Human telomerase reverse transcriptase and glucose-regulated protein 78 increase the life span of articular chondrocytes and their repair potential

Masato Sato,Miya Ishihara,Toshihiro Nagai,Nagatoshi Kaneshiro,Joji Mochida,Genya Mitani,Hidetoshi Tahara,Kazuo Shin-Ya,Jeong Ik Lee

doi:10.1186/1471-2474-13-51

Abstract

BackgroundLike all mammalian cells, normal adult chondrocytes have a limited replicative life span, which decreases with age. To facilitate the therapeutic use of chondrocytes from older donors, a method is needed to prolong their life span.MethodsWe transfected chondrocytes with hTERT or GRP78 and cultured them in a 3-dimensional atelocollagen honeycomb-shaped scaffold with a membrane seal. Then, we measured the amount of nuclear DNA and glycosaminoglycans (GAGs) and the expression level of type II collagen as markers of cell proliferation and extracellular matrix formation, respectively, in these cultures. In addition, we allografted this tissue-engineered cartilage into osteochondral defects in old rabbits to assess their repair activity in vivo.ResultsOur results showed different degrees of differentiation in terms of GAG content between chondrocytes from old and young rabbits. Chondrocytes that were cotransfected with hTERT and GRP78 showed higher cellular proliferation and expression of type II collagen than those of nontransfected chondrocytes, regardless of the age of the cartilage donor. In addition, the in vitro growth rates of hTERT- or GRP78-transfected chondrocytes were higher than those of nontransfected chondrocytes, regardless of donor age. In vivo, the tissue-engineered cartilage implants exhibited strong repairing activity, maintained a chondrocyte-specific phenotype, and produced extracellular matrix components.ConclusionsFocal gene delivery to aged articular chondrocytes exhibited strong repairing activity and may be therapeutically useful for articular cartilage regeneration.

Highlights

Like all mammalian cells, normal adult chondrocytes have a limited replicative life span, which decreases with age
Young rabbit (YRA) chondrocytes proliferated faster than Old rabbit (ORA) chondrocytes during the entire observation period, but their growth rate gradually decreased until they ceased at about 40 d and 60 d after the initiation of culture, respectively
Unlike control cells, which stopped proliferating after 10-20 population doubling level (PDL), ORA + human telomerase reverse transcriptase (hTERT) and YRA + hTERT cells continued proliferating for about 35 and 50 PDL, respectively

Summary

Introduction

Normal adult chondrocytes have a limited replicative life span, which decreases with age. As a result, immortalized human cells, such as epithelial and fibroblast cells [6] and chondrocytes [7,8], have been used as models of cellular aging. Goldring [7] showed that primary human chondrocytes can be immortalized with retroviral transfection of 4 genes, including simian vacuolating virus 40 large T antigen and telomerase; stable transfection of hTERT in chondrocytes that are cultured in a monolayer allows maintenance of the proliferative capacity but not the chondrocyte phenotype. Piera-Velazquez et al [8] showed that exogenous expression of hTERT in chondrocytes that are cultured on polyhydroxyethylmethacrylate coated dishes increases their life span and maintains their chondrocyte phenotype.

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