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 1 M and 3 M 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 1 M solutions were minimally toxic. The 3 M 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.     

Cryoprotective agent toxicity interactions in human articular chondrocytes

Background

Vitrification 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.

Methods

Chondrocytes 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 solution’s toxicity.

Results

Multiple-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.

Conclusions

Multiple-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.     

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.     

The effect of additive compounds on glycerol-induced damage to human chondrocytes

High concentrations of cryoprotective agents are required for cryopreservation techniques such as vitrification. Glycerol is a common cryoprotective agent used in cryopreservation protocols but this agent is toxic at high concentrations. This work is an attempt to mitigate the toxic effects of high concentrations of glycerol on intact chondrocytes in human knee articular cartilage from total knee arthroplasty patients by simultaneous exposure to glycerol and a variety of additive compounds. The resulting cell viability in the cartilage samples as measured by membrane integrity staining showed that, in at least one concentration or in combination, all of the tested additive compounds (tetramethylpyrazine, ascorbic acid, chondroitin sulphate, glucosamine sulphate) were able to reduce the deleterious effects of glycerol exposure when examination of membrane integrity took place on a delayed time frame. The use of additive compounds to reduce cryoprotectant toxicity in articular cartilage may help improve cell recovery after cryopreservation.     

Antioxidant additives reduce reactive oxygen species production in articular cartilage during exposure to cryoprotective agents

High concentrations of cryoprotective agents (CPA) are required during articular cartilage cryopreservation but these CPAs can be toxic to chondrocytes. Reactive oxygen species have been linked to cell death due to oxidative stress. Addition of antioxidants has shown beneficial effects on chondrocyte survival and functions after cryopreservation. The objectives of this study were to investigate (1) oxidative stress experienced by chondrocytes and (2) the effect of antioxidants on cellular reactive oxygen species production during articular cartilage exposure to high concentrations of CPAs. Porcine cartilage dowels were exposed to a multi-CPA solution supplemented with either 0.1 mg/mL chondroitin sulfate or 2000 μM ascorbic acid, at 4 °C for 180 min (N = 7). Reactive oxygen species production was measured with 5 μM dihydroethidium, a fluorescent probe that targets reactive oxygen species. The cell viability was quantified with a dual cell membrane integrity stain containing 6.25 μM Syto 13 + 9 μM propidium iodide using confocal microscopy. Supplementation of CPA solutions with chondroitin sulfate or ascorbic acid resulted in significantly lower dihydroethidium counts (p < 0.01), and a lower decrease in the percentage of viable cells (p < 0.01) compared to the CPA-treated group without additives. These results indicated that reactive oxygen species production is induced when articular cartilage is exposed to high CPA concentrations, and correlated with the amount of dead cells. Both chondroitin sulfate and ascorbic acid treatments significantly reduced reactive oxygen species production and improved chondrocyte viability when articular cartilage was exposed to high concentrations of CPAs.     

Effect of different cryoprotectants and vitrificant solutions on the hatching rate of turbot embryos (Scophthalmus maximus)

Vitrification could provide a promising tool for the cryopreservation of fish embryos. However, in order to achieve a vitrifiable medium, a high concentration of permeable cryoprotectants must be employed, and the incorporation of high molecular weight compounds should also be considered. The toxicity of these permeable and non-permeable agents has to be assessed, particularly when high concentrations are required. In the present study, permeable and non-permeable cryoprotectant toxicity was determined in turbot embryos at two development stages (F stage-tail bud and G stage-tail bud free). Embryos treated with pronase (2mg/ml, 10 min at 22 degrees C) were incubated in dimethyl sulfoxide (Me2SO), methanol (Meth.) or ethylene glycol (EG) in concentrations ranging from 0.5 to 6M for periods of 10 or 30 min, and in 5, 10, and 15% polyvinylpyrrolidone (PVP), 10, 15, and 20% sucrose or 0.1, 1, and 2% X-1000 for 2 min. The embryos were then washed well and incubated in seawater until hatching. The toxicity of permeable cryoprotectants increased with concentration and exposure time. There were no significant differences between permeable cryoprotectants. However, embryos tolerated higher concentrations of Me2SO than other cryoprotectants. Exposure to permeable cryoprotectants did not affect the hatching rate except at G stage with X-1000 treatment and 20% sucrose. Taking into account the cryoprotectant toxicity and the vitrification ability of cryoprotectant mixtures, three vitrification solutions (V1, V2, and V3), and one protocol for stepwise incorporation were designed. The tested solutions contained 5M Me2SO+2M Meth+1M EG plus 5% PVP, 10% sucrose or 2% X-1000. The hatching rate of embryos that had been exposed to the the vitrification solutions was analyzed and no significant differences were noticed compared with the controls. Our results demonstrate that turbot embryos can be subject to this cryoprotectant protocol without deleterious effect on the hatching rate.     

Effect of cryoprotectants on the viability and function of unfrozen human polymorphonuclear cells

High concentrations of membrane permeable cryoprotectants are necessary to protect human polymorphonuclear leukocytes from osmotic stress injury during freezing, but there are reports that some cryoprotectants are chemically toxic. Cells were exposed to various concentrations of glycerol, dimethyl sulfoxide, or ethylene glycol for 5 min to 2 hr at 37, 22 or 0 degree C, adding or removing the cryoprotectant either slowly or rapidly. Assays included cell number recovery, membrane integrity, phagocytosis, microbicidal ability, and chemotaxis. We conclude that (1) 1 and 2 M concentrations generally are not toxic if they are added and removed slowly at 22 degrees C; (2) addition and removal of glycerol at 0 degree C was injurious even at 1 M; (3) slow addition and removal allowed better recovery than rapid addition or removal; (4) salt concentration in cryoprotectant solutions should be adjusted to isotonic on the basis of moles per liter of solution, rather than moles per kilogram of water; (5) the toxicity reported by other investigators can be largely explained by osmotic stress or dilution shock rather than chemical toxicity; and (6) ethylene glycol is the easiest cryoprotectant to add to and remove from these cells.     

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.     

Cryopreservation and biophysical properties of articular cartilage chondrocytes

In order to successfully cryopreserve articular cartilage chondrocytes, it is important to characterize their osmotic response during the cryopreservation process, as the ice forms and the solutes concentrate. In this study, experimental work was undertaken to determine the osmotic parameters of articular cartilage chondrocytes. The osmotically inactive volume of articular cartilage chondrocytes was determined to be 44% of the isotonic volume. The membrane hydraulic conductivity parameters for water were determined by fitting a theoretical water transport model to the experimentally obtained volumetric shrinkage data; the membrane hydraulic conductivity parameter L(Pg) was found to be 0.0633 microm/min/atm, and the activation energy E, 8.23 kcal/mol. The simulated cooling process, using the osmotic parameters obtained in this study, suggests a cooling rate of 80 degrees C/min for the cryopreservation of the articular cartilage chondrocytes of hogs. The data obtained in this study could serve as a starting point for those interested in cryopreservation of chondrocytes from articular cartilage in other species in which there is clinical interest and there are no parameters for prediction of responses.     

Japanese flounder (Paralichthys olivaceus) embryos are difficult to cryopreserve by vitrification

The first successful cryopreservation of fish embryos was reported in the Japanese flounder by vitrification [Chen and Tian, Theriogenology, 63, 1207-1219, 2005]. Since very high concentrations of cryoprotectants are needed for vitrification and fish embryos have a large volume, Japanese flounder embryos must have low sensitivity to cryoprotectant toxicity and high permeability to water and cryoprotectants. So, we investigated the sensitivity and the permeability of Japanese flounder embryos. In addition, we assessed the survival of flounder embryos after vitrification with solutions containing methanol and propylene glycol, following Chen and Tian's report. The embryos were relatively insensitive to the toxicity of individual cryoprotectants at lower concentrations, especially methanol and propylene glycol as their report. Although their permeability to water and cryoprotectants could not be measured from volume changes in cryoprotectant solutions, the embryos appeared to be permeable to methanol but less permeable to DMSO, ethylene glycol, and propylene glycol. Although vitrification solutions containing methanol and propylene glycol, which were used in Chen and Tian's report, were toxic to embryos, a small proportion of embryos did survived. However, when vitrified with the vitrification solutions, no embryos survived after warming. The embryos became opaque during cooling with liquid nitrogen, indicating the formation of intracellular ice during cooling. When embryos had been kept in vitrification solutions for 60 min after being treated with the vitrification solution, some remained transparent during cooling, but became opaque during warming. This suggests that dehydration and/or permeation by cryoprotectants were insufficient for vitrification of the embryos even after they had been over-treated with the vitrification solutions. Thus, Chen and Tian's cryopreservation method lacks general application to Japanese flounder embryos.     

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.     

Baculovirus transduction of rat articular chondrocytes: roles of cell cycle

BACKGROUND: We have previously demonstrated highly efficient baculovirus transduction of primary rat articular chondrocytes, thus implicating the possible applications of baculovirus in gene-based cartilage tissue engineering. However, baculovirus-mediated gene expression in the chondrocytes is transient. METHODS: In this study, we attempted to prolong the expression by supertransduction, but uncovered that after long-term culture the chondrocytes became more refractory to baculovirus transduction. Therefore, the correlation between baculovirus-mediated enhanced green fluorescent protein (EGFP) expression and cell cycle was investigated by comparing the cycling chondrocytes and chondrocytes rich in quiescent cells, in terms of EGFP expression, virus uptake, cell cycle distribution, nuclear import and methylation of viral DNA. RESULTS: We demonstrated, for the first time, that baculovirus-mediated transduction of chondrocytes is correlated with the cell cycle. The chondrocytes predominantly in G2/M phase were approximately twice as efficient in EGFP expression as the cycling cells, while the cells in S and G1 phases expressed EGFP as efficiently as the cycling cells. Notably, the chondrocyte populations rich in quiescent cells resulted in efficient virus uptake, but less effective nuclear transport of baculoviral DNA and higher degree of methylation, and hence poorer transgene expression. CONCLUSIONS: These findings unravel the practical limitations when employing baculovirus in cartilage tissue engineering. The implications and possible solutions are discussed.     

Dehydration rates of meniscus and articular cartilage in vitro using a fast and accurate laser-based coordinate digitizing system

When used in in vitro studies, soft tissues such as the meniscus and articular cartilage are susceptible to dehydration and its effects, such as changes in size and shape as well as changes in structural and material properties. To quantify the effect of dehydration on the meniscus and articular cartilage, the first two objectives of this study were to (1) determine the percent change in meniscal dimensions over time due to dehydration, and (2) determine the percent change in articular cartilage thickness due to dehydration. To satisfy these two objectives, the third objective was to develop a new laser-based three-dimensional coordinate digitizing system (3-DCDS II) that can scan either the meniscus or articular cartilage surface within a time such that there is less than a 5% change in measurements due to dehydration. The new instrument was used to measure changes in meniscal and articular cartilage dimensions of six cadaveric specimens, which were exposed to air for 120 and 130 min, respectively. While there was no change in meniscal width, meniscal height decreased linearly by 4.5% per hour. Articular cartilage thickness decreased nonlinearly at a rate of 6% per hour after 10 min, and at a rate of 16% per hour after 130 min. The system bias and precision of the new instrument at 0 degrees slope of the surface being scanned were 0.0 and 2.6 microm, respectively, while at 45 degrees slope the bias and precision were 31.1 and 22.6 microm, respectively. The resolution ranged between 200 and 500 microm. Scanning an area of 60 x 80 mm (approximately the depth and width of a human tibial plateau) took 8 min and a complete scan of all five sides of a meniscus took 24 min. Thus, the 3-DCDS II can scan an entire meniscus with less than 2% change in dimensions due to dehydration and articular cartilage with less than 0.4% change. This study provides new information on the amount of time that meniscal tissue and articular cartilage can be exposed to air before marked changes in size and shape, and possibly biomechanical, structural and material properties, occur. The new 3-DCDS II designed for this study provides fast and accurate dimensional measurements of both soft and hard tissues.     

The effects of cryoprotectants on sperm motility of the Chinese pearl oyster,Pinctada fucata martensii

Cryopreservation has been widely employed to preserve genetic material of aquatic animals. Although of common use in bivalves, resulting effects due to the toxicity of the cryoprotectants dimethyl sulfoxide (DMSO), propanediol (PG), methanol (MET) and ethylene glycol (EG), upon sperm motility in the Chinese pearl oyster, Pinctada fucata martensii, has remained undocumented. This study endeavors to identify the least toxic among the effective cryoprotectant agents by observing and comparing their toxic effects on sperm motility under varying concentrations and duration of exposure. Sperm samples were exposed during controlled experiments, for 1, 3, 6, 9, 12 and 15 min durations, to each of the listed cryoprotectants at 5, 10, 15, and 20% (volume:volume) concentrations. Sperm motility was observed to diminish when exposed to all cryoprotectant solutions, and observations demonstrated that toxicity increased relative to both concentration and equilibration time. After 6 min of exposure to the cryoprotectants, sperm motility was seen to have diminished significantly in DMSO at just 5% concentration, and in MET, PG and EG at 10% concentrations, respectively (the values of the lowest observed effect concentrations). The relationship between the quantity of immotile sperm and the cryoprotectant concentration was described using the logarithmic regression equation. MET exhibited the lowest effective concentration required to inhibit sperm motility by 50% (EC₅₀), followed by EG, PG and DMSO, in order. Therefore, MET proved most toxic under the test conditions for sperm of P. fucata martensii, whereas DMSO, PG and EG were observed as comparatively safer, suggesting that DMSO, PG and EG warrant further study in the application of cryopreservation of Chinese P. fucata martensii sperm.     

The effect of oxygen tension on human articular chondrocyte matrix synthesis: Integration of experimental and computational approaches

Significant oxygen gradients occur within tissue engineered cartilaginous constructs. Although oxygen tension is an important limiting parameter in the development of new cartilage matrix, its precise role in matrix formation by chondrocytes remains controversial, primarily due to discrepancies in the experimental setup applied in different studies. In this study, the specific effects of oxygen tension on the synthesis of cartilaginous matrix by human articular chondrocytes were studied using a combined experimental‐computational approach in a “scaffold‐free” 3D pellet culture model. Key parameters including cellular oxygen uptake rate were determined experimentally and used in conjunction with a mathematical model to estimate oxygen tension profiles in 21‐day cartilaginous pellets. A threshold oxygen tension (pO₂ ≈ 8% atmospheric pressure) for human articular chondrocytes was estimated from these inferred oxygen profiles and histological analysis of pellet sections. Human articular chondrocytes that experienced oxygen tension below this threshold demonstrated enhanced proteoglycan deposition. Conversely, oxygen tension higher than the threshold favored collagen synthesis. This study has demonstrated a close relationship between oxygen tension and matrix synthesis by human articular chondrocytes in a “scaffold‐free” 3D pellet culture model, providing valuable insight into the understanding and optimization of cartilage bioengineering approaches. Biotechnol. Bioeng. 2014;111: 1876–1885. © 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

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.     

Dose–injury relationships for cryoprotective agent injury to human chondrocytes

Vitrification of articular cartilage (AC) could enhance tissue availability but requires high concentrations of cyroprotective agents (CPAs). This study investigated relative injuries caused by commonly used CPAs. We hypothesized that the in situ chondrocyte dose–injury relationships of five commonly used CPAs are nonlinear and that relative injuries could be determined by comparing cell death after exposure at increasing concentrations. Human AC samples were used from four patients undergoing total knee arthroplasty surgery. Seventy μm slices were exposed in a stepwise protocol to increasing concentrations of 5 CPAs (max = 8 M); dimethyl sulfoxide (Me₂SO), glycerol (Gly), propylene glycol (PG), ethylene glycol (EG), and formamide (FM). Chondrocyte viability was determined by membrane integrity stains. Statistical analysis included t-tests and nonlinear least squares estimation methods. The dose–injury to chondrocytes relationships for all CPAs were found to be nonlinear (sigmoidal best fit). For the particular loading protocol in this study, the data identified the following CPA concentrations at which chondrocyte recoveries statistically deviated significantly from the control recovery; 1 M for Gly, 4 M for FM and PG, 6 M for Me₂SO, and 7 M for EG. Comparison of individual means demonstrated that Gly exposure resulted in the lowest recovery, followed by PG, and then Me₂SO, FM and EG in no specific order. The information from this study provides an order of damage to human chondrocytes in situ of commonly used CPAs for vitrification of AC and identifies threshold CPA concentrations for a stepwise loading protocol at which chondrocyte recovery is significantly decreased. In general, Gly and PG were the most damaging while DMSO and EG were among the least damaging.     

Do changes in the mechanical properties of articular cartilage promote catabolic destruction of cartilage and osteoarthritis?

Osteoarthritis (OA) is a joint disease characterized by cartilage degeneration, a thickening of subchondral bone, and formation of marginal osteophytes. Previous mechanical characterization of cartilage in our laboratory suggests that energy storage and dissipation is reduced in osteoarthritis as the extent of fibrillation and fissure formation increases. It is not clear whether the loss of energy storage and dissipation characteristics is a result of biochemical and/or biophysical changes that occur to hyaline cartilage in joints. The purpose of this study is to present data, on the strain rate dependence of the elastic and viscous behaviors of cartilage, in order to further characterize changes that occur in the mechanical properties that are associated with OA. We have previously hypothesized that the changes seen in the mechanical properties of cartilage may be due to altered mechanochemical transduction by chondrocytes. Results of incremental tensile stress-strain tests at strain rates between 100%/min and 10,000%/min conducted on OA cartilage indicate that the slope of the elastic stress-strain curve increases with increasing strain rate, unlike the reported behavior of skin and self-assembled collagen fibers. It is suggested that the strain-rate dependence of the elastic stress-strain curve is due to the presence of large quantities of proteoglycans (PGs), which protect articular cartilage by increasing the apparent stiffness. The increased apparent stiffness of articular cartilage at high strain rates may limit the stresses borne and prolong the onset of OA. It is further hypothesized that increased compressive loading of chondrocytes in the intermediate zone of articular cartilage occurs as a result of normal wear to the superficial zone or from excessive impact loading. Once the superficial zone of articular cartilage is worn away, the tension is decreased throughout all cartilage zones leading to increased chondrocyte compressive loading and up-regulation of mechanochemical transduction processes that elaborate catabolic enzymes.     

Study of cryopreservation of articular chondrocytes using the Taguchi method

This study evaluates the effect of control factors on cryopreservation of articular cartilage chondrocytes using the Taguchi method. Freeze-thaw experiments based on the L₈(2⁷) two-level orthogonal array of the Taguchi method are conducted, and ANOVA (analysis of variables) is adopted to determine the statistically significant control factors that affect the viability of the cell. Results show that the type of cryoprotectant, freezing rate, thawing rate, and concentration of cryoprotectant (listed in the order of influence) are the statistically significant control factors that affect the post-thaw viability. The end temperature and durations of the first and second stages of freezing do not affect the post-thaw viability. Within the ranges of the control factors studied in this work, the optimal test condition is found to be a freezing rate of 0.61 ± 0.03 °C/min, a thawing rate of 126.84 ± 5.57 °C/min, Me₂SO cryoprotectant, and a cryoprotectant concentration of 10% (v/v) for maximum cell viability. In addition, this study also explores the effect of cryopreservation on the expression of type II collagen using immunocytochemical staining and digital image processing. The results show that the ability of cryopreserved chondrocytes to express type II collagen is reduced within the first five days of monolayer culture.