The modulation of the oxidative stress response in chondrocytes by Wip1 and its effect on senescence and dedifferentiation during in vitro expansion Byung-Hyun Cha a , Ji-Seon Lee b , Sung Won Kim c , Hyuk-Jin Cha b, ** , Soo-Hong Lee a, * a Department of Biomedical Sciences, CHA University, Republic of Korea b Department of Life Science, College of Natural Science, Sogang University, Seoul, Republic of Korea c Department of Otolaryngology-Head and Neck Surgery, Seoul St. Mary’s Hospital & The Catholic University of Korea, Republic of Korea article info Article history: Received 27 October 2012 Accepted 13 December 2012 Available online 8 January 2013 Keywords: Chondrocyte Oxidative stress modulator Wip-1 Senescence Dedifferentiation Cartilage tissue development abstract Obtaining a sufficient number of cells ex vivo for tissue regeneration, which are appropriate for cartilage repair, requires improved techniques for the continuous expansion of chondrocytes in a manner that does not change their innate characteristics. Rapid senescence or dedifferentiation during in vitro expansion results in loss of chondrocyte phenotype and the formation of fibrous cartilage replacement tissue, rather than hyaluronic cartilage, after transplantation. As demonstrated in the current study, wild- type p53-inducible phosphatase (Wip1), a well-established stress modulator, was highly expressed in early-passage chondrocytes, but declined rapidly during in vitro expansion. Stable Wip1-expressing chondrocytes generated by microporation were less susceptible to the onset of senescence and dedif- ferentiation, and were more resistant to oxidative stress. The increased resistance of Wip1 chondrocytes to oxidative stress was due to modulation of p38 mitogen-activated protein kinase (MAPK) activity. Importantly, chondrocytes expressing Wip1 maintained their innate chondrogenic properties for a longer period of time, resulting in improvements in cartilage regeneration after transplantation. Chondrocytes from Wip1 knockout (Wip1 / ) mice were defective in cartilage regeneration compared with those from wild-type mice. Thus, Wip1 expression represents a potentially useful mechanism by which a chondrocyte phenotype can be retained during in vitro expansion through modulation of cellular stress responses. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Cartilage is an important tissue that forms a flexible cushion- like layer at active joints to alleviate compression and frictional stress. The primary cellular component of cartilage is the chon- drocyte, and the flexibility of the tissue is entirely dependent on the level of complexity of the extracellular matrix (ECM), which includes chondrocyte-specific glycoproteins such as collagen II (Col II). Once cartilage is damaged, spontaneous regeneration is limited due to the lack of blood vessels surrounding and infiltrating the cartilage tissue. Therefore, chondrocyte implantation is proposed as one approach to tissue recovery after cartilage damage [1,2]. As with nearly all cell-based therapies, successful cartilage regenera- tion requires techniques for the expansion of clinical-grade chon- drocytes to obtain a sufficient number of cells for transplantation [3]. To this end, ex vivo monolayer cultures of isolated chondrocytes have been widely adopted due, in large part, to their simplicity. However, chondrocytes cultured under monolayer conditions undergo senescence and dedifferentiation, resulting in the loss of chondrocyte properties [4]. This results in the formation of unde- sirable fibrous cartilage at the transplantation site [5]. Senescence and dedifferentiation of chondrocytes in monolayer cultures is accompanied by fibroblastic morphological changes and decreased expression of chondrocyte-specific glycoproteins such as Col II, proteoglycans, and glycoproteins [5]. As is observed in other cell types, this loss of chondrocyte-specific properties is thought to be due to stress responses triggered in response to the ex vivo culture conditions [6,7]. Oxidative stress due to the increased production of reactive oxygen species (ROS) upon abnormal mechanical loading [8], damage, or aging [9] results in the onset of senescence or * Corresponding author. Department of Biomedical Science, CHA University, 502 Acecord Building 3rd Floor, Yatop-dong, Bundang-gu, Gyunggi-do 463-840, Republic of Korea. Tel.: þ82 31 8017 9415; fax: þ82 31 8017 9892. ** Corresponding author. College of Natural Sciences, Dept. of Life Sciences, Sogang University, Seoul, Republic of Korea. Tel.: þ82 2 705 4761; fax: þ82 2 704 3601. E-mail addresses: hjcha@sogang.ac.kr (H.-J. Cha), soohong@cha.ac.kr (S.-H. Lee). Contents lists available at SciVerse ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2012.12.009 Biomaterials 34 (2013) 2380e2388