Alkaline phosphatase color development kit

PDLSCs were separately transferred into 96-well plates, and per well has a density of 1 × 10⁵ cells. After 21 days of induction, ALP activity was measured using the ALP assay kit (Jiancheng Technology Co., Ltd., Nanjing, China). Cellular ALP was visualized by using Alkaline Phosphatase Color Development Kit (Beyotime, Shanghai, China) and the methodology is the same as the manufacturer’s protocol.

The alkaline phosphatase (ALP) activity was examined to evaluate the differentiation of BMSCs. An osteogenic medium was applied, consisting of α-MEN with 10% FBS, 1% PS, 100 nM dexamethasone, 50 mg/L ascorbic acid, and 10 mM Na-b-glycerophosphate. The medium was used after the BMSCs were incubated on the samples for 24 h and exchanged every two days. BSMCs were seeded on the Ti discs (CaP, LF-L, LF-M, and LF-H) at a cell density of 2 × 10⁴ cells/mL and cultured for 5 and 7 days. Then, the ALP activity of the BMSCs was measured qualitatively and quantitatively. For qualitative investigation, the samples were fixed with 4% PFA and stained with an alkaline phosphatase color development kit (Beyotime, Shanghai, China). The cells were observed by a stereomicroscope (OLYMPUS, Shinjuku, Japan). For quantitative evaluation, the BMSCs were rinsed by PBS twice and 1% Triton X-100 was added to obtain the cell lysates. Subsequently, an alkaline phosphatase assay kit (Jiancheng, Nanjing, China) was added and the results were measured with a spectrophotometer at 520 nm. Finally, the results were normalized to the total protein content measured by a BCA protein assay kit (Beyotime, Shanghai, China). The experiments were conducted in triplicate.

Hexadecyl trimethyl ammonium bromide (CTAB, 98%), tetraethyl orthosilicate (TEOS), triethyl phosphate (TEP), calcium nitrate tetrahydrate (Ca(NO₃)₂.4H₂O), ammonium hydroxide (NH₃.H₂O), and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China.). Sodium alginate (ALG, the viscosity of ALG (1%, Brookfield LV, 20°C) is 350–550 cP), 3-aminopropyltrimethoxysilane (APTES), ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS, 98%), and sodium alginate (98%) were purchased from Aladdin Scientific Co., Ltd. (Shanghai, China). Acridine orange/ethidium bromide was purchased from BestBio Co., Ltd. (Shanghai, China). The MTT kit and Alkaline Phosphatase Color Development Kit were purchased from Beyotime Co., Ltd. (Shanghai, China). Alizarin Red S (ARS) staining was purchased from Solarbio Co., Ltd. (Beijing, China).

PDLSCs were seeded into 96-well plates with a density of 1 × 10⁵ cells/well. Fourteen days after induction, ALP activity was detected through the ALP assay kit (Jiancheng Technology Co.). ALP activity was determined at 405 nm using p-nitro-phenyl phosphate as a substrate. Cellular ALP was also visualized by using Alkaline Phosphatase Color Development Kit (Beyotime) according to the manufacturer’s protocol.

Cells were cultured in 12-well plates with osteogenic induction medium for 3 or 5 days. For ALP staining, cells were fixed with 4% paraformaldehyde for 20–30 min. Cells were then washed by double distilled water (ddH₂O) three times and stained by Alkaline Phosphatase Color Development Kit (Beyotime, Shanghai, China). To measure ALP activity, cells were lysed with lysis buffer consisted of 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, and 150 mM NaCl. ALP activity was determined by ALP activity assay (Beyotime). Finally, the conversion color of p-nitrophenyl phosphate was measured after 3 and 5 days of culture in an osteogenic medium at 405/650 nm.