Garson K. Law

Cryopreservation of articular cartilage

Cryopreservation has numerous practical applications in medicine, biotechnology, agriculture, forestry, aquaculture and biodiversity conservation, with huge potentials for biological cell and tissue banking. A specific tissue of interest for cryopreservation is the articular cartilage of the human knee joint for two major reasons: (1) clinically, there exists an untapped potential for cryopreserved cartilage to be used in surgical repair/reconstruction/replacement of injured joints because of the limited availability of fresh donor tissue and, (2) scientifically, successful cryopreservation of cartilage, an avascular tissue with only one cell type, is considered a stepping stone for transition from biobanking cell suspensions and small tissue slices to larger and more complicated tissues. For more than 50years, a great deal of effort has been directed toward understanding and overcoming the challenges of cartilage preservation. In this article, we focus mainly on studies that led to the finding that vitrification is an appropriate approach toward successful preservation of cartilage. This is followed by a review of the studies on the main challenges of vitrification, i.e. toxicity and diffusion, and the novel approaches to overcome these challenges such as liquidus tracking, diffusion modeling, and cryoprotective agent cocktails, which have resulted in the recent advancements in the field.

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°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.

Cryoprotective agent toxicity interactions in human articular chondrocytes.

BACKGROUNDnVitrification is a method of cryopreservation by which cells and tissues can be preserved at low temperatures using cryoprotective agents (CPAs) at high concentrations (typically ≥6.0 M) to limit the harmful effects of ice crystals that can form during cooling processes. However, at these concentrations CPAs are significantly cytotoxic and an understanding of their toxicity characteristics and interactions is important. Therefore, single-CPA and multiple-CPA solutions were evaluated for their direct and indirect toxicities on chondrocytes.nnnMETHODSnChondrocytes were isolated from human articular cartilage samples and exposed to various single-CPA and multiple-CPA solutions of five common CPAs (dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), glycerol (Gy) and formamide (Fm)) at both 6.0 and 8.1 M concentrations at 0 °C for 30 min. Chondrocyte survival was determined using a fluorescent cell membrane integrity assay. The data obtained was statistically analyzed and regression coefficients were used to represent the indirect toxicity effect which a specific combination of CPAs exerted on the final solutions toxicity.nnnRESULTSnMultiple-CPA solutions were significantly less toxic than single-CPA solutions (P<0.01). The indirect toxicity effects between CPAs were quantifiable using regression analysis. Cell survival rates of approximately 40% were obtained with the four-CPA combination solution DMSO-EG-Gy-Fm. In the multiple-CPA combinations, PG demonstrated the greatest degree of toxicity and its presence within a combination solution negated any benefits of using multiple lower concentration CPAs.nnnCONCLUSIONSnMultiple-CPA solutions are less cytotoxic than single-CPA solutions of the same total concentration. PG was the most toxic CPA when used in combinations. The highest chondrocyte survival rates were obtained with the 6.0 M DMSO-EG-Gy-Fm combination solution.

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.

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.

Permeation of several cryoprotectant agents into porcine articular cartilage.

OBJECTIVEnOsteochondral 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.nnnDESIGNnDowels 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.nnnRESULTSnDiffusion 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.nnnCONCLUSIONnThe 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.

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.

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°C (without ice formation) compared to membrane integrity stains (MIS). Osteochondral dowels (10xa0mm in diameter) were harvested from sexually mature pigs within 24xa0h of sacrifice and randomized into three groups: (1) untreated control, (2) one day storage at −10°C (in cryoprotectant solution to prevent ice formation), and (3) seven day storage at −10°C (in cryoprotectant solution). Fluorescent MISs (Syto 13 and ethidium bromide) were used on 70xa0μm 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 70xa0μm thickness per sample to obtain reliable readings using a spectrophotometer at 450xa0nm. All samples underwent repeated measures of membrane integrity and metabolic activity obtained after 0, 3, 24, 48, 72, and 144xa0h incubation in growth media. WST-1 consistently overestimated cell recovery with results greater than fresh controls. After hypothermic storage for 7xa0days, the WST-1 measurement demonstrated decreased mitochondrial activity that recovered by 48xa0h. 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 48xa0h incubation may be the most accurate determination of the number of live chondrocytes in situ.

Storage of porcine articular cartilage at high subzero temperatures.

Objective: Transplantation of osteochondral allograft tissue can treat large joint defects but is limited by tissue availability, surgical timing, and infectious disease transmission. Fresh allografts perform the best but requirements for infectious disease testing delay the procedure with subsequent decrease in cell viability and function. Hypothermic storage at lower temperatures can extend tissue banking time without loss of cell viability and, therefore, increase the supply of allograft tissue. This study investigated the effects of different cryoprotectant solutions on intact AC at various subzero temperatures. Design: 10xa0mm porcine osteochondral dowels were immersed for 30xa0minutes in various combinations of solutions [(XVIVO, propylene glycol (51% w/w), sucrose (46% w/w)] cooled to various subzero temperatures (−10, −15, and −20xa0°C), and held for 30xa0min. After warming, 70xa0μm slices were stained with membrane integrity dyes, viewed under fluorescence microscopy and cell recovery calculated relative to fresh controls. Results: Results demonstrated excellent cell recovery (>75%) at −10°C provided ice did not form. Excellent cell recovery (>70%) occurred at −15°C in solutions containing 51% propylene glycol but formation of extra-matrix ice in other solutions resulted in significant cell loss. All groups had <6% cell recovery at −20°C and propylene glycol did not provide a protective effect even though extra-matrix ice did not form Conclusions: These results suggest that extra-matrix ice plays an important role in cell damage during cryopreservation. Excellent cell recovery can be obtained after storage at subzero temperatures if ice does not form. Hypothermic preservation at high subzero temperatures may extend AC storage time in tissue banks compared to current techniques.