Evaluation of five additives to mitigate toxicity of cryoprotective agents on porcine chondrocytes

Wu, Kezhou; Laouar, Leila; Dong, Rachael; Elliott, Janet A.W.; Jomha, Nadr M.

doi:10.1016/j.cryobiol.2019.02.004

Cited by 14 publications

(7 citation statements)

References 59 publications

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“…19 Additives such as chondroitin sulfate, ascorbic acid, or glucosamine have been shown to improve porcine chondrocyte survival after exposure to a high molarity CPA cocktail solution. 20 Therefore, we present the vitrification of intact porcine femoral condyles using an optimized approach based on an established 7-hour protocol. Our aim is to develop a successful protocol for long-term storage of intact femoral allografts via vitrification for clinical repair of articular cartilage defects.…”

Section: Introductionmentioning

confidence: 99%

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Vitrification of Intact Porcine Femoral Condyle Allografts Using an Optimized Approach

Laouar

Elliott

et al. 2020

CARTILAGE

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Objective Successful preservation of articular cartilage will increase the availability of osteochondral allografts to treat articular cartilage defects. We compared the effects of 2 methods for storing cartilage tissues using 10-mm diameter osteochondral dowels or femoral condyles at −196°C: (a) storage with a surrounding vitrification solution versus (b) storage without a surrounding vitrification solution. We investigated the effects of 2 additives (chondroitin sulfate and ascorbic acid) for vitrification of articular cartilage. Design Healthy porcine stifle joints ( n = 11) from sexually mature pigs were collected from a slaughterhouse within 6 hours after slaughtering. Dimethyl sulfoxide, ethylene glycol, and propylene glycol were permeated into porcine articular cartilage using an optimized 7-hour 3-step cryoprotectant permeation protocol. Chondrocyte viability was assessed by a cell membrane integrity stain and chondrocyte metabolic function was assessed by alamarBlue assay. Femoral condyles after vitrification were assessed by gross morphology for cartilage fractures. Results There were no differences in the chondrocyte viability (~70%) of 10-mm osteochondral dowels after vitrification with or without the surrounding vitrification solution. Chondrocyte viability in porcine femoral condyles was significantly higher after vitrification without the surrounding vitrification solution (~70%) compared to those with the surrounding vitrification solution (8% to 36%). Moreover, articular cartilage fractures were not seen in femoral condyles vitrified without surrounding vitrification solution compared to fractures seen in condyles with surrounding vitrification solution. Conclusions Vitrification of femoral condyle allografts can be achieved by our optimized approach. Removing the surrounding vitrification solution is advantageous for vitrification outcomes of large size osteochondral allografts.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Vitrification of Intact Porcine Femoral Condyle Allografts Using an Optimized Approach

Laouar

Elliott

et al. 2020

CARTILAGE

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“…Notably, we have previously demonstrated that AA provides significant chondroprotection to AC during cryoprotectant exposure/removal and during short-term hypothermic storage. [25][26][27][28][29] We have also shown that using clinical grade AA (i.e., the pharmaceutical equivalent of this additive) provided similar chondroprotective effects to chondrocytes when compared with research grade AA, allowing our research group to translate AA supplementation into vitrification protocols intended for human cartilage. 27 More recently, our group developed a 2-step, dual-temperature, multi-cryoprotectant loading protocol that successfully vitrified AC cubes (pig and human) and showed that the cartilage maintained high cell viability and metabolic function immediately after vitrification and re-warming.…”

Section: Introductionmentioning

confidence: 94%

“…16,18,25 We have investigated the inclusion of supplemental additive compounds as a means to reduce CPA toxicity by mitigating oxidative stress. [26][27][28][29] Various antioxidant compounds such as chondroitin sulfate or ascorbic acid (AA) have been evaluated for their ability to protect cartilage during high exposure to CPA. Notably, we have previously demonstrated that AA provides significant chondroprotection to AC during cryoprotectant exposure/removal and during short-term hypothermic storage.…”

Section: Introductionmentioning

confidence: 99%

Chondrocyte Viability of Particulated Porcine Articular Cartilage Is Maintained in Tissue Storage After Cryoprotectant Exposure, Vitrification, and Tissue Warming

Crisol

Congdon

et al. 2023

CARTILAGE

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Objective Vitrification of articular cartilage (AC) is a promising technique which may enable long-term tissue banking of AC allografts. We previously developed a 2-step, dual-temperature, multi-cryoprotectant agent (CPA) loading protocol to cryopreserve particulated AC (1 mm3 cubes). Furthermore, we also determined that the inclusion of ascorbic acid (AA) effectively mitigates CPA toxicity in cryopreserved AC. Prior to clinical translation, chondrocytes must remain viable after tissue re-warming and before transplantation. However, the effects of short-term hypothermic storage of particulated AC after vitrification and re-warming are not documented. This study evaluated the chondrocyte viability of post-vitrified particulated AC during a 7-day tissue storage period at 4 °C. We hypothesized that porcine particulated AC could be stored for up to 7 days after successful vitrification without significant loss of cell viability, and these results would be enhanced when cartilage is incubated in storage medium supplemented with clinical grade AA. Design Three experimental groups were examined at 5 time points: a fresh control (only incubated in medium), a vitrified − AA group, and a vitrified + AA group ( N = 7). Results There was a mild decline in cell viability but both treatment groups maintained a viability of greater than 80% viable cells which is acceptable for clinical translation. Conclusion We determined that particulated AC can be stored for up to 7 days after successful vitrification without a clinically significant decline in chondrocyte viability. This information can be used to guide tissue banks regarding the implementation of AC vitrification to increase cartilage allograft availability.

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“…[4,9] Literature also states that incubation of cells/tissue in additives, such as ascorbic acid, glucosamine, chondroitin sulfate or their inclusion in cryo-preservative media reduces cryoprotectant toxicity induced cellular injury thereby improving cell viability on retrieval. [11,12] Till date, there are no reports on the membrane function of cryopreserved chondrocytes, comparing them with freshly isolated cells. One of the methods to assess membrane function is to study its ion channel population and their characteristics.…”

Section: Introductionmentioning

confidence: 99%

An electrophysiological comparison of freshly isolated caprine articular chondrocytes versus cryopreserved chondrocytes

Kachroo

Kanthakumar

2021

IJPP

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Objectives: Cryopreserved chondrocytes find numerous applications in reconstructive surgery, tissue engineering, and cell-based therapy. Cryopreserved chondrocytes may behave differently due to a change in cell biology. To assess phenotype maintenance, the electrophysiological profile of the cells can be studied. In this study, a comparison between freshly isolated and cryopreserved chondrocytes was made by recording ionic currents using patch clamp. Materials and Methods: Caprine articular chondrocytes were harvested and cryopreserved for 7–15 days and divided into two groups. Percentage cell viability was assessed, following which both fresh and cryopreserved cells were subjected to analysis in whole cell configuration using depolarizing voltage steps. Results: Outwardly, rectifying currents were recorded in both groups. Comparison of current densities at all potentials above the threshold, revealed no significant difference between fresh and cryopreserved chondrocytes. Conclusion: As electrophysiological properties of cryopreserved chondrocytes appear to be maintained, they may be readily utilized in place of fresh cells.

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Evaluation of five additives to mitigate toxicity of cryoprotective agents on porcine chondrocytes

Cited by 14 publications

References 59 publications

Vitrification of Intact Porcine Femoral Condyle Allografts Using an Optimized Approach

Vitrification of Intact Porcine Femoral Condyle Allografts Using an Optimized Approach

Chondrocyte Viability of Particulated Porcine Articular Cartilage Is Maintained in Tissue Storage After Cryoprotectant Exposure, Vitrification, and Tissue Warming

An electrophysiological comparison of freshly isolated caprine articular chondrocytes versus cryopreserved chondrocytes

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