Orthopaedic Surgery

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  1. Cryoprotectant agent toxicity in porcine articular chondrocytes. [Download]

    Title: Cryoprotectant agent toxicity in porcine articular chondrocytes.
    Creator: Jomha, N. M.
    Description: 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.
    Subjects: statistical analysis, viability, chondrocytes, articular cartilage, cryoprotectant agents, porcine, cryopreservation
    Date Created: 2010
  2. Permeation of several cryoprotectant agents into porcine articular cartilage [Download]

    Title: Permeation of several cryoprotectant agents into porcine articular cartilage
    Creator: Jomha, N.M.
    Description: 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 (10 mm diameter) were immersed in solutions containing 6.5 M concentrations of four commonly used CPAs [dimethyl sulfoxide (Me2SO), propylene glycol (PG), ethylene glycol (EG) and glycerol] for different times (1 second, 1, 2, 5, 10, 15, 30, 60, 120, 180 minutes, 24 hours) at three different temperatures (4, 22, and 37 C). The cartilage was isolated and the amount of CPA within the matrix was determined. Results: Diffusion coefficients (Me2SO = 2.4 - 6.2 x 10-6 cm2/s; PG = 0.8 - 2.7 x 10-6 cm2/s; EG = 1.7 - 4.2 x 10-6 cm2/s; and glycerol = 0.8 - 2.4 x 10-6 cm2/s) and activation energies (Me2SO = 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.
    Subjects: activation energy, diffusion coefficient, cryoprotectant agents, cryopreservation, vitrification, porcine, permeation, articular cartilage
    Date Created: 2014/09/26
  3. Vitrification of intact human articular cartilage. [Download]

    Title: Vitrification of intact human articular cartilage.
    Creator: Jomha, N. M.
    Description: 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.
    Subjects: cartilage tissue engineering, transplantation, human, arthritis, vitrification, articular cartilage
    Date Created: 2012
  4. Errata to "Permeation of several cryoprotectants in porcine articular cartilage" [Download]

    Title: Errata to "Permeation of several cryoprotectants in porcine articular cartilage"
    Creator: Jomha, N.M.
    Description: Some minor errors in our published manuscript need to be addressed
    Subjects: errata
    Date Created: 2014/10/08
  5. Statistical prediction of the vitrifiability and glass stability of multi-component cryoprotective agent solutions. [Download]

    Title: Statistical prediction of the vitrifiability and glass stability of multi-component cryoprotective agent solutions.
    Creator: Weiss, A. D.
    Description: 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.
    Subjects: vitrification, ethylene glycol, articular cartilage, dimethyl sulphoxide, logistic regression, glass stability, glycerol, cryoprotective agents, propylene glycol, formamide
    Date Created: 2010
  6. Dose-injury relationships for cryoprotective agent injury to human chondrocytes. [Download]

    Title: Dose-injury relationships for cryoprotective agent injury to human chondrocytes.
    Creator: Fahmy, M. D.
    Description: 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.
    Subjects: toxicity, vitrification, injury, cryopreservation, cryoprotective agents, human, dose, chondrocytes
    Date Created: 2014
  7. Clinical efflux of cryoprotective agents from vitrified human articular cartilage. [Download]

    Title: Clinical efflux of cryoprotective agents from vitrified human articular cartilage.
    Creator: Yu, H.
    Description: In previous research, we successfully cryopreserved intact human articular cartilage on its bone base with high chondrocyte viability using a vitrification protocol that entailed sequential exposure to several cryopreserving agents (CPAs) at lowering temperatures resulting in a high final concentration of CPA. The CPA must be removed from the cartilage at warming due to its toxicity to cells in the cryopreserved tissue and the post-transplant adjacent tissues. The current experiment explores the relationship between removal solution volume and time required for complete removal of CPA from bone–cartilage samples. Osteochondral dowels of 10 mm diameter from five patients undergoing total knee arthroplasty were vitrified using our protocol resulting in 6.5 M CPA within the matrix. In the primary experiment, the warmed dowels were immersed in 10 mL of X-VIVO for 30 min and this was repeated 5 times (the last wash being 5 min only). Removal solution osmolality was recorded at various times and compared to controls of pure X-VIVO. Changes in removal solution osmolality over time were normalized to tissue volume. In a secondary experiment, the procedure was repeated using double the volume of removal solution (20 mL X-VIVO). Results showed a rapid change in the osmolality of the removal solution indicating a rapid efflux of CPA from cartilage. The efflux rate decreased with time and during subsequent immersions until equilibrium was reached during the 4th immersion indicating effectively complete removal of CPA. Doubling the amount of removal solution demonstrated the effective removal of CPAs by the third immersion. The results of this study yield a practical relationship between the amount of removal solution and the time and number of immersions required to remove CPA from the transplantable tissue.
    Subjects: vitrification, dilution, cryoprotective agents, efflux, articular cartilage
    Date Created: 2013
  8. Cryoprotective agent toxicity interactions in human articular chondrocytes. [Download]

    Title: Cryoprotective agent toxicity interactions in human articular chondrocytes.
    Creator: Almansoori, K. A.
    Description: 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.
    Subjects: cryoprotectant agents, toxicity, cryopreservation, interactions, vitrification, chondrocytes
    Date Created: 2012