Canine samples_37_only.xlsx

Data collected from Canine primary osteoblasts. See methodology below.The excised femoral head was cut in the frontal plane with an IsoMet® Low Speed Diamond Saw (Buehler, Coventry, UK) to facilitate access to subchondral, trabecular, and cortical bone types. Subchondral bone was prepared by removing the overlying cartilage with a scalpel and then isolated using rongeurs. Trabecular bone was collected from the epiphysis avoiding the growth plate area. Cortical bone was collected from the femoral neck region. The pieces of bone from each type were cut into 1 mm3 explants and vortexed separately, at least six times in PBS containing 10% AB/AM to remove bone marrow, blood and other debris. The following isolation procedure was used. Briefly, the bone pieces were incubated with 1% trypsin for 10 minutes at 37°C. The trypsin was discarded and fragments were then washed in Dulbecco’s modified Eagle’s medium (DMEM) and in PBS. Bone pieces were incubated in 0.2% collagenase type II for 30 minutes at 37°C; the supernatant was discarded and fragments were washed in PBS and then in DMEM (low glucose, pyruvate, no glutamine, no phenol red). The fragments were then seeded in 75 cm2 culture flasks in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL AB/AM as a basal media at 37ºC, 5% CO2. Cultures were left for 7 days without media change and thereafter, a half media change was performed every 4 days until confluent. At confluence, cells were trypsin-digested and sub-seeded in six replicates in basal media in 12 or 6-well plates at 50000 cells/well, and the media was changed twice a week. Cells from the different bone types were seeded as described above and maintained in basal media until confluence. At 24 hours post-confluence cells were changed into mineralizing media which consists of basal media plus 2 mM β-glycerophosphate and 200 µg/mL of ascorbate. Media was changed twice a week until day 40 after confluence. Cell number and viabilityCrystal violet binding to DNA was used to calculate the number of viable cells in culture, based on the principle that dead cells are no longer attached to the cell culture plastic. Briefly, cells from the different bone types were seeded into 12-well plates at 50000 cells/well, with six replicates per region, per dog sample. Cells were maintained in basal media for 6 days before assessing cell number. At day six, cells were washed with PBS and fixed with 4% paraformaldehyde. Cells were stained with 0.1% crystal violet in distilled water for 30 minutes at room temperature. Cells were subsequently washed with distilled water to remove excess dye and the bound stain was eluted from cells with 1 mL of 10% acetic acid. The quantity of nuclear stain was assessed by transferring 200 µL of the acetic acid solution to a 96 flat well plate and the absorbance was measured at 595 nm in a Tecan M200 Pro Plate Reader. A standard curve was prepared with known serial dilutions of canine cells that were stained. Absorbance was used as a direct measure of cell nucleus staining, and cell number was extrapolated from the standard curve. Tissue non-specific alkaline phosphatase (TNAP) activity assayCell lysates were collected 24 hours after confluence or, at the same time, plates were fixed for analysis from each individual well. In distilled water, cells were scraped off the plate and the cell suspension was centrifuged. A modified version of the SensoLyte® pNPP Alkaline Phosphatase Assay Kit (AnaSpec, Belgium) was used, where 50 μL of cell lysate supernatant was incubated with the pNPP chromogenic liquid substrate and absorbance was measured at 37°C, 405 nm at regular intervals in a Tecan M200 Pro Plate Reader. The cell lysate was also used to measure protein content with the Bradford reagent according to the manufacturer’s instructions. Mineralization analysisCells which had been maintained in mineralizing media in 6-well plates for 40 days were fixed and analysed. Plates were washed in PBS, fixed for 5 minutes at room temperature with 2.5% Gluteraldeheyde and then washed again with PBS and with 70% ethanol. When dry, plates were scanned at 2400 dots per inch (dpi) on a high-resolution flat-bed scanner for analysis of mineralized nodule formation. ImageJ (http://rsbweb.nih.gov/ij/) was used to determine the surface area of mineralized bone nodules.