Tissue engineered cartilage constructs have potential clinical applications in human healthcare. Their effective utilization is, however, hampered by the lack of an optimal cryopreservation procedure that ensures their availability as and when required at the patient’s bedside. Cryopreservation-induced stress represents a major barrier towards the cryopreservation of such tissue constructs, and they remain a scientific challenge despite the significant progress in the long-term storage and banking of isolated chondrocytes and thin cartilage tissue slices. These stresses are caused by intra- and extracellular ice crystallization, cryoprotectant (CPA) toxicity, suboptimal rates of cooling and warming, osmotic imbalance, and altered intracellular pH that might cause cellular death and/or a disruption of extracellular matrix (ECM). This paper reviews the cryopreservation-induced stresses on tissue engineered cartilages and discusses how they influence the integrity of the tissue during its long-term preservation. We have also reported how various antioxidants, vitamins, and plant extracts have been used to inhibit and overcome the stress during cryopreservation and provide promising results. Based on the reviewed information, the paper has also proposed some novel ways which might help in increasing the postthawing cell viability of cryopreserved cartilage.
Defects and diseases of articular cartilage are common ailments in humans. Osteoarthritis, the most common form of arthritis involving the inflammation of the articular cartilage, is observed in 60–70% of the people above the age of 65 [
Preservation of tissue engineered articular cartilage is also essential for their widespread commercialisation so that they can be provided to patients as and when required. The growing need and limited availability of viable transplantable cartilaginous tissues have necessitated the development and optimization of the preservation techniques and banking of tissue engineered cartilage constructs. Preservation of tissue engineered constructs also enables the tissue to undergo extensive immunological testing, size/contour matching, and optimal timing for supply whenever demanded by patient or surgeon [
There are various techniques of cryopreservation such as preserving at 4°C to −80°C and storing by a controlled rate freezing, or by plunging in LN2, by vitrification (Table
Existing methods for the cryopreservation of tissue engineered constructs.
Preservation method | Storage temperature | Storage medium | Duration of storage | Advantage | Disadvantage | Reference |
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Domestic freezer | 4°C | Culture medium often without any addition of CPA | 14–28 days | No ice crystal formation, |
Short storage period which is insufficient to carry out immunological and biomechanical assays which are to be carried before transplantation, decrease in cellular activity with an increase in time | Bae et al., 2009 [ |
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Deep freezer | −80°C | CPA or without CPA | 30–60 days | No complex device required, low running cost | Critical temperature for preservation of the 3D construct and the sensitive cells seeded on it, uncontrolled freezing, ice crystals formation | Bakhach, 2009 [ |
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Controlled rate freezing | A stepwise method can be up to −150°C | CPAs are added | As long as desired when sample is plunged in LN2 | Low concentration of CPA, cooling is carried out at a constant cooling rate so as to cause minimum damage to the tissue, allows the cells to dehydrate in equilibrium with the partially frozen cryoprotectants and other extra solutions present in the suspension | Optimized protocols required for each biological product, needs a skilled and experienced person to handle the entire process so that cooling is optimized enough to allow water to efflux out from the cell during extracellular ice formation |
Enneking, 1991 [ |
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Liquid nitrogen LN2 | Ultra low temperature |
CPAs are added | As long as desired with reliable LN2 back up | Rapid cooling method, simple handling, mechanically reliable | Risk of contamination via LN2, LN2 is costly; therefore, a high running cost as regular supply of LN2 is required | Salai et al., 1997 [ |
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Vapour phase LN2 | Temperature below −150 | CPAs are added | As long as desired with reliable LN2 back up | Eliminate the risk of cross-contamination from other samples and microbes | Vapour phase varies from protocol to protocol and the geometry of the tissue, there is often temperature fluctuation within the vapour phase, volume occupied within the container is nonplanned, regular supply of LN2 is required |
Tissue engineering Clemens van blitterswijk—chapter 13 by Lilia Kuleshova and Dietmar Hutmacher [ |
Commercial media used in cryopreservation of cartilage.
Many researchers have addressed the complexity and difficulty in the cryopreservation of tissue engineered cartilages which continues to remain a challenge [
In many cases, it is believed that when a cell commits to cell death, first apoptosis is activated and continues via the classical apoptotic pathway till the stress experienced by the cell exceeds the normal limits or the energy reserves become too low for apoptosis to continue. It is at this point that the cell switches from apoptotic to necrotic pathway (causing secondary necrosis) [
The physiochemical environment within the engineered cartilage is determined by various parameters such as matrix hydration, interstitial ion concentration, fixed charge density, and activity coefficients for specific ions within the extracellular matrix (ECM) of the cartilage [
In healthy cartilage water, along with other extracellular components, a gel-like matrix is formed which helps the cartilage to perform its function of shock absorption. In diseases such as osteoarthritis much of the water is lost from the cartilage. Similar loss of water is also observed during the cryopreservation of cartilages. The formation of extra-cellular ice crystals during freezing compresses the tissue leading to the exudation of water from the tissue. Release of water from the cells and the ECM of the cartilage strongly affects the transportation of ions and interferes with the physiochemical environment of the tissue. With the increasing loss of water the concentration of proteoglycans inside the tissue goes on increasing; as a result, the fixed charge density and associated interstitial ions also increase [
An immense loss of water from the tissue also causes a breakdown in the proteoglycan aggregates to which chondroitin and keratin sulphate GAG chains are attached making the tissue saggy [
Tissue engineered cartilaginous scaffolds already have various problems associated with biocompatibility, biodegradability, and self-assembling. Such constructs during the process of freezing and thawing further undergo an immense stress from which the fragile constructs are not able to shield themselves especially from the changes in osmotic pressure which makes the subtle tissue on the scaffold susceptible to swelling and cellular deformation [
During extracellular ice formation, the extracellular fluid becomes acidic, and the water from the tissue squeezes out to maintain the concentration gradient, reducing the net content of free water within the tissue. The flow of intra cellular and intramatrix water out of the tissue generates an increased osmotic pressure within the tissue which may lead to the formation of fissures. These fissures may later develop into sites of cell necrosis after thawing. Initially, it is only in the inner and middle layers of the cartilage, which gradually develops towards the articular surface.
It has been also reported by various researchers that extracellular pH is a significant regulator of cartilage matrix metabolism and activity of various enzymes. A slight change in the extracellular pH (less than 6.9) may significantly inhibit the matrix synthesis rate up to 50% [
Acidic pH conditions also activate various degradative enzymes. Example of such an enzyme is cathepsin K that shows an increased proteolytic activity in acidic environment. Cathepsin K can cleave the triple helix of collagen type I and II. Cathepsin K is also the only enzyme of the cytkosine protease family known to cleave the telopeptide domains as well as the
CPA addition or removal is one of the most crucial steps in the cryopreservation of cells, natural tissue, or engineered constructs. The presence of CPA induces a series of solution effects and chemical toxicity that cause chemical stress to the cell. CPAs usually have a concentration higher than that of the intracellular fluid when added to the tissue also causing a hypertonic shock for the cell resulting in an osmotic stress on the cells and the extracellular matrix. The cell shrinks necessitating the membrane to contract. Hypertonic shock triggers the activation of many ion channels and transporters (PMCA, NCX, NKCC etc.,) which may change the biosynthetic activity of the cell. They pump in large concentrations of Ca, Na, K, and Cl inside the cell, thereby increasing the ionic concentration of the cell. The intracellular pH of the cell decreases leading to an acidic condition inside the cell, and pH is also altered during the removal of the CPA resulting in an increase of pH. Furthermore due to cellular shrinkage multiple ion channels of the chondrocytes are known to undergo conformational changes. Abnormal morphology of the chondrocytes is a sign of cartilage degradation and cellular apoptosis. Extreme physiological conditions such as osmotic pressure, cellular shrinkage, and the increase in intra cellular Ca seem reasonable during the process of cryopreservation though they are the symptoms of cartilage degradation. Even intracellular cryoprotectants which remain in the cells after thawing are known to be toxic as they disrupt the acid-based equilibrium of the cell. Thus, CPA can damage the cell either due to the osmotic stress it renders on the cell, or due to the toxicity of the cryoprotectant itself.
Physiochemical stress can be prevented by controlling the ice crystallization and creating an osmotic balance within the cell and its surrounding ECM. Zheng et al. [
Chondrocytes are the only cell type present in the cartilage and are responsible for the synthesis and degradation of the articular cartilage matrix. For tissue engineered cartilage to be successful for clinical applications, the chondrocytes in the engineered cartilage should be able to synthesize normal ECM which primarily contains large aggregating proteoglycans such as aggrecan, type II collagen, and water. The type II collagen network provides strength to the cartilage while hydrated proteoglycans provide compressive resistance [
However, freezing and thawing is known to affect the biochemical properties of the engineered cartilage. Cryopreservation usually results in changes at the molecular, cellular, and tissue levels besides causing changes in hydrostatic pressure gradient and fluid flow which in turn can modify the synthesis and/or degradation of the macromolecules present in the extracellular matrix [
Ice crystallisation results in the increase of the ionic concentration of ECM and, hence, changes the chemical environment of the cell and its ECM. Various transport pumps present on the cell membrane that are sensitive to ionic and osmotic changes are denatured, and thus, membrane integrity gets destroyed. The intracellular organelles also get denatured due to changes in the ionic concentration, leading to cell membrane deformation and the initiation of cell death pathway. Extracellular ice is also reported to destroy the cell receptors present on the membrane of the chondrocytes. Formation of ice leads to the increase in the concentration of salts that denatures many proteins and lipoproteins. Integrins (
Biochemical stress may also be induced by the CPAs themselves. Many CPAs can block the ion channels and can imbalance the physiological ion homeostasis. Certain CPAs such as DMSO block the Ca+ and Na2+ ion channels within the chondrocyte membrane and alter the mithocondrial membrane potential to cause the mithocondrial dysfunction. These events aid in the slowing down of the cellular processes related to energy production, free radical detoxification, and maintenance of the chondrocytes. Mitochondrial dysfunction may also cause apoptosis, aging, and other cytopathic conditions leading to tissue destruction [
During cryopreservation of tissue engineered cartilaginous constructs, usually uneven cooling occurs due to complex nature of the scaffold. In such a condition, when the temperature is reduced from 35 to 15°C or 0 to −80°C, a series of chemical changes occur in the components of the ECM and the cellular membrane causing cell membrane lesions. This extreme change in temperature from normal physiological temperature to subzero causes a thermal shock to the tissue. It also causes ionic and hydraulic imbalance in the cell. Even in the absence of any intra- or extracellular ice this shock may cause a graft failure.
Furthermore, changes in the template of temperature cause various alterations in the ionic composition of the extracellular medium of the chondrocytes. Imbalance in the concentration of anions such as acetate, chloride, nitrate sulphate, and phosphate anions is another contributing reason for cell membrane damage [
Cryopreservation protocols, thus, need to be developed if the biochemical properties of the chondrocytes and the extracellular matrix are to be preserved. One approach is to add various nutrients and growth factors to the culture medium to provide a fast recovery of the cryopreserved chondrocytes [
Cryopreservation-induced mechanical stress on tissue engineered cartilage is one of the least studied areas in the field of cryopreservation. It can result from the physiochemical and biochemical events that occur during the process of cryopreservation. There are a few studies that have recently reported the microstructural changes and ECM swelling of the tissue along with the unequal distribution of interstitial fluid after thawing [
Although the mechanism is yet to be fully understood [
Mechanical injuries are obviously occuring due to intra- or extracellular ice crystal formation. Ice crystallization is known for its activation of stressors such as osmotic shock, hypothermia, ischemia, and ionic dysregulation which trigger necrosis. In cartilage cryopreservation, ice crystals have been reported to disrupt the extracellular matrix of the articular cartilage [
In certain cases when rate of growth of ice is dependent on the rate of diffusion of solutes away from the ice front, a slight decrease in the temperature may lead to an amplified supercooling of the inner mass of the tissue leading to sudden unplanned nucleation at various positions within the matrix and also leads to the formation of a lesion.
The addition of cryoprotectants and their removal are two crucial procedures followed during the cryopreservation of tissue engineered cartilage constructs [
During CPA loading, there is an increase in the ionic concentration of the fluid outside the cells. Chondrocytes osmotically respond to the increasing concentration of extracellular fluid by ejecting out most of the interstitial fluid present. Movement of the intracellular fluid out of the cell leads to cell shrinkage and ultimately dehydrates the cells. This also results in an increase of the intracellular pH, and as most of the interstitial fluid flows out of the cations, the fixed charges get concentrated within the chondrocytes [
The reverse mechanism occurs during CPA unloading. The ionic concentration of the fluid outside the tissue is far less compared to the concentration of the interstitial fluid. The ionic concentration gradually increases as more and more faces a gradual increase as more and more fluids from the surrounding tissue penetrate into the cell to osmotically balance the fluid conditions across the membrane [
Cartilage is an avascular tissue, and as a result the oxygen transport of matured cartilage is reduced compared to vascularised tissues. Despite the minimal consumption of oxygen by cartilage, oxygen is an essential requirement for the chondrocytes to survive and carry out other metabolic activities. Freezing the cartilage resists the delivery of oxygen and essential nutrients, removal of waste, and cell to cell communication. Under low oxygen conditions the ATP level of the cells, glycolysis, and matrix production also fall, damaging the cell physicochemical reactions. Low oxygen conditions are also known to damage the glucose transporters that cause changes in the synthesis of the extracellular matrix [
Another kind of oxidative stress is caused by the reactive oxygen radicals released during the freezing and thawing process. Reactive oxygen species (ROS) inhibit the synthesis of the proteoglycan by the chondrocytes, collagen and hyaluronic acid, and ECM degradation and initiates the release of LDH by damaging the cell membrane and damaging various transporters and pathways and depressing its metabolic pathway for energy production. A damage caused to chondrocyte membrane during freezing and thawing also transfers the cellular membrane injury to the mitochondria through the cytoskeleton. The mitochondria in turn releases ROS in high amount causing chondrocyte death and ECM degradation. ROS scavengers superoxide dismutase is reduced in the cryopreserved cartilage. Levels of ROS have been found to be greater in the cartilage of the people suffering from joint diseases [
Excess of ROS and oxidative stress trigger the mitochondrial or intrinsic apoptotic pathway of cell death. These stresses stimulate the opening of mitochondrial permeability transition pore (MPT). The opening of MPT causes loss in mitochondrial membrane potential and finally leading to the release of various proteins into the cytosol. Cytochrome c is an example of one such protein released by mitochondria, which may cause the release of apoptogenic proteins, such as cytochrome c, from mitochondria [
A number of cryoprotectants have been discovered since 1949, when Polge Smith and Parks discovered the cryoprotective nature of glycerol for bull sperms [
By the time a tissue engineered cartilage construct is subjected to thawing, the tissue has already been comprised with various critical temperatures and have gone under various stresses. Therefore, CPA must be mixed with other agents that may render a protective effect on the cells and tissues during freezing and thawing. Such agents are called extenders. Citrates, FBS, FCS, and BSA are examples of such compounds. These substances are added in the CPA along with buffers like PBS and HEPES in a particular amount, such that the concentration of the resultant solution is similar to that of the physiological solutions [
To overcome this problem, substances which act as free radical scavengers or inhibit generations of oxidants or compounds that induce the production of antioxidants are used. Scavengers of nonradical oxidants include catalase, n-acetylcysteine, and thiols; free radicals such as superoxide, hydroxy radicals, hydrogen peroxide, hypochlorite radicals, nitric oxide, and singlet oxygen are neutralized by the free radical scavengers such as ascorbic acid, superoxide dismutase (SOD), vitamins A, E and C, glutathione, carotenoid, flavonoid, polyphenols, and saccharide; and minerals such as selenium are mixed along with the cryoprotectants to defend the cartilage against oxidation or oxidative injury and to allow the tissue to undergo multiple free thaw cycles.
Cryopreservation of tissue engineered cartilage is an emerging field of tissue engineering and its importance is increasingly realized owing to its potential clinical importance and the vast commercialization scope. It is envisaged to be indispensible when tissue engineering becomes a common practice among scientists, researchers, and clinicians. However, a cryopreservation-induced stress remains to be a major hurdle in the effective application of the tissue engineered cartilages, and overcoming these stresses may enhance the success of cartilage cryopreservation. The problem faced during the cryopreservation of tissue engineered cartilage constructs is not limited to the maintenance of chondrocyte viability but, is also faced in maintaining functionality.