Biomaterials 25 (2004) 2949–2956 Proliferation and bone-related gene expression of osteoblasts grown on hydroxyapatite ceramics sintered at different temperature Chaoyuan Wang a,b, *, Yourong Duan a , Boban Markovic c , James Barbara d , C. Rolfe Howlett d , Xingdong Zhang a , Hala Zreiqat d a Engineering Research Center in Biomaterials, Sichuan University, Chengdu, Sichuan Province 610064, China b Yangtze Fisheries Institute, Chinese Academy of Fishery Science, Jingzhou, Hubei Province 434000, China c Chemical Safety and Toxicology Laboratories, School of Safety Science, UNSW, Sydney 2052, Australia d Bone Biomaterial Unit, Department of Pathology, School of Medical Science, UNSW, Sydney 2052, Australia Received 11 July 2003; accepted 22 September 2003 Abstract Human osteoblast-like cells SaOS-2 (ATCC HTB85) were seeded onto three kinds of hydroxyapatite (HA) ceramics sintered at different temperature (1200  C, 1000  C and 800  C). Scanning electron microscopy (SEM) was conducted to detect the surface microstructure. Cells were cultured on these substrates for 6 and 12 days and cell proliferation rate and mRNA expression for osteocalcin, osteonectin, type I collagen and alkaline phosphatase and protein production for osteocalcin, bone sialoprotein and osteonectin were detected with quantitative in situ hybridization and immunocytochemistry techniques. SEM revealed that crystal particle size was affected by sintering temperature. Result showed that cell proliferation rate on HA ceramics sintered at 1200  C was the highest. Osteonectin and type I collagen mRNA expression was not altered by sintering temperature. After 12 days in culture, bone sialoprotein, osteocalcin and osteonectin proteins levels were significantly (po0:05) higher when SaOS-2 cells were cultured on HA sintered at 1200  C, compared to the other two surfaces, suggesting that HA sintered at high temperature may be a better candidate for in vivo implantation. This result provides valuable information concerning the clinic application of HA ceramics sintered at different temperature. r 2003 Elsevier Ltd. All rights reserved. Keywords: Hydroxyapatite ceramics; Quantitative in situ hybridization; Protein; Cell proliferation; mRNA; Gene expression 1. Introduction Due to their excellent biocompatibility and bioactiv- ity, HA ceramics have been widely used in bone grafting and dental devices as bone substitute and since the discovery of their osteoinductivity, have attracted much research interest in the field of biomaterial in the past decade [1–4]. HA ceramics have the ability to induce mesenchymal cells to differentiate toward osteoblasts rendering it as a potential scaffold material for bone tissue engineering [5–7]. Substantial amount of work has been done to explore how sintering temperature may influence HA ceramic properties and its bioresponses after implantation. Sintering temperature can change the crystallinity of HA ceramics and its bioactivity, and thus, when sintered at different temperature, they possess different biome- chanical strength and other physical properties [8], osteogenicity [9] and initial bonding behavior with bone [10,11]. Sintering temperature of HA ceramics can also affect physiological changes in cells, as demonstrated by the ionic concentration changes in monocytes in vitro [12]. When the biological behavior of sintered and unsintered HA were compared, totally different proper- ties in protein absorption has been reported [13]. At the molecular biology level, different alkaline phosphatase and osteocalcin protein expression were reported with different sintering temperatures [14]. When they were implanted in vivo, different biological responses were also observed [9]. Although many studies have explored how sintering temperature may influence the osteoblast behavior, little is known about the molecular levels that determines the interaction between the ARTICLE IN PRESS *Corresponding author. Tel.: +86-716-8230185. E-mail address: wangcyscu2001@yahoo.com (C. Wang). 0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2003.09.088