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Cell Transplantation, Vol. 19, pp. 681–689, 2010 Printed in the USA. All rights reserved. Copyright  2010 Cognizant Comm. Corp.

Biological and Biomechanical Evaluations of Osteochondral Allografts Preserved in Cold Storage Solution Containing Epigallocatechin Gallate Jung Yoon Bae,* Dong-Wook Han,† Shigeyuki Wakitani,‡ Masashi Nawata,§ and Suong Hyu Hyon* *Department of Medical Simulation Engineering, Research Center for Nano Medical Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan †Department of Nanomedical Engineering, College of Nanoscience & Nanotechnology,Pusan National University, Busan, Korea ‡Department of Orthopedic Surgery, Osaka City University Graduate School of Medicine, Osaka, Japan §Department of Orthopaedic Surgery, Marunouchi Hospital, Matsumoto, Japan

The beneficial effects of (−)-epigallocatechin-3-O-gallate (EGCG) on the nonfrozen preservation of mammalian cells and tissues are generally not well understood. A storage solution containing EGCG was employed to test the hypothesis that EGCG is capable of extending the storage duration for the cold preservation of articular cartilages. Human articular cartilages were preserved in a storage solution composed of serum-free RPMI-1640 medium with 1% antibiotic-antimycotic solution and 1 mM EGCG at 4°C for 1, 2, and 4 weeks. The chondrocyte viability (CCK-8 assay), biochemical and immunohistochemical composition [glycosaminoglycans (GAG) and (type II) collagen], and biomechanical property (compressive elastic modulus) were assessed. The chondrocyte viability of the cartilages preserved with EGCG was significantly well maintained for at least 2 weeks with high content of GAG and total collagen. These beneficial effects of EGCG were confirmed by the immunohistochemical observations of well-preserved cartilaginous structures and delayed denaturation of the extracellular matrix in preserved cartilages. There was no significant difference in the compressive elastic modulus (MPa) between the cartilages preserved with and without EGCG. These results suggest that EGCG may play an effective role in preserving osteochondral allografts, which can be exploited in devising strategies for the long-term preservation of other tissues under cold storage conditions. Key words: Osteochondral allografts; Articular cartilages; Epigallocatechin gallate; Cold preservation

INTRODUCTION

chondral allograft resurfacing allows for the implantation of geographically matched, mature articular cartilage with viable chondrocytes, while avoiding donor site morbidity. Despite its clinical success (2,7), the fresh osteochondral allografts have great limitations with respect to the short storage time in which the cartilage properties are well retained (22). With advances in the surgical methods and the development of immunosuppressive agents, the number of tissue or organ transplants has increased substantially in recent years (6,24). Ideally, the tissues should be transplanted immediately from the donor to the recipient. However, immediate transplantation is not always possible, and, therefore, the problem of tissue preservation becomes very important for ensuring a successful transplantation. Hence, it is essential to develop storage solutions that can maintain the viability and function of the tissues or organs for longer periods. One of the most

Articular cartilage injury occurs frequently; however, articular cartilage cannot self-regenerate when there is limited intrinsic healing capacity (9,12). Although the natural history of the isolated cartilage lesions remains unknown, it is generally believed that articular cartilage injury may predispose the involved joint to an accelerated degeneration (31,35). This problem is magnified by the relative frequency of cartilage injuries. The limited ability of articular cartilage to repair after injury has led to the investigation of new therapeutic methods to enhance osteochondral regeneration (33,34). The unsuitability of total joint replacements for young, active individuals has provided the stimulus to search for alternative treatments in the field of biologic resurfacing of joints. For example, the use of fresh allografts has become popular for the treatment of articular cartilage lesions. Fresh osteo-

Received June 1, 2009; final acceptance April 21, 2010. Online prepub date: June 3, 2010. Address correspondence to Suong-Hyu Hyon, Department of Medical Simulation Engineering, Research Center for Nano Medical Engineering, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Tel: +81 75 751 4125; Fax: +81 75 751 4141; E-mail: [email protected]

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important requirements for success in clinical tissue transplantation is the use of tissues with a large number of viable donor cells. However, the current methods can result in a substantial loss of function and can lead to the damage and destruction of the cells and tissues. In the case of fresh allografts, surgical implantation must be performed within 1 week of graft harvest in order to maximize its viability (11), but appropriate serological testing, implemented to minimize the chances for the disease transmission, requires a minimum of 2 weeks. Therefore, the prolongation of the storage period has been of long-standing interest in the storage of articular cartilage grafts. Our study focused on (−)-epigallocatechin-3-O-gallate (EGCG), the predominant catechin from tea, because it has a wide range of pharmacological activities, including antioxidant, anticancer, antiproliferative, anti-inflammatory, and antimicrobial effects (1,25, 28). In addition to these biological activities of EGCG, its beneficial preservative effects on mammalian cells and tissues were also examined in order to design a cellor tissue-preserving medium/solution at physiological temperatures as described in our previous studies (3, 13,23). In the present study, a storage solution containing EGCG was employed in order to test the hypothesis that EGCG is capable of extending the storage duration for the cold preservation of articular cartilages. Our data suggest that EGCG-based storage solution can be exploited to devise strategies for the long-term preservation of other tissues under cold storage conditions. MATERIALS AND METHODS Cartilage Specimen Collection and Cold Preservation The human articular cartilages were obtained from the hip and the meniscus of eight patients (52–74 years of age) who were undergoing a total knee arthroplasty at Marunouchi Hospital, Matsumoto, Japan. The cartilage specimens (15–20 mm in diameter and 2–2.5 mm in thickness) were procured by osteotome from the donor under sterile conditions and were placed in saline for 0.5–1 h until the end of surgery. Immediately after surgery, the specimens were transferred in a storage solution [serum-free RPMI-1640 medium (Sigma-Aldrich Co., St. Louis, MO) with 1% antibiotic-antimycotic solution (including 10,000 units penicillin, 10 mg streptomycin, and 25 µg amphotericin B per ml, Sigma-Aldrich Co.)] with or without 1 mM EGCG (TEAVIGOTM, DSM Nutritional Products Ltd., Basel, Switzerland) and kept at 4°C. The specimens were then delivered to the senior investigator (Prof. Hyon) within 1 day after procurement. In addition, the fresh cartilages were delivered in a complete medium with 10% fetal bovine serum (FBS; Sigma-Aldrich Co.) at room temperature after the procurement from the donor. Because of this necessary pro-

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cessing delay between the procurement of tissue from the donor and its subsequent delivery, 1 day was set as the data point for the fresh specimen. The cartilage specimens of the pretreated groups in the biochemical tests for the chondrocyte viability and collagen content were rinsed three times with phosphate-buffered saline (PBS, pH 7.4) containing 1% antibiotics after 1-day incubation with the EGCG storage solution and, thereafter, the storage solutions were changed into serum-free medium without EGCG. All the procedures involving human subjects received prior approval from Marunouchi Hospital, Osaka City University Graduate School of Medicine and the Institutional Review Board of Institute for Frontier Medical Sciences, Kyoto University, and all the subjects provided their written informed consent. Immediately after receiving the cartilage tissues, the specimens were placed in either 20 ml of a storage solution with or without EGCG and then stored at 4°C for 1, 2, and 4 weeks without changing the storage solution (Fig. 1). At the end of each storage period, the biochemical, immunohistochemical, and biomechanical analyses were performed as described below. Chondrocyte Viability The number of viable cells was quantified indirectly using a highly water soluble tetrazolium salt [WST-8; 2(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazolium, monosodium salt] (Dojindo Lab., Kumamoto, Japan) that was reduced to formazan dye by mitochondrial dehydrogenase. The viability of the chondrocytes in the cartilages was found to be directly proportional to the metabolic reaction products obtained in WST-8 (17). According to the manufacturer’s instructions, the specimens following cold preservation were incubated with WST-8 in the last 4 h of the incubation period (24 h) at 37°C in the dark. The fresh cartilages (hip cartilages) and the cartilages preserved without any treatment (menisci) were regarded as the controls. The absorbance was determined at 450 nm in a microplate reader (VersaMaxTM, Molecular Device Co., Sunnyvale, CA). Hydroxyproline Content (Total Collagen Content) The hydroxyproline content of the tissue digest was determined as described in a previous report (30). Briefly, the papain digests were hydrolyzed with equal volumes of 6N hydrochloride at 115°C for 16–24 h. Chloramine-T hydrate (Sigma) and p-dimethylaminobenzaldehyde (Sigma) were added to hydrolyze the specimens, and the absorbance, immediately after the addition of the dye, was measured at 560 nm with a spectrophotometer. The hydroxyproline content of the specimens was determined by using the specimens that were preserved without EGCG as the standard.

OSTEOCHONDRAL ALLOGRAFTS PRESERVED EGCG SOLUTION

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Figure 1. The experimental scheme of this study. The cartilage specimens were procured, transferred in a storage solution with or without EGCG and kept at 4°C. The specimens were then delivered to the senior investigator (Prof. Hyon) within 1 day of procurement. Immediately after receiving the cartilage specimens, the specimens were refreshed with either 20 ml of a storage solution with or without EGCG and then stored at 4°C for 1, 2, and 4 weeks. At the end of each storage period, the specimens were transferred in a complete medium and incubated in a CO2 incubator for 1 day. After the incubation, the biochemical, immunohistochemical, and biomechanical analyses were performed as described in Materials and Methods.

Histological and Immunohistochemical Analyses At the completion of the predetermined preservation and incubation, each cartilage specimen was rinsed with phosphate-buffered saline (PBS, pH 7.4) and was immediately fixed with 2% glutaraldehyde, 2% paraformaldehyde, and 0.2% CaCl2, followed by embedding in paraffin. The tissue blocks were sectioned into 5-µm thickness, stained with either hematoxylin and eosin (H&E, for general evaluation) or with Safranin-O/fast green (for GAG content and distribution), and were immunostained with a rabbit monoclonal antibody (Ab) against type II collagen. The prepared sections were examined using an optical/fluorescence microscope (Biozero-8000, Keyence, Osaka, Japan). Biomechanical Analysis At each data point, cartilage specimens were removed from the storage solutions and placed immediately in a −80°C freezer until the biomechanical testing was completed. At the time of testing, each specimen was thawed at room temperature (25°C) in PBS containing proteinase inhibitors. A cartilage compression disk with an intact articular surface measuring 10 mm in diameter × 1 mm in thickness was made from each specimen. In order

to determine the compressive elastic modulus, this cartilage disk was then subjected to dynamic viscoelastic compression (Rheogel E-4000, UBM Co. Ltd., Japan) with 0.5% strain. All the data were collected at a frequency of 10 Hz. Statistical Analyses All variables were tested in three independent storages for each experiment, and each experiment was repeated twice (n = 6). The quantitative data were expressed as the mean ± SD. The statistical comparisons were carried out with a one-way analysis of variance (ANOVA, SAS Institute Inc., Cary, NC, USA), and were followed by the Bonferroni test for the multiple comparisons. A value of p < 0.05 was considered to be statistically significant. RESULTS Effect of EGCG on the Chondrocyte Viability of Cartilages The preservation of the hip cartilage specimens without EGCG resulted in a significant time-dependent decrease in the cell viability (p < 0.05) (Fig. 2). After 1 week the cell viability was already close to 39%, and

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Figure 2. Chondrocyte viability in hip cartilage (A) and meniscus (B) preserved with or without EGCG. The cell viability was measured with a WST-8 assay as described in Materials and Methods. The results are reported as the mean ± SD (n = 6) and the data were analyzed by the Bonferroni test (A: *p < 0.05 vs. fresh control, #p < 0.05 vs. nontreated control, and §p < 0.05 between co- and pretreated; B: *p < 0.05 between co- and pretreated).

was reduced to approximately 15% after 2 and 4 weeks. When the specimens were preserved by the cotreatment with 1 mM EGCG, the loss in viability was only 32% after 1 week and 54% after 2 weeks, thus indicating that EGCG suppresses the time-dependent cellular reduction in viability. Although this decrease in the viability accelerated with an increase in the incubation time, there was a significant difference between the cartilages preserved with and without the cotreatment with EGCG after 1 week (p < 0.05). However, the protective activity of the cotreated EGCG was relatively less effective after 4

weeks, thus demonstrating the viability to be reduced to approximately 40%. The viability of the specimens that were preserved by a pretreatment with 1 mM EGCG for 1 day was 54% after 1 week and 42% after 2 weeks. However, this viability was significantly reduced to 17% after 4 weeks. In the preservation of the meniscus specimens, the viability of the specimens preserved by the cotreatment with 1 mM EGCG was significantly higher after 2 weeks in comparison to the nontreated controls (p < 0.05) (Fig. 2B).


Effect of EGCG on Contents of Collagen in Cartilages The collagen content of the cartilages correlated well with their viability (Fig. 3). After 2 weeks of preservation by the cotreatment with EGCG, the collagen content was more than 2.5 times in hip cartilage and 2.2 times in the meniscus in comparison to the nontreated controls. For the preservation by a pretreatment with EGCG, the collagen content was measured as 228% in the hip cartilage and 190% in the meniscus, which was significantly higher in comparison to the preservation without EGCG (p < 0.05). There was a significant difference between the pre- and the cotreatments of EGCG in the hip cartilage (p < 0.05), but this difference was not observed in the meniscus. However, the collagen content of the meniscus with pretreated EGCG was still significantly higher in comparison to the storage without EGCG (p < 0.05). The storage with EGCG improved the preservation of the chondrocyte metabolism, in contrast to the storage without EGCG. Effect of EGCG on the Matrix Structure of Cartilages The routine histological and immunohistochemical examinations were performed on the hip cartilage specimens preserved with or without EGCG (Fig. 4). Throughout the H&E staining, it was difficult to differentiate between the cartilages that were preserved with and without EGCG (Fig. 4A). However, there was a numerical trend in favor of the specimens that were stored with EGCG for 2 weeks. The specimens stored with EGCG stained positively with Safranin-O, thus indicat-

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ing an abundant presence of GAG (Fig. 4B). In contrast, the preservation without EGCG resulted in an appreciable decrease in the positive staining for Safranin-O. The peripheral region of each cartilage had a higher cell density and stained more intensely for GAG in comparison to the central region. The cells in the lacunas of the fresh and EGCG-preserved cartilages were well maintained with an original chondrocytic phenotype in comparison to those cells preserved without EGCG. An immunohistochemical examination with monoclonal Ab against type II collagen showed the presence of this macromolecule in the cartilages preserved with EGCG, and as before, the cell density and staining intensity were greatest in the periphery of the specimens (Fig. 4C). In each of the specimens examined, type II collagen was primarily found intracellularly. The comparison of the cartilage tissues that stained positive for both GAG and type II collagen within the same specimen showed that the Safranin-O positive tissues had an usual association with type II collagen, an association typically seen in normal articular cartilage. Effect of EGCG on the Biomechanical Property of Cartilages Mechanical testing evaluated the function of the existing cartilage matrix, and in contrast to the GAG and collagen content, the biomechanical property appreciably increased with time. After the storage with and without EGCG for as long as 2 weeks, there were no significant changes, but the compressive elastic modulus was

Figure 3. The ECM composition of meniscus and hip cartilage preserved with or without EGCG after 2 weeks. The total cartilage collagen levels were determined as described in Materials and Methods. The results are reported as the mean ± SD (n = 6) and the data were analyzed by the Bonferroni test (*p < 0.05 vs. nontreated control and #p < 0.05 between co- and pretreated).

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Figure 4. The ECM structure of the hip cartilages preserved with or without EGCG after 2 weeks. The cartilage specimens were stained with either H&E (A) or Safranin-O/fast green (B, for GAG content and distribution), and immunostained with rabbit monoclonal Ab against type II collagen (C). The photographs shown in this figure are representative of six independent experiments, showing similar results.

slightly increased in the specimens preserved with EGCG (Fig. 5). DISCUSSION In this study, we found that articular cartilage stored with EGCG solution was capable of maintaining its ECM properties well. The chondrocyte viability of the cartilages preserved with EGCG was significantly well maintained with high cell viability, high content of total collagen, histological and immunohistochemical appearance of well-preserved cartilaginous structures, delayed denaturation of extracellular matrices (ECM), and almost the same compressive modulus in comparison to the nontreated controls. In the comparison of the storage of the cotreated and 1-day pretreated EGCG, the cartilages preserved by the cotreatment with EGCG had higher cell viability and collagen content after storage. Although the storage with pretreated EGCG had a less preservative effect than cotreated EGCG, there was a significant difference between cartilages preserved with and without pretreated EGCG. These results suggest that EGCG may actively penetrate into the cartilaginous

layer and may protect the matrix structure as well as the chondrocyte viability. The interest in fresh osteochondral allografts has increased remarkably because of numerous good clinical outcomes for the treatment of articular injury (4,10,36). However, there are significant practical limitations, such as the need to perform the surgical implantation within 1 week of graft harvest in order to maximize its viability (29). However, appropriate serological testing implemented to minimize the chances for disease transmission requires a minimum of 2 weeks. Therefore, a prolongation of the storage period of articular cartilage grafts has been of long-standing interest. Lactate Ringer’s solution has been used traditionally for the storage of fresh osteochondral allografts. However, this storage medium lacks the essential nutrients required for a sustained metabolic function of the chondrocytes (5). Lactate Ringer’s solution is elemental in constituency, with physiological concentrations of sodium, potassium, chloride, and bicarbonate, but it contains no nutrients to support the metabolism of chondrocytes. It was shown that culture medium was superior at preserving the chondrocyte via-


bility and the metabolic activity, and was therefore preferred over Lactate Ringer’s solution (5,18). In an effort to provide nutrients and prolong chondrocyte viability, the storage solution was changed to a standard culture medium containing amino acids, glucose, and inorganic salts. In most tissue banks, tissues are now stored in culture medium prior to transplantation. EGCG, the predominant catechin from tea, has a wide range of pharmacological activities, including antioxidant, anticancer, antiproliferative, anti-inflammatory, and antimicrobial effects (1,19,28). It is known that EGCG, due to its amphipathic properties, easily binds to ECM, lipid membranes, and any type of intracellular proteins (21,27). Therefore, it seems that this compound enables internalization into the cell cytosol and translocation into the nucleus, which leads to a modulation of the exogenous signals directed to genes that are required for survival and apoptosis. Although the exact mechanism of the internalization of EGCG into cells has not been elucidated, it was reported that EGCG was bound to the membrane was incorporated into the cytosol and the nucleus of cancer cell lines, such as the PC-9 and HT-29 cells (11,26). In addition, a recent study has demonstrated that the expression of the metastasis-associated 67 kDa laminin receptor may confer EGCG responsiveness to cancer cells at physiologically relevant concentrations, thus suggesting that the gallate moiety of EGCG may be critical for receptor binding and subsequent activity (32). Moreover, the observation that nucleic acids

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extracted from catechin-treated cells were colored implied that because both galloyl and catechol groups of EGCG were essential for DNA binding, both groups seemed to hold the strands of DNA via their branching structures (20). EGCG was also reported to have different effects on the proliferation of cancer versus normal cells; that is, EGCG has a cytotoxic effect on cancer cells, but a hibernatory effect on normal cells (8). Therefore, we examined in previous studies the cytopreservative effects on mammalian cells and tissues in order to design a cell- or tissue-preserving medium at physiological temperature (14–16). In this study, we performed the preservation of human menisci and hip cartilages in culture medium with or without EGCG in order to investigate the preservative effects of EGCG on the osteochondral grafts. The preservation of the cartilages resulted in a timedependent decrease in the cell viability, but storage with EGCG induced a delay in the decrease of the cell viability (Fig. 2). Because maintaining cell viability is highly important for transplantation, EGCG may be a useful storage agent for preserving osteochondral grafts. Another issue to be solved in cartilage preservation is the decrease in the ECM content and the compressive modulus that occurs with an increase in the storage time. These findings correlated highly with chondrocyte viability. Therefore, the collagen content of cartilage preserved with co- or pretreated EGCG was significantly higher in comparison to the nontreated controls (Fig. 3).

Figure 5. The biomechanical property of meniscus preserved with or without EGCG after 2 weeks. The compressive elastic modulus was determined by dynamic viscoelastic compression as described in Materials and Methods. The results are reported as the mean ± SD (n = 6) and the data were analyzed by the Bonferroni test.

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In contrast, in this study there was no significant difference for the compressive elastic modulus between the cartilages preserved with and without EGCG (Fig. 5). There are a number of explanations for these inconsistencies, including the measurement methods of the compressive modulus. We have to do further investigations and more biomechanical studies on the preservation of cartilage. Histologically, staining of the hip cartilage matrix, preserved in the medium without EGCG for 2 weeks, was lost mainly from the perichodrocyte area, thus suggesting that the chondrocytes that lost their viability had digested the matrix by themselves. However, the storage of the hip cartilages enabled the high-quality preservation of the cartilaginous structures and the delay of the denaturation of the extracellular matrices (Fig. 4). With respect to the storage in the culture medium without EGCG, there was a significant decline in the viability of the cartilaginous structure within 2 weeks and the following were observed in the histological specimen: increased disorganization, hypocellularity, pyknotic nuclei, and empty lacunae. The cartilaginous structure of hip cartilage stored with 1 mM EGCG was well retained until 2 weeks. The results of this study showed that the storage of articular cartilages in culture medium containing with EGCG provides an enhanced clinical effect on osteochondral storage in comparison to the storage in only culture medium. EGCG may play an effective role in preserving osteochondral allografts, and can be exploited in devising strategies for the long-term preservation of other tissues under cold storage conditions. This finding has important clinical implications because it suggests that the storage of osteochondral grafts has considerable relevance to the treatment of defects in human cartilages and provide the basis for the development of repair technology in articular injury. Further research on a more long-term follow-up basis and a detailed study of optimal conditions is necessary to establish the clinical use in orthopedic surgery. REFERENCES 1. Ahmed, S.; Pakozdi, A.; Koch, A. E. Regulation of interleukin-1beta-induced chemokine production and matrix metalloproteinase 2 activation by epigallocatechin-3-gallate in rheumatoid arthritis synovial fibroblasts. Arthritis Rheum. 54:2393–2401; 2006. 2. Aubin, P. P.; Cheah, H. K.; Davis, A. M.; Gross, A. E. Long-term followup of fresh femoral osteochondral allografts for posttraumatic knee defects. Clin. Orthop. Relat. Res. 391(Suppl.):S318–327; 2001. 3. Bae, J. Y.; Kanamune, J.; Han, D. W.; Matsumura, K.; Hyon, S. H. Reversible regulation of cell cycle-related genes by epigallocatechin gallate for hibernation of neonatal human tarsal fibroblasts. Cell Transplant. 18:459–469; 2009. 4. Bae, J. Y.; Matsumura, K.; Wakitani, S.; Kawaguchi, A.;

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