To observe the level of two bone formation biomarkers (alkaline phosphatase and osteocalcin) during the osseointegration period compared with control group using GCF from the first molar of the contralateral side of implant position, the sample collection of GCF was performed before the surgical procedure, immediately after the surgical operation and after 1, 2, 3, 4, 6, 8, 10, and 12 weeks. The PICF sample collection was performed immediately after the surgical operation and after 1, 2, 3, 4, 6, 8, 10, and 12 weeks by a single trained operator. PICF samples were collected from the buccal side of the implant healing abutment, and GCF samples were collected from the buccal side of the contralateral nonsurgical tooth. The GCF/PICF collecting techniques were modified from the method of Ciantar and Caruana [23]. Before collecting the samples, the sites were isolated with cotton rolls, supragingival plaque was removed by probe, and tissues were kept dry by gentle air flow. Paper strips (Periopaper, Oraflow, Smithtown, NY, USA) were inserted into the sulcus until resistance was felt and left in place for 30 s. The volume of fluid was immediately measured by Periotron 8000 (Periotron, Oraflow). The paper strips containing the GCF/PICF sample were kept in a 1.5-ml Eppendorf tube. The tube was labeled and kept on ice and then was transferred to −80 °C for storage until the analysis was performed.

GCF/PICF in the Periopaper strip was eluted by adding 320 μl quantity of phosphate-buffered saline (PBS) into the sample tube and incubated at 4 °C, overnight. The eluted protein solution from each gingival fluid sample was used for the biochemical analysis.

Total protein in the gingival fluid sample was measured by the Bradford analysis [24]. Briefly, a 10 μl volume of sample or protein standard (0–500 μg/ml) was added into each well of a 96-well microplate and then 200 μl of Bradford working reagent were added to each well. The microplate was shaken for 5 min. After that, the absorbance was measured at 620 nm. The concentrations of total protein in the samples were detected and calculated from a standard curve.