RFA assessments

At implant placement and after 1, 2, 3, 4, 6, 8, 10, and 12 weeks, RFA assessments were performed using the Osstell® ISQ (Integration Diagnostics AB, Goteborg, Sweden) according to the manufacturer’s instructions. A Smartpeg™ (type 47) (Integration Diagnostics AB) was screwed to the implant using a Smartpeg mount. After Smartpeg mount removal, the RFA assessment was performed with the measurement probe on the handheld Osstell® ISQ instrument. The measurement probe was held close to the top of the SmartPeg without touching it until the instrument emitted a beeping sound and the implant stability quotient (ISQ) value presented. Two measurements were performed, one from the buccal direction and one from the mesial direction. The two ISQ values were recorded.

GCF and PICF sample collection

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. 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.