Transduction of Anti-cell Death Protein FNK Suppresses Graft Degeneration After Autologous Cylindrical Osteochondral Transplantation

Noriki Nakachi ¹, Sadamitsu Asoh ¹, Nobuyoshi Watanabe ¹, Takashi Mori ¹, Takashi Matsushita ¹, Shinro Takai ¹ and Shigeo Ohta ^1*

¹ Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School, Kawasaki-city, Kanagawa, Japan (NN,SA,SO); Department of Orthopaedic Surgery, Teikyo University School of Medicine, Tokyo, Japan (NN,TMatsushita,ST); Institute of Medical Science, Department of Pathology, Saitama Medical Center/Saitama Medical University, Kamoda, Kawagoe, Saitama, Japan (TMori); and Department of Orthopaedic Surgery, Kyoto Kujo Hospital, Kyoto, Japan (NW)

^* To whom correspondence should be addressed. E-mail: ohta{at}nms.ac.jp.

Submitted on September 12, 2008
Accepted on 14 October 2008

	Abstract

This study demonstrates that artificial super anti-apoptotic FNK protein fused with a protein transduction domain (PTD-FNK) maintains the quality of osteochondral transplant by preventing chondrocyte death. Cylindrical osteochondral grafts were obtained from enhanced GFP (EGFP)-expressing transgenic rats, in which living chondrocytes express green fluorescence, and submerged into medium containing PTD-FNK, followed by transplantation into cartilage defects of wild-type rats by impact insertion simulating autologous transplantation. The tissues were histologically evaluated by hematoxylin and eosin, and Safranin-O staining. At 1 week, chondrocyte alignment was normal in the PTD-FNK treatment group, whereas all grafts without PTD-FNK treatment showed mixed cluster cell distribution. At 4 weeks, all grafts with PTD-FNK treatment showed almost normal matrix, whereas two grafts without PTD-FNK treatment showed fibrocartilage. Notably, all grafts with PTD-FNK retained high intensity of Safranin-O staining, but all grafts without PTD-FNK largely lost Safranin-O staining. PTD-FNK significantly suppressed a decrease in the survival rate and the density of EGFP-positive cells at 1 and 2 weeks, and this tendency continued at 4 weeks. The results of TUNEL staining showed that PTD-FNK inhibited cell death, indicating that PTD-FNK protects chondrocyte death and suppresses graft degeneration.

Key Words: apoptosis, Bcl-x_L, EGFP transgenic rat, mosaicplasty, osteochondral transplantation, protein transduction, Safranin O, TUNEL, articular cartilage

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