Nano bioactive glass (nBG) was synthesized using a modified sol-gel method [14, 15]. Briefly, 16.8 mL tetraethyl orthosilicate (TEOS) was mixed with calcium nitrate in a water/ethanol (2:1) solution. The pH of solution was adjusted to 2 by addition of citric acid (1 M). Stirring was continued until a clear solution (solution A) was obtained. A solution of 2% polyethylene glycol (PEG) (MW: 2000) and diamonium hydrogen orthophosphate was prepared and by addition of ammonia, its pH was adjusted to 10 (solution B). Solutions A and B were mixed under stirring for 10 h to obtain a homogeneous gel. It was washed and filtered with deionized double distilled water. The obtained white gel was dried and lyophilized using an electrical oven at 60 °C for 8 h, and finally calcined for 10 h at 650 °C.

A solution of casein (10% w/v) was prepared at pH 8 (pH was adjusted with NaOH) and then an appropriate amount of trypsin (0.2 w/w relative to casein) was dissolved in the solution and stirred for 2 h at 50 °C. The pH of solution was adjusted to 4.6 by addition of HCl. It was centrifuged and the pH of the obtained precipitate was adjusted to 9 by addition of NaOH. Then, CaCl2 (1 mol), Na2HPO4 (1 mol) and NaF (200 mmol) were slowly added to the solution during continuous shaking to obtain the final concentration of calcium chloride (100 mM Ca (II)), sodium phosphate (60 mM phosphate) and sodium fluoride (12 mM fluoride) [16, 17]. The solution was filtered using a microfilter (0.1–0.2 μm) and the obtained precipitate was spray dried. The obtained white powder containes 50% CPP and 40% ACP [16, 17]. Finally, CPP-ACP@nBG and CPP-ACP@F were prepared by addition of 0.5% w/w nBG or sodium flouride to the CPP-ACP paste.

X-Ray diffraction pattern (XRD) was used to investigate the crystal structure of nBG. Panalytical Xpert PRO X Ray Diffractometer (Panalytical, Netherlands) model Xpert Pro MPD with wavelength 1.5405 Å and power 40KV/30mA was used to study the structure and crystal phase of the nBG by scanning in the range of 15 to 80 degrees.

Fourier transform infrared spectroscopy (FTIR) was used to assess the functional groups and chemical structure and bonds of the synthesized nBG. FTIR spectra of the synthesized nBG were recorded by an FT-IR Spectrometer (PerkinElmer, USA) model Spectrum400 in the frequency range of 400–4000 cm− 1. The morphology, size distribution and shape of nBG particles were assessed by transmittance electron microscope (TEM) model Philips XL30 ESEM (Netherlands).

The study protocol was approved by the Ethics Committee of Hamadan University of Medical Sciences (No# IR.UMSHA.REC.1398.843). Premolars that had been extracted due to orthodontic or prosthetic treatment, were collected and kept in chloramine (0.5%). A written consent form was obtained from all patients over the age of 16 and from legal guardians of patients under the age of 16 prior to extraction of their teeth. A sharp blade was used to clean soft tissue and calculus around the extracted teeth and were subsequently polished. The premolars were examined by stereo microscope (Olympus, Shinjuku, Tokyo, Japan) and 32 premolars without cracks or lesions were selected. The crown of the teeth was removed from the CEJ area by a diamond saw (Micro slice 2, Metal Research, Cambridge, UK) and then the crowns were cut into facial and palatal halves. Each half was mounted in acrylic resin (Acropars, Kaveh, Tehran, Iran) exposing 4 x 4 mm of enamel surface. Polishing procedure incorporated on the exposed enamel, by using of 600-grit, 800-grit, 1200-grit silicon carbide papers subsequently and were thoroughly rinsed with deionized double distilled water after each gritting. The mounted samples were placed in deionized double distilled water to prevent the dehydration.

For demineralization, all mounted samples were immersed in the demineralizing solution (2.2 mM CaCl2 • 2H2O, 2.2 mM KH2PO4, 50 mM sodium acetate) for 96 h at 37 °C and pH of 4.4 [18].

After washing with deionized double distilled water, the samples were pH cycled in demineralization (2.2 mM CaCl2 • 2H2O, 2.2 mM KH2PO4, 50 mM sodium acetate) and remineralization (20 mM 4-(2-hydroxyethyl) -1 piperazineethanesulfonic acid (HEPES), 1.5 mM CaCl2, 0.9 mM KH2PO4, 150 mM KCl) solutions. The pH cycling procedure constituted cycling in the demineralizing solution at pH of 4.4 for 30 min and then for 10 min in remineralizing solution at pH of 7.0 at room temperature. This cycle was repeated 6 times a day for 8 days and the samples were kept in deionized double distilled water during the night [18]. The samples were randomly divided into 4 groups as follows. G1: commercial CPP-ACF@P (MI paste plus); G2: synthetic CPP-ACP@F; G3: synthetic CPP-ACP@nBG and G4: control, in which demineralization process was conducted, however, no reminrelization process was undertaken.

In the experimental groups, the paste (0.1 g of material was mixed with 3 mL of deionized double distilled water for 1 min to form a paste) was applied directly on the surface of the demineralized enamel using a micro-brush for 4 min (twice a day at 8 am and 4 pm for 28 days) at room temperature. Then, the samples were washed using a micro-brush and deionized double distilled water and kept in deionized double distilled water for 48 h at room temperature [19].

To evaluate the remineralization of enamel surfaces and measure the surface microhardness, a Vickers microhardness test was used. Microhardness was measured at 3 points on the surface of each sample, and the mean microhardness was calculated. Indentation was generated with a force of 500 g for 5 s and was calculated by the use of a microhardness tester microscope (Micrometer 1, Buehler, Lake Bluff, IL, USA).

A scanning electronic microscope (Hitachi S-450, 20 kV, Japan) was used to assess the surface morphology of enamel samples. Samples were dehydrated using ethanol and were subsequently coated with a layer of gold prior to analysis.

Data were analyzed using SPSS version 21 software (IBM Corp., Armonk, NY, USA). Kolmogorov-Smirnov test was used to evaluate the normality of microhardness data distribution. Due to the normality of data, one-way analysis of variance (ANOVA) and Tukey post hoc tests were used for comparison between the studied groups. The confidence level was set as 95% (α = 0.05).

The minimum sample size required in this study was determined as 16 (64 samples in 4 groups) [18, 20]. The reliability of the test was 95% and the test power was 80%. The expected difference was considered in the average of \(\mu_1-\mu_2=30\) and the standard deviation was \(\sigma=30\).