Alp activity detection kit

After 7 days of osteogenic induction, ALP staining of hPDLSCs-CA and hUCMSC-CA was performed by an ALP color development kit (Beyotime, Shanghai, China) and ALP activity was quantified by an ALP activity detection kit (Jiancheng Bioengineering, Nanjing, China). Quantitative real-time PCR (RT-qPCR) was performed to examine the mRNA expression of ALP, Runx2 and OCN as well as extracellular matrix-related gene, fibronectin, integrin-β and collagen type I. In addition, BSP and OPN, which are strongly expressed in the native periodontium in bone and cementum, especially acellular cementum, were also detected by the RT-qPCR. Briefly, total cellular RNA was isolated from hPDLSCs-CA and hUCMSCs-CA using Trizol Reagent (Invitrogen) according to the manufacturer's standard instructions. Reverse transcription of mRNA and PCR reaction were performed as described previously 40 (link). Primer sequences used in this experiment are listed in Table 1. Also, expression of BSP, OPN, fibronectin, collagen type I, OCN and osterix were detected by immunofluorescence in both cell-aggregates during the induction period. The experimental process and analysis method were as previously described 32 (link). The experiment was repeated at least three times.

BMSCs were plated in 12-well plates (5,000 cells/cm²). Osteogenesis was induced by the OM, which is complete IMDM supplemented with dexamethasone (0.1 nM), β-glycerophosphate (10 mM), and ascorbic acid (50 μM). We changed culture media every 3 days. The calcium deposition was evaluated through Alizarin Red staining. The levels of ALP were measured by using ALP activity detection kit (Nanjing Jiancheng, A059-2-2) according to the manufacturer’s instructions.

A cell suspension of 1 mL was seeded on each sample at a density of 1×10⁴ cells/mL. After culturing for 7 and 14 days in the absence and presence of osteogenic supplements, the cells were washed and fixed. ALP staining was conducted with an ALP color development kit (Beyotime Institute of Biotechnology), and ALP activity was determined by an ALP activity detection kit (Jiancheng Bioengineering Institute, Nanjing, People’s Republic of China), according to the manufacturer’s suggested protocols.

For osteogenic differentiation, the cells (P4) were seeded in 6-well plates at a density of 4×10⁴ cells/per well, which aimed to quantify mineralized nodule formation in vitro. The cells were incubated in medium supplemented with 10% FBS. The medium was then switched to osteogenic differentiation medium, which contains 0.1 mM dexamethasone, 10 mM β-glycerophosphate, 50 mg/mL ascorbate phosphate (all from Sigma Aldrich), and 10% FBS. The medium was changed every 2 days. Mineralization was detected and quantified using an Alizarin Red, and 4 wells were analyzed for each group. The quantification assay was performed using an ALP activity detection kit (Jiancheng Bioengineering, Nanjing, China). The cells were fixed with 4% paraformaldehyde for 20 min and stained with 2% Alizarin Red (PH 4.2) (Kermel, Tianjin, China). The mineralized nodules were dissolved by hexadecyl pyridinium chloride and isopropanol, and quantitative absorbance was measured at 560nm for statistical analysis.