Experimental Groups and Preparation of Samples.
Eight Ti discs (diameter, 2.5 mm; central hole diameter, 1.0 mm) were cut from grade 2, commercially pure titanium rods (ASTM F67 Unalloyed titanium for surgical implant applications, Northwest Nonferrous Metal Institute, Xian, P.R.China). They were divided into two groups: the modified sandblasted surface group and the smooth surface group. Discs in the former group were sandblasted with 0.15- to 0.21-mm corundum (AL2O3) on circumferential surfaces and modified by oxalic acid attack. The circumferential surface of the latter group was polished through a series of silicon carbide papers to 800-grit. Before use, degreasing of all discs was performed by cleaning in trichloroethylene and ethanol under ultrasonics for 10 minutes each and passivation was performed by immersing in 40% nitric acid for 30 minutes. They were then rinsed with distilled water five times and finally sterilized by autoclaving at 120°C for 30 minutes.

Scanning Electron Microscopy of Ti Disc Circumferential Surface.
The topography of Ti-disc circumferential surfaces was observed by scanning electron microscopy (S-520, Hitachi Co., Japan) on one sample from each group after samples were cleaned, passivated, and sterilized.

Cell Culture.
Primary osteoblast-like cells were derived from human fetal calvaria following the explant method described by Li et al. Calvaria were isolated from six-month-old human fetuses after legal abortion and washed in Dulbeco modified Eagle medium (DMEM) (Sigma Chemical, St. Louis, MO) supplemented with 300 U/mL penicillin and 300 U/mL streptomycin. After scraping of the periosteal sides and removal of sutures, calvarial plates were cut into small pieces and then plated into flasks cultured with DMEM containing 10% fetal bovine serum, 50 mg/mL L-ascorbic acid at 37°C, and 5% CO2. The medium was changed the next day and every three days thereafter. When reaching confluence at the 12th culture day, the outgrowth cells were subcultured with 0.25% trypsin. They were identified as osteoblast-like cells and well characterized (Fig. 1).
The seventh passage osteoblasts were seeded into three wells of a six-well plate in DMEM supplemented with 10 nmol/L dexamethasone and 10 mmol/L Naglycerophosphate as well as 10% fetal bovine serum and 50 mg/mL L-ascorbic acid as above. Until the seventh culture day, the osteoblasts had been reaching confluence.

Plating of Ti Discs.
When the confluent cellular layer had formed, the six prepared Ti discs (three per group) were carefully put onto it at an interval of 1 cm; both groups within the same well and the triplicate samples in separate wells. The cells were fed every three days. On each application, intensive care was taken to prevent discs from moving.

Follow-up Observation with Inverted Phase-Contrast Microscope.
The culture lasted one month after disc plating. On the first day, cells in the cellular layer migrated, attached, and oriented to the circumferential surfaces of Ti discs. As the culture continued, the migrating and attaching cells increased and a three-dimensional interface between discs and osteoblasts was formed. These events were observed and followed in an inverted phase-contrast microscope (YMT-Z, Olympus Co., Japan). The refractile area surrounding the discs was the portion of interfacial orienting and attaching cells in this three-dimensional situation under the phase contrast condition (original magnification, 340).

Histologic Observation.
After the one-month culture, discs associated with the surrounding cells were harvested and processed by rinsing with phosphate-buffered saline, fixing with 4% paraformaldehyde, rinsing again, dehydrating serially with ethanol of increasing concentrations, and embedding in Epon 812. After separation of the embedded surrounding cells from circumferential surfaces of Ti discs by cryofracture and trimming of the derived specimen, they were re-embedded with Epon 812 in a horizontal plane. The specimens were then trimmed again, sliced with ultramicrotome, mounted on a glass slide, and finally stained with methylene blue-basic fuchsin staining method. The sections were 1.0-mm thick. Observation was made with an Olympus microscope (H-2, Olympus Co., Japan) and photographed (Kodak color film 100°, Eastman Kodak, Rochester, NY).

Transmission Electron Microscopy.
Specimens of one-month–cultured interfacial cells were processed as above except that the fixation and sectioning were done by the routine method for transmission electron microscopy. The specimens were fixed with 4% paraformaldehyde for 10 minutes, postfixed with 1% osmic acid for 30 minutes, and ultrasectioned with a thickness of 0.02 mm. Observations were made with the JEM-2000EX transmission electron microscope (JEC Co., Japan).