Evaluation of the attachment, proliferation, and differentiation of osteoblast on a calcium carbonate coating on titanium surfaceYi Liua,1, Tao Jianga,1, Yi Zhoua, Zhen Zhanga, Zhejun Wanga, Hua Tongb, Xinyu Shenb, Yining Wanga,aKey Laboratory for Oral Biomedical Engineering, Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079, PR China bCollege of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, PR Chinaa b s t r a c ta r t i c l ei n f oArticle history: Received 9 September 2010 Received in revised form 11 January 2011 Accepted 6 March 2011 Available online 11 March 2011Keywords: Calcium carbonate Coating Surface topography In vitro OsteointegrationTitanium has been reported to have some limitations in dental and orthopaedic clinical application. This study described a coating process using a simple chemical method to prepare calcium carbonate coatings on smooth titanium (STi) and sandblasted and acid-etched titanium (SATi), and evaluated the biological response of the materials in vitro. The surfaces of STi, SATi, calcium carbonate coated STi (CC-STi) and calcium carbonate coated SATi (CC-SATi) were characterized for surface roughness, contact angles, surface morphology and surface chemistry. The morphology of MG63 cells cultured on the surfaces was observed by SEM andImmuno-fluorescence staining. Cell attachment/proliferation was assessed by MTT assay, and cell differentiation was evaluated by alkaline phosphatase (ALP) activity. MG63 was found to attach favorably to calcium carbonate crystals with longer cytoplasmic extensions on CC-STi and CC-SATi, resulting in lower cell proliferation but higher ALP activity when compared to STi and SATi respectively. Moreover, CC-SATi is more favorable than CC-STi in terms of biological response. In conclusion, the calcium carbonate coatings on titanium were supposed to improve the osteointegration process and stimulate osteoblast differentiation, especially in early stage. And this method could possibly be a feasible alternative option for future clinical application. 2011 Elsevier B.V. All rights reserved.1. IntroductionTitanium has been widely used in dental and orthopaedic implants due to its high biocompatibility, excellent mechanical properties and chemical stability 1,2. The biocompatibility of titanium is attributedto its surface oxide film that spontaneously forms when it is exposedto oxygen 3. However, this film with a thickness of 310 nm is usually very dense and stable, which makes titanium bioinert. The bioinert titanium can only be integrated with bone passively and unable to induce bone apposition 4. In order to improve the bioactivity of titanium and its alloys, various bioactive coatings have been developed, such as calcium phosphate, bioactive glasses and biologically functional molecules. Currently, plasma spraying is one of the most popular techniques for the preparation of calcium phosphate coatings 5. Short-term clinical and experimental studies have shown that plasma-sprayed hydroxyapatite (HA)-coated implants are able to result in strongerfixation and faster bone growth rate comparedto noncoated implants. However, there are still some concerns about the long term clinicalapplication of the plasma-sprayed HA-coated implants. Coating delamination and wear debris are reported as major problems associated with these coatings in some long term clinical observations as they cause osteolysis in the surrounding tissue 6,7. To overcome the drawbacks of plasma-sprayed HA-coated implants, various resorbable coatings, such as biodegradable bone- like carbonate apatite (BCA), octacalcium phosphate (OCP) 8,9, biodegradable Hydroxyapatite 10,11 and resorbable calcium phos- phate 11,12 have been developed. Development of the resorbable coatings is based on the thought that an optimal coating should dissolve in a rate similar to bone formation, and should completely disappear after completion of the bone-healing process 9. The total resorption of the coating may reduce the problems of plasma-sprayed HA-coated implants, which have been mentioned above. Besides calcium phosphate, calcium carbonate is also an important biomaterial which has been widely used in bone surgery 13,14. There are three crystalline and one amorphous phase of anhydrous calcium carbonates in nature, which are calcite, aragonite, vaterite and amorphous calcium carbonate. It has been demonstrated that calcium carbonate is a biocompatible and osteoconductive material either in the form of aragonite 13,1517 or in the form of calcite 18. Therefore, calcium carbonate might be an applicable candidate to prepare resorbable coating with improved biodegradation rates due to its higher solubility compared to apatite 19. Recently, Wang et al. 20 successfully fabricated a biologically nacre coating on dentalMaterials Science and Engineering C 31 (2011) 10551061 Corresponding author at: Key Laborat