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 analysis

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.