Jaw bone samples were obtained from five healthy individuals aged 17–25-years-old during orthognathic surgery of the mandible after obtaining informed consent and the approval of the Ethics Committee Board of Prince of Songkla University, Songkhla, Thailand (EC6609-046). Primary osteoblast cells were isolated by explantation culture, as described in previous studies [14]. Briefly shown in Fig. 1A, the alveolar bone pieces were thoroughly washed in phosphate buffered saline (PBS) to remove any soft tissue and periosteum and vortexed. The specimens were transferred to culture dishes (Corning, Glendale, AZ, USA) and then incubated in α-Modified minimal essential medium (αMEM; Gibco BRL, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco BRL), 1% penicillin/streptomycin (Gibco BRL), and 1% fungizone solution (Gibco BRL) at 5% CO2 and 37 °C. Cells from the third to fourth passages were used for the experiments.

For the bone differentiation, the cells were cultured in the α-MEM supplemented with 10% FBS, 50 mM ascorbic acid, 100 mM dexamethasone and 10 mM β-glycerophosphate for 7–21 days.

A A diagram presenting isolation of primary alveolar osteoblast cells. B Compressive force application model, performed in a 24-well plate

The protocol of mechanical force application in this study was modified from previously described method [15, 15]. To stimulate compressive force, osteoblasts in 24-well plates were subjected to continuous compressive force varying from 1.0 to 4.0 g/cm2 using an acrylic cylinder containing a metal ball as shown in Fig. 1B. Control cells were not exposed to compressive force. All experiments were performed in triplicate for each sample.

CGRP peptide treatment was adapted from the protocol of previous studies [17]. To synchronize the osteoblasts at the G0 phase and study the effect of CGRP treatment on bone formation, the osteoblasts were incubated in serum-free DMEM culture media in 24-well plates for 24 h at 37 °C. The synchronized cells were then divided into five groups and exposed to different conditions: Group 1 served as the negative control, and were cultured in DMEM. Group 2 were treated with DMEM containing 25 ng/mL CGRP. Group 3 were treated with DMEM containing 50 ng/mL CGRP. Group 4 were treated with DMEM containing 100 ng/mL CGRP. Lastly, Group 5 were treated with DMEM containing 200 ng/mL CGRP.

The MTT assay was utilized to assess the viability and proliferation of the osteoblast cells over a period of up to 3 days. MTT reagent (Sigma-Aldrich, Inc., St. Louis, MO, USA) was added to each sample, and the cells were incubated at 37 °C in a humidified atmosphere with 5% CO2 for 3 h to allow the formation of MTT formazan. After incubation, the cells were washed with PBS and the formazan was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Inc.). The absorbance of each solution was measured at a wavelength of 570 nm using a microplate reader (Bio-Rad, Hercules, CA, USA) in triplicate. The viability and proliferation of osteoblast cells was expressed as relative change in comparison to the control  [17].

Calcium deposits of differentiated osteoblasts were identified by Alizarin red staining. After 7–21 days of culture with differentiation medium, cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 20 min, followed by three washes with phosphate-buffered saline (PBS). The fixed cells were stained for 5 min with alizarin red (Sigma-Aldrich) and viewed using Zen software version 2.6 blue edition (Carl Zeiss, Oberkochen, Germany).

The expression of RAMP1, RUNX2, osteocalcin, p38 and p-p38 in osteoblasts were evaluated by western blot analysis. After the experiments, the cells were lysed using lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA). The protein concentration was quantified using the BCA protein assay kit (Pierce™, Waltham, MA, USA). Each protein sample was separated by 10% SDS-PAGE and transfered onto PVDF membranes. Then, the blots were blocked with 5% skimmed milk solution and 3% BSA solution (for phosphor proteins) for 60 min at room temperature. The blots were incubated with polyclonal rabbit anti-RAMP1 (1:500; Abcam, Cambridge, MA, USA), polyclonal rabbit anti-RUNX2 (1:500; Abcam), polyclonal rabbit anti- osteocalcin (1:500; Abcam), polyclonal rabbit anti-p38 (1:1000; Cell Signaling, Denvers, MA, USA) or polyclonal rabbit anti-p-p38 (1:1000; Cell Signaling) overnight at 4 °C. Then, the membranes were washed with Tris-buffered saline for 5 min three times and incubated with HRP-conjugated goat anti-rabbit IgG (1:5,000; Invitrogen, Carlsbad, CA, USA) or goat anti-mouse IgG (1:3,000; Invitrogen) for 60 min at room temperature. Protein band densities were detected and analyzed by a ChemiDoc XRS System (Bio-Rad, Hercules, CA, USA).

The data are expressed as the mean ± standard deviation of measurements obtained from osteoblasts isolated from five independent donors. Each measurement was performed in triplicate. One-way analysis of variance (ANOVA) was conducted to compare the mean values between different groups in cell viability, percentage of mineralization, and protein expression of RAMP1, RUNX2, osteocalcin, p38 and p-p38 in alveolar osteoblasts. Statistical significance was determined as P < 0.05.