Address for correspondence : Hong-Ryul Jin, MD, PhD, Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University College of Medicine, Seoul National University Boramae Medical Center, 20 Boramae-ro 5-gil, Dongjak-gu, Seoul 07061, Korea
Tel : +82-2-870-2441, Fax : +82-2-870-3866, E-mail : doctorjin@daum.net

Introduction

In rhinoplasty, the nasal septum, the ribs, and the auricular cartilages have been widely used for graft purposes in various parts of the nose. Because re-harvesting of those cartilages is often necessary in revision surgery, some surgeons have attempted to cryopreserve the remained harvested cartilage at the time of primary septorhinoplasty to reduce associated morbidity.
The first report of ex-vivo cartilage preservation before transplantation was published in 1881 by Prudden,1) who immersed the cartilage in 95% alcohol. Since then, investigators have attempted to preserve functional chondrocytes, both isolated and in a matrix form, in an attempt to transplant viable tissue that is capable of functioning like intact cartilage.2) Till recently, incremental advances have been made focusing on the effective cryoprotective agent as well as the mechanisms of cell cryoinjury and cryopreservation temperature in several decades.3,4)
Because there is always a need for cartilage reuse in clinical practice, many rhinosurgeons still believe that cryopreservation of remaining cartilage is safe and these tissues can be effectively used later. Even though there have been numerous studies investigating the effect of cartilage cryopreservation which showed variable results,5,6,7) there are few studies reporting the clinical implication of those experimental results in the field of septorhinoplasty. This study was conducted to provide an overview on current knowledge in the cryopreservation or hypothermic preservation of cartilage through systemic literature review and to propose a best option in the ex-vivo preservation of cartilage.

Method

A critical review of the published literature using PubMed, EMBASE, and Cochrane Central Register of Controlled Trials (CENTRAL) with particular emphasis on studies which include cartilage cryopreservation was performed. Using databases, articles published from 1956 through 2011 for all available studies reporting cryopreservation of cartilage were investigated. The keywords used in this search included "cryopreservation," "nasal cartilage," "articular cartilage," "chondrocytes," and "rhinoplasty" alone and in various combinations using the Boolean operator "AND." The initial research identified 193 articles; all these titles were accurately screened by 3 authors to exclude articles not related to this topic, leaving a total 52 articles. Of which, 33 studies about ex-vivo preservation of cartilage were extracted by senior author (H.R.J.)

Cryopreservation: General Principle

Cryopreservation may be defined as the maintenance of biologics at sub-freezing temperatures, below -80℃ and typically below -140℃.2) The principle of cryopreservation dictates prevention of injury to the cells or tissues due to the toxic effects of cryopreservative solution and avoidance of intracellular ice formation. Cryopreservation method begins with hypothermic exposures which persist through the period of active extracellular ice growth until the glassy-state (vitrification). Vitrification action involves the exposure of a biologic to high concentrations of cryoprotective agents for short periods of time in order to quickly dehydrate the cell. As a cryopreservative agent, 10% dimethyl sulfoxide (DMSO) is widely used for the chondrocytes without being cytotoxic.8) Addition of cryoprotectant to the cells depresses the temperature at which intracellular ice is formed and thus allows cooling rates to be reduced for more efficient water loss.3) Many investigators suggest the use of two-stage cooling technique to achieve greater viability, in which the initial cooling is performed slowly up to -30℃ or -40℃, then rapidly cooled to -80℃.4,9) The majority of cell lines must be cooled -1 to -3℃/ min until -80℃ in the refrigerator or -196℃ in the liquid nitrogen tank.

Cryopreservation of Intact Cartilage Slice

The preservation of cartilage by freezing and storage at low temperatures for subsequent use in surgical procedures has been extensively practiced in the field of rhinoplasty in recent years.3,4,5,6,9) However, despite the early encouraging results with frozen cartilage allotransplantation in humans and experimental animals, many articles proved that cryopreservation irreversibly damages the cartilage cells.5,6,7) Findings demonstrated that viability rate of chondrocyte is 3-20% in intact cartilage slice and they are unable to produce new cartilage (Table 1). Particularly, it was noticed that cell survival was only confined to the superficial layer of the cartilage while intermediate or deep layer of the cartilage showed almost no cell recovery under any freezing condition.3,7,10,11,12) There are several reasons for the lack of cell survival in deeper than the superficial layer. The first is the timing of freezing and thawing. Surface cells are frozen more rapidly than cells deep in the matrix and are also thawed more quickly. This hinders penetration of cryopreservative agent into deeper part of cartilage. Besides, even though DMSO is well known for its extremely rapid penetration of skin and other tissues of the body, its rate of penetration and concentration throughout a cartilaginous matrix is still unknown. Cartilaginous matrix consists of highly charged proteoglycans macromolecules in a collagenous weave that is diffused with water. When one freezes cells in a matrix, DMSO must penetrate this matrix and the cell membrane, which suggests poor diffusion of these agents through the dense matrix of the cartilage.13) Thus viability can vary among the cartilage layers. For similar reason, the viability of septal cartilage is generally known to be poorer than articular cartilage although they are both hyaline cartilage. Unlike articular cartilage, nasal septal cartilage contains perichondrium in spite of proper cleansing, a relatively homogeneous population of cartilage cells and lower cellular density.14) These characters are thought to be the factor reducing viability of septal cartilage more than articular cartilage.
Water in the form of ice, both extracellular and intracellular, is one of the major causes of cell damage during freezing too.15) One can easily imagine the damage to cells when temperatures fall from 37℃ to -196℃. This causes up to about 95% loss of intracellular water, a considerable increase in electrolyte concentrations in both intra and extracellular media which modifies and denatures proteins and lipoprotein complexes and possible ice crystal formation in the intracellular spaces that deform and compress cells. Necrosis from ice formation, osmotic changes and apoptotic processes directly decrease viability.3,16,17,18,19)
Schachar, et al.20) investigated the fate of the cryopreserved cartilage after transplantation. Their experiment demonstrated the survival and function of transplanted cartilage by quantitative assessment of metabolic and biochemical parameters and concluded that the chondrocytes in osteochondral dowels cryopreserved in 7.5% DMSO and cooled at 1℃/min down to -70℃ maintained some functional activity although it was significantly less than the untreated control group at 12 months after transplantation. These cryopreserved dowels also demonstrated a thinner cartilage after 12 months indicating some breakdown of the cartilage matrix.
Requirements for acceptable storage of cartilage grafts should include preserved composition of the extracellular matrix (ECM) and metabolic activity such that the integrity of the graft may be maintained in the long term after transplantation. But the cryopreservation of cartilage continues to present a challenge, because the storage methods have the disadvantage of diminishing or eliminating cell viability.

Cryopreservation of Isolated Chondrocytes

Many studies of cartilage cryopreservation have based their results on isolated chondrocytes and not on the whole tissue.3,19,21,22) In 1965, Smith pioneered work on freezing chondrocytes from a variety of species including the human and succeeded in preserving isolated cells in cartilage by freezing to -79℃ with DMSO from 4℃ to -20℃ at a rate of 1℃/rain and from -20℃ to -79℃ at a rate of 4℃/rain. After storage for 7 days at -79℃, cell survival was estimated to be as high as 95%.23) Since then, many investigators have successfully isolated cartilage cells after enzymatic digestion and the cell survival rate was reported as 80-90% (Table 2).6,24,25)
Among many protocols for effective cryopreservation, Rendal-Vázquez, et al.25) proved that controlled freezing using a freezing rate of -1℃/min to a temperature of -40℃, 2℃/min to -60℃, and 5℃/min to -150℃ was the best method to preserve cellular functions. Although viable chondrocytes have been recovered, it is important to maintain their ability to synthesize ECM. Type II collagen and proteoglycan are the most abundant proteins of both nasal and articular cartilage and their content is vital not only because it is a marker of the hyaline phenotype but also because its concentration is directly related to the tensile strength of the tissue.26) Studies have demonstrated that the presence of viable chondrocytes is the fundamental basis for the reconstructive procedure, as chondrocytes must be present to maintain the production of collagen and proteoglycan to prevent degeneration over time.27,28) Enneking and Campanacci29) reported on frozen osteochondral allograft retrievals, which were entirely devoid of viable chondrocytes. In their study, the integrity of the acellular cartilage matrix was maintained in retrievals up to 2 to 3 years, but degeneration was seen in the longer term. This suggests that the ECM can withstand mechanical joint forces for a short period but that viable chondrocytes are necessary to sustain the matrix in the long-term.
Cartilage tissue has limited capacity for repair and little capacity for regeneration. As there are no blood vessels in cartilage, there is no source for undifferentiated cells to develop new tissue. Thus, any injury during cryopreservation, can have a significant deleterious effect on the quality of cartilage causing chondrocyte cell death, leading to a nonviable graft that may be resorbed gradually. Because live chondrocytes are necessary to maintain the integrity of the cartilage matrix, a dead homograft would gradually undergo resorption.30) Irradiation causes the formation of free radicals which leads to fragmentation of collagen, thereby probably accelerate homograft resorption according to Donald's study.30,31) Therefore, live autografts are the most desirable material because of their long-term survival without extensive resorption.11)

Hypothermic Preservaton of Cartilage

Several studies have analyzed the effects of hypothermic storage (4℃) on chondrocyte viability and articular cartilage mechanical properties with the use of culture media agent or lactated Ringer's solution rather than cryopreservative agent (Table 3).3,15,32) Most studies showed that chondrocyte viability and density, preservation of glycosaminoglycan content and biomechanical properties undergoes significant decreases from day 7.18,19) Brockbank, et al.33) assessed ECM and chondrocytes of porcine articular cartilage during refrigerated storage at 4℃ for 1 month and concluded that storage in culture medium provided good cartilage viability and metabolic function for 7 days, while the chondrocyte assessment values were <30% of fresh controls at 28 days. To analyze the effects of prolonged storage time at warm (23℃) and cold (4℃) temperatures, Hicks, et al.34) preserved human septal cartilage in bacteriostatic isotonic sodium chloride solution and then assessed the viability of the chondrocytes within the cartilage using confocal laser scanning microscopy every 5 days. After 1 week, cell survival in all specimens was essentially unchanged from the day of harvest. After 1 month, however, survived cells of specimens were 54% and 70% at 23℃ and 4℃, respectively. Thus, preservation of cartilage at 4℃ is more recommendable than 23℃ if hypothermic storage is needed. When comparing lactated Ringer's solution and culture media for storage solution, culture media provided significantly better preservation of the cartilage with viability and metabolic activity remaining essentially unchanged from baseline for as many as 14 days.17)

Conclusion

Cryopreservation of isolated chondrocytes using proper protocol successfully retrieved 80-90% viable cells with intact cellular function. On the contrary, short-term and long-term storage of intact cartilage with the current cryopreservation protocol showed very low viability of the cells. The tissue remained non-viable and was not able to originate new cartilage. Thus, such cartilage will be subject to resorption processes and not practical for reconstruction of parts of the skeleton subject to mechanical stress.
Currently, the best way preserving cartilage tissue ex-vivo after harvesting is preserving the tissue at 4℃ in culture media, because numerous studies proved that the tissue showed good viability with metabolic activity for 1-2 weeks. However application of this preservation method is not clinically practical because using culture media-preserved cartilage for revision within 2 weeks after surgery rarely occurs.

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