Although epigallocatechin-3-O-gallate (EGCG), the predominant catechin from tea, has various pharmacological and biological activities including anti-carcinogenic, anti-thrombotic and anti-inflammatory effects, relatively a little is known about its beneficial effects on the non-frozen preservation of mammalian cells and tissues. In the present study, a storage solution containing EGCG was employed to testify the hypothesis that cold preservation of osteochondral allografts was attributed to EGCG-mediated reversible regulation of cell cycle.

Human articular cartilages were obtained from knee joints of 10 patients (58 – 86 years old) undergoing total knee arthroplasty at Marunouchi Hospital, Matsumoto, Japan. Cartilage specimens were procured by osteotome under sterile conditions from the donor and placed in saline for 1 hr until the end of surgery. Immediately after surgery, the specimens were transferred in a storage solution (serum-free RPMI 1640 media with 1% antibiotic-antimycotic solution without or with 1 mM EGCG) and kept at 4°C. The specimens were then delivered to the senior author (Prof. Hyon) within 1 day from procurement. Additionally, fresh cartilages were delivered in a complete media with 10% fetal bovine serum at room temperature after procurement from the donor. Because of this necessary processing delay between tissue procurement from the donor and its delivery, 1 day was set as the data point for the fresh specimen. All 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 subjects providing written informed consent.

On receipt of the cartilage tissues, the specimens were replaced with either 20 ml of a storage solution without or with EGCG and then stored at 4°C for 1, 2 and 4 wk. At the end of each storage period, chondrocyte viability (CCK-8 assay), biochemical and immunohistochemical composition [glycosaminoglycans (GAG) and (type II) collagen], and biomechanical property (compressive elastic modulus) were assessed. The regulatory effects of EGCG on cell cycle distribution as well as expression levels of cyclins (CCNs) and nuclear factor-κB (NF-κB) were also investigated in articular chondrocytes.

Chondrocyte viability of cartilages preserved with EGCG was significantly well-maintained for at least 2 weeks with high contents of GAG and total collagen. These beneficial effects of EGCG were confirmed by histological and immunohistochemical observations showing well-preserved cartilaginous structures and delayed denaturation of extracellular matrices. The compressive elastic modulus of cartilages preserved with EGCG was almost in the same range as that of fresh ones. Moreover, the penetration of FITC-conjugated EGCG into the matrices of cartilages and its incorporation into the cytosol of cells in lacunae were observed. Increased cell population at the G0/G1 phase by EGCG returned to the normal level after EGCG removal, whereas decrease at the G2/M phase did not. Negatively regulated expression of CCND1, CCNE2 or NF-κB in EGCG-treated cells was restored by removing EGCG, but not CCNA2 and CCNB1. It is suggested that EGCG play effective roles in preserving articular cartilages by reversibly regulating cell cycle at G0/G1 phase and NF-κB expression.