Titanium discs were made out of commercially pure titanium grade 4 and zirconia discs were made out of yttrium-stabilized tetragonal poly-crystals (Y-TZP). All discs had a diameter of 12 mm and a thickness of 3 mm. The sandblasted/acidetched titanium discs were purchased (Warantec Co., Seoul, Korea). The zirconia discs were sandblasted with 125-µm Al₂O₃ powders at 3.5 bar for 1 minute. After surface treatment, all the discs were washed in distilled water, cleaned in an ultrasonic bath, and sterilized by EO-gas.

The surface topography of the discs was examined by confocal laser scanning microscopy (LSM 5 Pascal, Zeiss, Obercochen, Germany).

MC3T3-E1 cells are the non-transformed cell line established from newborn mouse calvaria. These cells exhibit the osteoblastic phenotype, as evidenced by the expression of ALPase activity [14], the synthesis of extracellular matrix (ECM) components such as osteocalcin and type-1 collagen [15], and their ability to mineralize the ECM.

MC3T3-E1 cells were purchased from Riken BioResource Center (Shiga, Japan). MC3T3-E1 cells were grown in alpha MEM medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco) at 37℃ in a humidified atmosphere of 5% CO₂. The culture medium was replaced twice a week. The cells were plated on discs of zirconia and titanium in 24-well tissue culture plates. One day after cell seeding, the commercially available demineralized bone matrix (DBM) gel (Rafugen, human demineralized bone matrix, Korea Bone Bank, Seoul, Korea) was added (25 mg/600 µL of culture medium) to the culture medium adjacent to the zirconia and titanium discs, and the DBM gel with BMP-2 (including BMP-2 of 50 µg/mL) (Korea Bone Bank) was added in the same manner. The discs were classified into 6 groups: zirconia discs with DBM gel, zirconia discs with the DBM gel including BMP-2, titanium discs with DBM gel, titanium discs with the DBM gel including BMP-2, zirconia discs without gel, and titanium discs without gel.

The kinetics of BMP-2 release from the DBM gel with BMP-2 was examined (Fig. 1). 50 mg of the DBM gel with BMP-2 was placed in a tube and 1 mL of phosphate buffered saline (pH 7.4) was added to the tube as a releasing medium. The tubes were placed in a shaking incubator at 37℃ and shaken at 15 rpm. The supernatants were decanted and replenished with fresh phosphate-buffered saline (PBS) at predetermined time intervals over a 1-week period. The concentrated BMP-2 released from the gel was assayed using a BMP-2 immunoassay kit (Quantikine BMP-2, R&D Systems Inc., Minneapolis, MN, USA).

Cells were seeded onto the materials in 24-multiwell plates at a final density of 1×10⁴ cells/cm². One day later, commercially available DBM gel alone and the DBM gel with BMP-2 were added to the culture medium (25 mg/600 µL culture medium/well). 24 hours after gel loading, the cells were fixed and stained with 4'-6-Diamidino-2-phenylindole for nuclei and phalloidin for actin filaments. The specimens were examined by Confocal laser scanning microscopy (Olympus-FV300, Olympus, Tokyo, Japan).

The cell viability was evaluated by measuring mitochondrial dehydrogenase activity with the MTS assay (Cell Titer 96 AQ Nonradioactive Cell Proliferation Assay, Promega Co., Madison, WI, USA) at 1, 4, and 7 days after gel loading. The assay measures the conversion of MTS into an aqueous soluble formazan product. 60 µL of reaction solution (in 600 µL culture medium per well) were added. The samples were incubated with the reagent for 3 hours in the CO2 incubator at 37℃ and the optical density was read at 490 nm by a spectrophotometric plate reader (THERMOmax, Molecular Devices Co., Sunnyvale, CA, USA).

Seven days after gel loading, the alkaline phosphatase activity was measured according to the protocol of the ALP activity assay kit (AnaSpec Inc., San Jose, CA, USA). Briefly, the culture medium was removed. The cells were rinsed twice with PBS and lysed in lysis buffer with 1% Triton X-100 at 4℃ for 10 minutes. The lysates were harvested and clarified by centrifugation at 13,000 rpm at 4℃ for 10 minutes. The supernatants were incubated with 3.7 mM 4-nitrophenyl phosphate in 100 mM diethanolamine, pH 9.8, containing 0.1% Triton X-100 at 37℃ for 30 minutes. The reaction was stopped with 100 mM NaOH. The amount of released 4-nitrophenolate was determined photometrically at 405 nm. Alkaline phosphatase activity was expressed as the concentration of p-nitrophenyl phosphate (in nmole) transformed per microgram of protein. The protein concentration was measured with a commercially available kit (SMART microBCA kit; iNtRON Biotechnology, Seoul, Korea).

The osteoblastic differentiation of MC3T3-E1 cells was evaluated by real-time PCR examination of ALPase, bone sialoprotein, type I collagen, runt-related transcription factor 2 (Runx-2), osteocalcin, and osterix. The cells were plated at a density of 5×10⁴ cells/cm² on Ti and Zr discs and cultured for 4 and 7 days after gel loading.

The total cellular RNA was isolated using Easy-BLUE Total RNA Extraction kits (iNtRON Biotechnology) according to the manufacturer's protocol. The first-strand of single-strand cDNA was synthesized from 1µg of RNA and used as a PCR template with a SuperScript First-Strand cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA). Quantitative real-time PCR (QRT-PCR) was carried out using the Applied Biosystems 7500 Real-time PCR system (Applied Biosystems, Foster City, CA, USA) with a reaction mixture containing SYBR Premix Ex Taq II (Takara Bio Co., Shiga, Japan), cDNA, and PCR forward and reverse primers. Oligonucleotide primers for ALPase, Runx-2, osteocalcin, type I collagen, osterix, BSP, and 18s rRNA were purchased from Macrogen (Seoul, Korea) (Table 1). The thermal cycling conditions were 95℃ for 15 seconds, 95℃ for 15 seconds, 60℃ for 15 seconds, and 72℃ for 33 seconds. The relative gene expression of the samples of interest was compared with that of the titanium discs, with each sample being normalized to 18s rRNA to correct for differences in RNA quality and quantity.

The results are expressed as mean±SD of triplicate determinations. Comparative studies of the means were performed using one-way analysis of variance with Bonferroni's multiple comparison test. A value of P<0.05 was considered statistically significant.

The surface roughness Ra (arithmetical mean deviation of the profile) of the titanium discs had an average of 1.04 µm, and that of the zirconia discs had an average of 1.34 µm. The roughness of the titanium discs treated by sandblasting and acid-etching did not differ from that of the sandblasted zirconia discs.

The morphology of adherent cells on the different surfaces was visualized by fluorescence staining of cytoskeleton actin filaments. 24 hours after gel loading (48 hours after cell seeding), confocal laser scanning microscopy images of MC3T3-E1 cells cultured on the different samples showed that the cells adhered properly on both zirconia and titanium surfaces (Fig. 2). Cells on both surfaces appeared to be well distributed and in contact with each other via cellular extensions. The MC3T3-E1 cells cultured on both zirconia and titanium surfaces showed well-organized actin fibers. The cells cultured with DBM gel or BMP-2 gel showed more well-organized actin fibers and cellular adhesions than did the cells cultured on the discs alone. However, there was no difference in cell morphology or adhesion between the cells on the zirconia and titanium surfaces.

To examine the proliferation of MC3T3-E1 cells, we carried out a measurement of MTS activity at 1, 4, and 7 days after gel loading. Fig. 3 shows the optical density of formazan produced by the cells on the zirconia and titanium disc after 1, 4, and 7 days. The cells on the zirconia disc proliferated continuously for 7 days, when they were cultured with DBM gel or BMP-2 gel. However, cell proliferation on the titanium disc increased for 4 days and then decreased until day 7, when they were cultured with BMP-2 gel, and their proliferation was significantly lower than that on the titanium discs with DBM gel and the zirconia discs with and without gel (P<0.05).

Cell proliferation on the titanium disc cultured with DBM gel increased continuously for 7 days, like that on the zirconia disc. At day 1 and day 4, no statistically significant differences were observed between the cells cultured on the zirconia and titanium discs. There were no statistically significant differences in cell proliferation among the specimens cultured with and without DBM gel or BMP-2 gel at day 1 and day 4.