Alp assay kit

ALP assay of BMSCs on membranes was detected after osteogenic culture for 14 days. After removing the medium and rinsing three times with PBS, regents were added following the instructions of the ALP assay kit (Sigma-Aldrich, United States). Finally, ALP staining was conducted and photographed. ALP activity was detected by transferring 50 µl of each sample to a 96-well plate, and the absorbance at 520 nm was measured with an automatic microplate reader.

The C2C12 cells were plated at 200,000 cells/well in 6-well plates and grown overnight in DMEM containing 10% FBS. On day 2, the culture medium was replaced with DMEM containing 2% FBS and the cells were treated with 0.5 uM or indicated concentration of compound for 24 hr in 2 ml culture medium. On day 3, the cells were treated with a final concentration of 50 ng/ml of BMP-2 with or without compound in DMEM medium containing 2% FBS for 72 hr. The cells were washed with phosphate-buffered saline (PBS) and lysed by the addition of lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM MgCl₂, and 0.5% Triton X-100). The cell lysates were centrifuged for 5 min at 13,000 × g. The supernatant was removed, and the aliquots were assayed for ALP activity and protein amount. The ALP activity was measured in triplicate using an ALP assay kit (Sigma-Aldrich, St. Louis, MO) in microtiter plates. The protein amount was determined with Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA) using bovine serum albumin (BSA) as a standard. The ALP activity (nmoles of p-nitrophenol per ml) was normalized to the protein amount (nmoles of p-nitrophenol per μg).

Maxillary alveolar bone was fixed with 4% paraformaldehyde for 2 days at 4°C and decalcified with 10% ethylenediaminetetraacetic acid (EDTA, pH 7.4) for 21 days. Samples were embedded in paraffin and 4‐μm‐thick sagittal plane slices prepared using an RM2235 microtome (Leica Microsystems GmbH). Masson's trichrome and haematoxylin and eosin (H&E) staining were performed respectively, following the manufacturer's instructions (Beijing Solarbio Science & Technology Co., Ltd.). Alkaline phosphatase (ALP) staining was performed using an ALP assay kit (Sigma‐Aldrich). Sections were scanned on a Scan Scope GL optical microscope (ZEISS, Axiocam 503 color) and collage was quantified by Image J.

ALP activity was detected by using an ALP assay kit (Sigma) following the manufacturer’s instructions. In brief, cells were mixed with an alkaline buffer solution (1.5 M, pH 10.3) containing 10 mM p-nitrophenyl phosphate as a substrate and NaOH solution (3 M) was used as stop solution. The optical density was measured at 405 nm with a microplate reader. ALP activity was normalized by the DNA content and expressed as nmol of p-nitrophenol produced per minute per mg of total DNA. The implanted samples were smashed in liquid nitrogen and lysed in 1 ml harvest buffer for 1 h, and then homogenized carefully to further lyse cells. After a centrifugation at 2000 rpm for 10 min, 10 ml supernatant were harvested for ALP activity assay.

At each time point, 4 mL ear margin venous blood stewed in the bio-tube for 30 min was centrifugalized at the speed of 2500 r/min. Then, the upper layer of the serum was removed and ALP activity of the lower layer was detected with ALP assay kit (Sigma-Aldrich, St. Louis, MO, USA). The measurement was performed using Beckman LX 20 Analyzer (Beckman Coulter, Inc., CA, USA).