ROCK inhibitor prevents the dedifferentiation of human articular chondrocytes Emi Matsumoto, Takayuki Furumatsu ⇑ , Tomoko Kanazawa, Masanori Tamura, Toshifumi Ozaki Department of Orthopaedic Surgery, Science of Functional Recovery and Reconstruction, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikatacho, Kitaku, Okayama 700-8558, Japan article info Article history: Received 18 February 2012 Available online 1 March 2012 Keywords: Chondrocyte Dedifferentiation Redifferentiation ROCK inhibitor SOX9 Type II collagen abstract Chondrocytes lose their chondrocytic phenotypes in vitro. The Rho family GTPase ROCK, involved in orga- nizing the actin cytoskeleton, modulates the differentiation status of chondrocytic cells. However, the optimum method to prepare a large number of un-dedifferentiated chondrocytes is still unclear. In this study, we investigated the effect of ROCK inhibitor (ROCKi) on the chondrogenic property of monolayer- cultured articular chondrocytes. Human articular chondrocytes were subcultured in the presence or absence of ROCKi (Y-27632). The expression of chondrocytic marker genes such as SOX9 and COL2A1 was assessed by quantitative real-time PCR analysis. Cellular morphology and viability were evaluated. Chondrogenic redifferentiation potential was examined by a pellet culture procedure. The expression level of SOX9 and COL2A1 was higher in ROCKi-treated chondrocytes than in untreated cells. Chondro- cyte morphology varied from a spreading form to a round shape in a ROCKi-dependent manner. In addi- tion, ROCKi treatment stimulated the proliferation of chondrocytes. The deposition of safranin O-stained proteoglycans and type II collagen was highly detected in chondrogenic pellets derived from ROCKi-pre- treated chondrocytes. Our results suggest that ROCKi prevents the dedifferentiation of monolayer-cul- tured chondrocytes, and may be a useful reagent to maintain chondrocytic phenotypes in vitro for chondrocyte-based regeneration therapy. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Articular cartilage has a poor intrinsic capacity for healing, and cartilage lesion from trauma or degeneration can result in morbidity and functional impairment [1]. Cartilage repair strategies that in- clude arthroscopic debridement, bone marrow stimulation, osteo- chondral mosaicplasty, and autologous chondrocyte implantation are available for the treatment of symptomatic chondral and osteo- chondral lesions [2]. In chondrocyte-based approaches for the treat- ment of cartilage defects, several issues involved in a loss of chondrocytic phenotype during culture, which is called dedifferen- tiation, and requiring two surgeries (chondral tissue harvest and cultured cell implantation) are considered [3]. Monolayer-cultured articular chondrocytes leads to a process of dedifferentiation whereby the cells acquire a fibroblastic morphology and lose their chondrocytic properties [4–7]. The expression of chondrocyte-spe- cific genes, such as a1(II) collagen (COL2A1), aggrecan, SRY-type high-mobility-group box (SOX) 9, and SOX 5/6, is gradually down- regulated during cell multiplication in monolayer culture conditions [4–7]. Several authors have reported that three-dimen- sional culture systems, such as cultures in agarose, alginate beads, and the other scaffolds, recover the chondrogenic phenotype of dedifferentiated chondrocytes and induce their redifferentiation in vitro [4–6]. However, three-dimensional culture is not a cell-pro- liferating method to prepare a sufficient number of chondrocytes. Since primary chondrocytes that can be isolated from the native car- tilage is limited, a monolayer culture technique that stimulates cel- lular proliferation without inducing their dedifferentiation or hypertrophic differentiation is important for a clinical application of chondrocyte-based therapy. Several biochemical factors such as fibroblast growth factor (FGF)-2, insulin, insulin-like growth factor, and bone morphogenetic protein (BMP)-2 synergistically promote chondrocyte proliferation in a three-dimensional atelocollagen gel culture [8,9]. After the cell number-increasing step (FGF-2/insulin- supplemented monolayer culture), the combination of BMP-2, insu- lin, and triiodothyronine has the optimal effect on chondrocyte redifferentiation in the sequential gel culture system [9]. However, ex vivo three-dimensional culture systems accompanied by contin- uous growth factor treatments are expensive and complicated. In this study, we investigated the effect of a reasonable reagent, ROCK inhibitor (ROCKi), on the status of cellular dedifferentiation, redif- ferentiation, and proliferation in monolayer-cultured human artic- ular chondrocytes. The Rho family GTPases including RhoA, Rac1, and Cdc42 are members of the Ras superfamily of small GTPases [10,11]. Rho GTP- ases regulate various biological functions such as cell motility and gene expression by organizing the actin cytoskeleton [12]. ROCK, one of the RhoA downstream effectors, has an important role in inducing stress fiber formation and assembly of focal adhesions 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.02.127 ⇑ Corresponding author. Fax: +81 86 223 9727. E-mail address: matino@md.okayama-u.ac.jp (T. Furumatsu). Biochemical and Biophysical Research Communications 420 (2012) 124–129 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc