Primary human osteoblasts (HOBs) were previously isolated from the minced trabecular ends of long bones of 18–20-week-old fetuses using a well-established model [28, 32,33,34,35,36], according to the National Health and Medical Research Council (NHMRC) guidelines and the project had the approval of the University of Sydney (USYD) human ethics committee (HEC) (approval number 01/02/40). These primary human osteoblasts are very well characterized with a mesenchymal appearance in monolayer culture as shown in Fig. 1A. They have high alkaline phosphatase activity, produce both osteoprotegerin (OPG) and receptor-activator of NFκB-Ligand (RANKL), and express the calcium-sensing receptor and Homer proteins [34]. When cultured on an appropriate substrate for prolonged periods, these cells form multilayers which secrete the osteocyte marker sclerostin [35]. All cultures used here are between passage 2 and 5. The effects of high glucose concentrations and osmolar controls (mannitol) on the HOBs used here have also been previously assessed [28]. Cultured primary cells were maintained in 10% fetal bovine serum (FBS) and low glucose DMEM (5 mM glucose with 3.5 g/L NaCHO3, 0.57 g/L Na2HPO4, and 0.006 g/L NaH2PO4, pH 7.4) (Invitrogen, Waltham, MA, USA).

A Phase contrast microscopy of primary human osteoblasts (HOBs) in monolayer culture at 10X. B Confocal microscopy images of a monolayer culture of primary human osteoblasts taken with × 10 objective (scale bar = 10 μm) stained for CaSR (green) or Homer (red). Nuclei (blue) were stained with DAPI

Cells were treated in multi-well plates with 1 µL/mL DMSO vehicle, 1 µM CN (MedChemExpress, Monmouth Junction, NJ, USA), 10 nM PTH 1–34 (Sigma-Aldrich, St Louise, MO, USA), or 1 µM CN+10 nM PTH 1–34 in either ‘physiological glucose’ (5 mM) or ‘high glucose’ (25 mM) DMEM. The treatment period was 7 days, with media changes being performed on alternate days. Studies were performed on triplicate samples (i.e., from three separate donors) and repeated n = 3 or n = 4 times. See Fig. 1A.

Primary human osteoblasts (HOBs) were grown on poly-l-lysine-coated coverslips for 7 days and were then fixed with 4% (w/v) paraformaldehyde followed by 100% ice-cold methanol and were processed with the following antibodies: anti-CaSR (Sigma, mouse monoclonal, clone HL1499), anti-Homer1 (Santa Cruz Biotechnology, rabbit polyclonal, clone H-174), and isotype controls. Coverslips were then washed and incubated with anti-rabbit Alexa Fluor 488 (1:750; Santa Cruz Biotechnology) and anti-mouse Cy3 (1:750; Life Technologies, Inc.) at room temperature for 60 min. To visualize the nuclei, coverslips were mounted with UltraCruz™ mounting medium containing DAPI. Slides were observed under a LSM510 Meta confocal laser microscope (Zeiss) at the Advanced Microscopy Facility (Bosch Research Institute, University of Sydney). See Fig. 1B.

The effects of the treatments on the rate of osteoblast proliferation were examined using the IncuCyte® Live-Cell Analysis System (Essen Bioscience, Ann Arbor, MI, USA). HOBs were seeded at a density of 0.03 × 106 cells/well in 24-well plates before treatments were added. The cells were then incubated at 37° C and 4% CO2 in the IncuCyte Analysis instrument. Throughout the 7-day treatment period, phase scans were automatically taken at 2-h intervals using a 10 × objective. Phase object confluency was then automatically calculated and recorded by the machine and used to plot a time course for confluency. To normalize for possible differences in the initial confluence percentage between the different samples, the time required to double the initial confluence value was used for subsequent statistical analysis.

At treatment day 7, HOBs were detached, incubated with trypan blue (Sigma-Aldrich), centrifuged, resuspended, and counted using a Neubauer Hemocytometer (Livingstone).

At treatment day 7, a Caspase-3 assay was performed as previously described [35]. In brief, HOBs were seeded at 1 × 103 in 96-well plates in physiological glucose media and allowed to attach. After 24 h, media were changed to either physiological or high glucose containing treatments as indicated. After 7 days, oxidative stress was then induced by the addition of 50 µM hydrogen peroxide and caspase activity assessed by a previously reported protocol using the caspase-3 substrate, Ac-DEVD-AFC [37]. Caspase-3 activity was used as a measure for apoptosis [38]. Cell numbers were controlled for by BCA assay (Thermo Scientific).

At treatment day 7, HOBs in 96-well plates were assayed for alkaline phosphatase activity using a colorimetric assay as previously published [32]. Briefly, cells were lysed in 1 vol of PBS pH 7.2 containing 0.1% v/v TX-100. To the lysate, 1 vol of 1 mg/mL p-Nitrophenyl Phosphate (Sigma) in 0.2 M glycine pH 9 was added and the plate incubated at RT for 15 min. A standard curve was reacted simultaneously on the same plate using calf intestinal ALP (New England Biolabs) under the same conditions. The formation of the yellow reaction product was measured at 405 nm using a Clariostar spectrophotometer (BMG Labtech). Values were corrected for total cell protein as measured by BCA assay.

At treatment day 7, HOBs were washed, fixed with 4% paraformaldehyde, then stained using 2% Alizarin Red made up freshly in distilled water, pH 4.2. Cell monolayers were washed with 10% acetic acid in distilled water for 30 min at room temperature. To quantify the stain, cells were transferred into tubes, heated at 95° C, immediately placed on ice for 5 min, and centrifuged for 15 min at 20,000 g at 4° C. The absorbance of each sample was quantified using a standard curve and corrected for total cellular protein as determined by BCA assay (Thermo Fisher Scientific).

HOBs lysates from 6-well plates were separated by SDS-PAGE (Bio-Rad Laboratories, Hercules CA, USA) and were transferred to methanol-activated PVDF membranes (Thermo Fisher Scientific, Waltham, MA USA) by wet tank transfer. Membranes were blocked with BSA (Thermo Fisher Scientific) then incubated with primary antibody targeting RUNX2 (rabbit polyclonal, 1:200), OPG (mouse monoclonal, 1:200), RANKL (goat polyclonal, 1:200), OCN (mouse monoclonal, 1:200), or CaSR (mouse monoclonal, 1:2000) (all Santa Cruz Biotechnology, Dallas, TX, USA). A primary antibody targeting the housekeeping protein, β-Tubulin (mouse monoclonal, 1:200, Santa Cruz Biotech), was also used to provide means for normalization. The membranes were then incubated with the appropriate HRP-conjugated secondary antibody (Santa Cruz Biotech). Bands were detected using a ChemiDoc Imaging System (Bio-Rad) following exposure of the membrane to a chemiluminescent HRP substrate (Merck).

Graphing and statistical analysis were performed using GraphPad Prism software version 7.02 (GraphPad Software, California USA). A non-parametric, two-way, 2 × 4 analysis of variance (ANOVA) and post hoc Tukey’s Multiple Comparison tests were conducted compare all of the treatment groups. Graphical representations of Western blot data show the mean fold change from vehicle treatment in physiological glucose conditions ± standard error of the mean (SEM).