of four commonly used CPAs in porcine articular cartilage. This information will be useful for developing effective vitrification protocols for cryopreservation of AC.

A novel method to measure cryoprotectant permeation into intact articular cartilage.

Successful cryopreservation of articular cartilage (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 the time required for cryoprotective agent (CPA) permeation into the matrix and high CPA toxicity. This study describes a novel, practical method to examine the time-dependent permeation of CPAs [dimethyl sulfoxide (DMSO) and propylene glycol (PG)] into intact porcine AC. Dowels of porcine AC (10 mm diameter) were immersed in solutions containing high concentrations of each CPA for different times (0, 15, 30, 60 min, 3, 6, and 24 h) at three temperatures (4, 22, and 37 degrees C), with and without cartilage attachment to bone. The cartilage was isolated and the amount of cryoprotective agent within the matrix was determined. The results demonstrated a sharp rise in the CPA concentration within 15-30 min exposure to DMSO and PG. The concentration plateaued between 3 and 6 h of exposure at a concentration approximately 88-99% of the external concentration (6.8 M). This observation was temperature-dependent with slower permeation at lower temperatures. This study demonstrated the effectiveness of a novel technique to measure CPA permeation into intact AC, and describes permeation kinetics of two common CPAs into intact porcine AC.

Adequate oral threonine is critical for mucin production and gut function in neonatal piglets.

In previous experiments, we found that the threonine requirement of neonatal piglets fed parenterally was 40% of that when fed intragastrically; we hypothesized that much of the oral supply of threonine is being used for mucin production. To investigate this hypothesis, intragastrically fed 2-day-old piglets were fed one of three treatments for 8 days: 1) a threonine-adequate diet (IG-A; 0.6 g threonine.kg(-1).day(-1) fed intragastrically); 2) a threonine-deficient diet (IG-D; 0.1 g threonine.kg(-1).day(-1) fed intragastrically); or 3) a threonine-deficient diet with adequate threonine delivered parenterally (IV-A; 0.5 g threonine.kg(-1).day(-1) fed parenterally plus 0.1 g threonine.kg(-1).day(-1) fed intragastrically). IG-D piglets experienced higher nitrogen excretion, higher plasma urea, and lower plasma threonine concentrations versus both of the other groups (P < 0.05), indicating profound threonine deficiency. Mucosal mass and total crude mucin content were lower in the colons of IG-D pigs (P < 0.05). Histopathological analysis showed lower numbers of acidic mucin-producing goblet cells in the duodenum and ileum of IG-D pigs. In IG-D pigs, acidic mucin subtypes were lower in the small intestine but higher in the colon, which corresponded with persistent diarrhea. The parenteral supply of threonine was adequate to maintain most outcome parameters, although IV-A pigs did have smaller colonic goblet cells with more acidic mucins compared with IG-A pigs. Overall, our results suggest that adequate dietary threonine was critical in the production of mucus and that a parenteral threonine supply can ameliorate most of the symptoms of oral threonine deficiency.

Evaluation of chondrocyte survival in situ using WST-1 and membrane integrity stains.

Evaluating chondrocytes in situ to document the effectiveness of cartilage preservation techniques has proven exceedingly difficult. This study was conducted to determine the effectiveness of WST-1 on porcine chondrocytes in situ after cooling to -10 degrees C (without ice formation) compared to membrane integrity stains (MIS). Osteochondral dowels (10 mm in diameter) were harvested from sexually mature pigs within 24 h of sacrifice and randomized into three groups: (1) untreated control, (2) one day storage at -10 degrees C (in cryoprotectant solution to prevent ice formation), and (3) seven day storage at -10 degrees C (in cryoprotectant solution). Fluorescent MISs (Syto 13 and ethidium bromide) were used on 70 microm slices. Representative images were digitized and green and red pixel numbers determined the percent recovery of intact cells. Mitochondrial activity (WST-1) was determined using 20 slices of 70 microm thickness per sample to obtain reliable readings using a spectrophotometer at 450 nm. All samples underwent repeated measures of membrane integrity and metabolic activity obtained after 0, 3, 24, 48, 72, and 144 h incubation in growth media. WST-1 consistently overestimated cell recovery with results greater than fresh controls. After hypothermic storage for 7 days, the WST-1 measurement demonstrated decreased mitochondrial activity that recovered by 48 h. MIS was most accurate when "absolute" cell recovery was compared to original controls, taking into account cell density. In conclusion, WST-1 can track metabolic activity of chondrocytes in situ over time but "absolute" cell recovery determined by MISs after 48 h incubation may be the most accurate determination of the number of live chondrocytes in situ.

Dimethyl sulfoxide toxicity kinetics in intact articular cartilage.

Osteochondral defects can degenerate into osteoarthritis and currently there are no good treatment alternatives available to most Orthopaedic surgeons. Osteochondral allografting can restore damaged joint surfaces but its clinical use is limited by poor access to high quality tissue. Vitrification of osteochondral tissue would allow the banking of this tissue but requires high concentrations of cryoprotective agents. This study was designed to ascertain dimethyl sulfoxide (DMSO) toxicity kinetics to chondrocytes in situ after exposure to DMSO at different temperatures recorded as a function of time. Porcine osteochondral dowels were exposed to 1, 3, 5, and 6M DMSO at 4, 22, and 37 degrees C for 0.5 min to 120 min. Chondrocyte recovery was determined by membrane integrity (Syto 13 and ethidium bromide) and mitochondrial (WST-1) assays. Results demonstrated that cell recovery was concentration, temperature and time dependent. At higher concentrations and temperatures, significant cell loss occurred within minutes. A rate constant calculated for chondrocyte death was dependent on temperature. 1 M DMSO appeared relatively non-toxic. This experiment established a method to examine systematically toxicity parameters for chondrocytes in situ and this data can be used to tailor vitrification protocols by limiting exposure temperature and time or lowering DMSO concentrations below toxic levels recorded.