Biomaterials 25 (2004) 4817–4824 Surface modification of poly(ethylene-co-vinyl alcohol): hydroxyapatite immobilization and control of periodontal ligament cells differentiation Kazuaki Matsumura a , Suong-Hyu Hyon a , Naoki Nakajima a , Hiroo Iwata a , Akira Watazu b , Sadami Tsutsumi a, * a Institute for Frontier Medical Sciences, Kyoto University, Shogoin, Sakyo-ku, Kyoto 606 8507, Japan b Institute for Structural and Engineering Materials, National Institute of Advanced Industrial Science and Technology, Chubu, Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463 8560, Japan Received 27 June 2003; accepted 22 November 2003 Abstract To reveal and control the differentiation and proliferation of the periodontal ligament (PDL) cells and to develop a highly organized hybrid implant possessing periodontium, hydroxyapatite (HAP) was immobilized on the poly(ethylene-co-vinyl alcohol) (EVA) by alternate soaking method following with carboxyl groups’ introduction through ozone exposure. Human PDL cells were cultured on the ozone-exposed EVA, collagen-immobilized EVA, HAP-immobilized EVA, HAP plate, tricalcium phosphate plate, and conventional tissue culture dish. Cell proliferation was highest on the collagen-immobilized EVA and lowest on the HAP- immobilized EVA. Alkaline phosphatase activity and osteocalcin secretion were highest on the HAP-immobilized EVA. These results suggest that PDL cells were differentiated toward bone-like cells on the HAP-immobilized EVA. r 2003 Elsevier Ltd. All rights reserved. Keywords: Poly(ethylene-co-vinyl alcohol); EVA; Periodontal ligament; Cementum; Regeneration; Implant; Hydroxyapatite 1. Introduction The periodontal ligament (PDL) lies between the cementum and the alveolar bone and plays quite important roles such as a shock absorber against the mastication impact and receptor of biting forces [1,2]. PDL is a fibrous connective tissue and contains heterogeneous cell populations including the fibroblast, osteoblast, blood vessel cells, nerve cells, and mesench- ymal stem cells [3–5]. Generally, the cells including all cells derived from PDL are called PDL cells. It is thought that these PDL cells have an ability to maintain the structure of the PDL and surrounding alveolar bone and cementum [6]. Usage of a guided tissue membrane for preventing the downgrowth of the gingival tissue is effective in the regeneration of the periodontal tissues in the case of periodontal disease [7,8], which suggests that the PDL cells are differentiated to suitable cell phenotypes in suitable places such as the cementoblasts in the cementum, fibroblasts in the fibrous tissue, and osteoblasts in the alveolar bone. Indeed, it was reported that the PDL has been regarded as a source of cementoblasts and osteoblasts [9]. PDL cells showed osteoblastic differentiation in response to dexametha- sone [10,11] and mechanical stress [12]. The bone-like properties were usually characterized by the upregula- tion of alkaline phosphatase activity [13,14] and osteocalcin mRNA [15,16]. However, the mechanism of PDL cell differentiation to bone-like cells is still unclear. Our previous reports showed the regeneration of PDL around an artificial implant by culturing PDL cells [17–19]. Briefly, this hybrid implant is explained as follows: Poly(ethylene-co-vinyl alcohol) (EVA) was coated on a titanium implant [17]. The adhesive strength between EVA and titanium was very high (about 15MPa) due to the electrostatic force and the water resistance was higher than other adhesives. ARTICLE IN PRESS *Corresponding author. Tel.: +81-75-751-4130; fax: +81-75-751- 4144. E-mail address: tsutsumi@frontier.kyoto-u.ac.jp (S. Tsutsumi). 0142-9612/$-see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2003.11.055