Nadr M. Jomha

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.

Cryopreservation of intact human articular cartilage

Damaged articular cartilage (AC) impairs joint function and many treatment techniques are being investigated to determine their long term results. Successful cryopreservation of AC can provide a reliable source of intact matrix with viable chondrocytes to maintain the cartilage over long periods of time. This study investigated the application of an established cryopreservation protocol to determine the recovery of intact chondrocytes from human AC. Ten millimeter diameter osteochondral dowels were harvested from two human donors. The cryopreservation protocol was performed and the samples were rapidly warmed from varying experimental holding temperatures (−10, −20, −30, −40°C), with and without plunging into liquid nitrogen, using 1 M dimethyl sulfoxide as cryoprotectant. The cartilage was stained with membrane integrity dyes and viewed under fluorescence microscopy. The percent of intact chondrocytes was compared to fresh controls. Low recovery of intact chondrocytes was recorded from all temperature levels with and without cryoprotectant. The results of this experiment demonstrated that the cryopreservation procedure used to achieve moderate success with intact sheep AC was not successful with intact human AC and further investigation is required.

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.

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.

Effects of introducing cultured human chondrocytes into a human articular cartilage explant model

Articular cartilage (AC) heals poorly and effective host-tissue integration after reconstruction is a concern. We have investigated the ability of implanted chondrocytes to attach at the site of injury and to be incorporated into the decellularized host matrix adjacent to a defect in an in vitro human explant model. Human osteochondral dowels received a standardized injury, were seeded with passage 3 chondrocytes labelled with PKH 26 and compared with two control groups. All dowels were cultured in vitro, harvested at 0, 7, 14 and 28xa0days and assessed for chondrocyte adherence and migration into the region of decellularized tissue adjacent to the defects. Additional evaluation included cell viability, general morphology and collagen II production. Seeded chondrocytes adhered to the standardized defect and areas of lamina splendens disruption but did not migrate into the adjacent acellular region. A difference was noted in viable-cell density between the experimental group and one control group. A thin lattice-like network of matrix surrounded the seeded chondrocytes and collagen II was present. The results indicate that cultured human chondrocytes do indeed adhere to regions of AC matrix injury but do not migrate into the host tissue, despite the presence of viable cells. This human explant model is thus an effective tool for studying the interaction of implanted cells and host tissue.

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.

Clinical efflux of cryoprotective agents from vitrified human articular cartilage

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 10mm diameter from five patients undergoing total knee arthroplasty were vitrified using our protocol resulting in 6.5M 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.