Relation between the stability of dental implants and two biological markers during the healing period: a prospective clinical study

Ten patients, who were partially edentulous in the mandibular posterior region for whom a single nonsubmerged implant was planned, participated in this study. All of them presented at the Center of Excellence for Dental Implantology, Faculty of Dentistry, Chiang Mai University, Thailand, between February and August 2015. The inclusion and exclusion criteria were shown in Table 1.

At the examination, the patients were informed about the study’s purposes, procedures, and possible risks and signed an informed consent. Scaling of all the teeth and oral hygiene instructions were performed for each patient.

All surgical procedures were completed by the same implant surgeon. After clinical and radiographic examination, the latter using periapical, panoramic and cone beam computed tomography images, and 2 g of amoxicillin for antibiotic prophylaxis were administered to the patients 1 hour before surgery. One PW Plus® implant (PW Plus, Nakhon Pathom, Thailand) per patient was placed in the mature bone at bone level under local anesthesia (4% articaine with epinephrine 1:100,000). Midcrestal and sulcular incisions around the teeth were performed at the implant site. After full-thickness mucoperiosteal flap elevation, implant bed preparation was started by using custom surgical stents. Ten PW Plus® implants were placed according to the manufacturer’s recommendations. After implant insertion and stability measurement, the smooth (polished) healing abutment was screwed to the implant and the flaps were sutured using 4-0 polypropylene suture material. As post-operative instructions, the patients were asked to abstain from mechanical plaque control at the surgical sites, 0.12% chlorhexidine mouthwash was prescribed for microbial control, and 400 mg ibuprofen for analgesia.

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.

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.

The level of OC was measured by using commercially available ELISA kits (Human Osteocalcin Quantikine ELISA Kit, R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions. A 100 μl volume of Assay Diluent RD1-117 (R&D Systems) was added into each well of the microplate and then 50 μl of standard (0–64 ng/ml) or sample was added to each well. The microplate was incubated for 2 h at room temperature on a horizontal orbital microplate shaker (Labnet International, Inc., Edison, NJ, USA) set at 500 rpm. After that, the solutions were aspirated and each well was washed with 400 μl of Wash Buffer (R&D Systems). This step was repeated three times for a total of four washes. Then, 200 μl of Human Osteocalcin Conjugate were added into each well, and the solutions were incubated for 2 h at room temperature on the shaker. After that, the solutions were aspirated and washed again. A 200 μl volume of substrate solution (tetramethylbenzidine) was added into each well, and the solutions were incubated for 30 min at room temperature on the bench-top. Next, 50 μl of stop solution (2 N sulfuric acid) were added into each well. The color in the wells changed from blue to yellow. After that, the color solution in each well was measured at 450/540 nm within 30 min. The OC level in each sample was calculated from a standard curve and normalized by total protein.

The level of the ALP activity was measured by colorimetric analysis. Briefly, 80 μl of sample solution were added into each well of a 96-well microplate, and then a mixture of 100 μl phosphate substrate solution (p-nitrophenyl phosphate) and 20 μl 1.5 M alkali buffer solution were added to each well. The microplate was incubated at 37 °C for 1 h. Next, 50 μl of stop solution (1 M NaOH) were added into each well. After that, the absorbance was measured at 620 nm. Known concentrations of ALP product (p-nitrophenol) were prepared with the dilutions ranging from 0 to 250 μM. The activity of ALP in the samples were calculated from a standard curve and normalized by total protein.

Data were analyzed by using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Repeated measures analysis of variance was performed to evaluate the change in the ISQ values at each measurement. The independent samples t test was used to investigate the differences in implant stability in the patients’ gender and bone quality. The Friedman test, followed by the Wilcoxon signed-rank test, was used to examine the differences in the crevicular fluid volume, the OC, or the ALP levels in each measurement. The Mann-Whitney U test was used to analyze the differences between the biomarker levels in the control and the test groups. The correlations between OC or ALP levels and ISQ values were calculated using the Pearson correlation coefficient. P values <0.05 were considered statistically significant.