Hexadecylpyridinium chloride monohydrate

All chemicals were purchased
and used without further purification.
Propan-2-ol (C₃H₈O, ≥99.5%), ethanol
(EtOH, ≥99.8%), methanol (MeOH, ≥99.5%), acetone (C₃H₆O, ≥99.5%), and hydrochloric acid (HCl,
37.0%) were purchased from VWR. Nitric acid (HNO₃, ≥65%),
sodium hydroxide pellets (NaOH, ≥98.0%), sodium borohydride
(NaBH₄, ≥97%), and l(+)-ascorbic acid (AA,
≥99%,) were purchased from Carl Roth. Hydrogen tetrachloroaurate
trihydrate (HAuCl₄·3H₂O, ≥99.9%),
hexadecyltrimethylammonium bromide (CTAB ≥99%), cetyltrimethylammonium
chloride solution (CTAC, 25 wt % in H₂O), hexadecylpyridinium
chloride monohydrate (CPC, 99.0–102.0%), (3-aminopropyl)triethoxysilane, N,N-diisopropylethylamine (DIPEA), and
poly(sodium 4-styrenesulfonate, M_w of
70000 g/mol) were purchased from Sigma-Aldrich. 3-(Triethoxysilyl)propylsuccinic
anhydride, 3-acetoxypropyltrimethoxysilane, n-butyltriethoxysilane, and heptadecafluoro-1,1,2,2-tetrahydrodecyl)triethoxysilane
were purchased from abcr. (α-Thiol, ω-bromo)-terminated
poly(acrylic acid) (PAA-SH, M_w of 8500
g/mol) was purchased from Polymer Source Inc. Mica sheets were obtained
from Micro to Nano with different shapes and a thickness of 0.15 to
0.21 mm and the highest grade V-1 quality. Double-polished Si wafers
(orientation [100] of 5 mm length and 7 mm width) from Siegert Wafer
were used for ellipsometry measurements. Milli-Q water (18.2 MΩ·cm)
was used in all experiments.

DPCs were seeded into 12-well plates and cultured in OS medium for 7 days. After fixation in 4% paraformaldehyde for 30 min, DPCs were stained with 1% alizarin red staining solution (ARS; Cyagen, Suzhou, China) for 5 min at room temperature and were then rinsed with distilled water. The matrix calcium deposition was scanned using an inverted phase-contrast microscope (Axio 40; Zeiss, Jena, Germany). To quantify the mineralized nodules, the stained cells were incubated in 0.1M hexadecylpyridinium chloride monohydrate (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. The absorbance of the supernatant was then measured at a wavelength of 562 nm.

Three Ф 32 mm titanium plates were taken from each group and placed into a 6-well plate. rBMSCs were inoculated on the titanium plates with a density of 6 × 10⁴/well. After incubating for 14 d in osteogenic medium, cells on samples were fixed and stained with the Alizarin red S (ARS) reagent (Cyagen, China) for 15 min at room temperature. After the samples were washed with DPBS three times, images were taken. Then, the mineralized nodules were eluted with 10% hexadecylpyridinium chloride monohydrate (Sigma, USA), and the O.D. value at 570 nm was measured for quantitative detection.

The human MSCs were treated with TRX in the osteogenic induction medium (OIM) for 7 days, then the cells were collected, qualitative, and quantitative examination of ALP activity were carried out according to the protocol of BCIP/NBT Alkaline phosphatase Color Development Kit (Beyotime, Shanghai, China). For the alizarin red S staining, the TRX-treated MSCs were incubated in OIM for 14 days, and washed with PBS twice and fixed with ice-cold 75% ethanol for 15 min. The MSCs were then stained with 2% Alizarin Red S staining solution (pH 4.2, Leagene, Beijing, China) for 30 min. The stained calcified nodules were dissolved with 10% Hexadecylpyridinium chloride monohydrate (Sigma-Aldrich) at room temperature and then the absorbance was detected at the wavelength of 562 nm.

CaO₂, MnO₂, polyvinyl alcohol (PVA), ascorbic acid, β-glycerophosphate, Alizarin red S, and hexadecylpyridinium chloride monohydrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). PLGA (with a lactide:glycolide molar ratio of 75:25) was acquired from Green Square Material (Taipei, Taiwan). The mouse preosteoblast MC3T3-E1 cells were purchased from the American Type Culture Collection (ATCC CRL-2593, Manassas, VA, USA), while cell culture reagents were procured from Thermo Fisher Scientific (Waltham, MA, USA). All other chemicals and reagents used were of analytical grade.

Cells were washed twice with cold PBS and fixed with 4% paraformaldehyde for 10 minutes. Then, they were stained with 30 mM Alizarin Red S (pH 4.2, Sigma) for 10 minutes at room temperature. then, images were obtained with a Canon camera. In order to quantify calcium deposition, Mineralization of calcium nodules were quantified by a method described previously21 (link). Briefly, after staining, the cells were washed three times with PBS, 10% Hexadecylpyridinium Chloride Monohydrate (Sigma-Aldrich, Louis, MO, USA) was added and incubated for 20 mins at room temperature, then, Absorbance of the supernatant was measured at 540 nm in triplicate using ThermoMultiskan EX plate reader (Thermo Scientific, Waltham, MA, USA). Finally, the cells was washed with PBS and lysed with RIPA buffer and protein content was measured, the calcium levels were normalized to the total protein content.

Mineral deposition was evaluated at 14 and 21 days in osteogenic media. DPSCs at a density of 1 × 10⁴ were seeded on top of hydrogels (n = 6/group) and incubated according to each time point. Then, hydrogels were fixed with 70% ethanol for 1 h at 4 °C, washed with distilled water, and incubated with Alizarin Red staining (40 mM; pH 4.2) for 20 min. After staining, hydrogels were washed with distilled water ten times. Then, nodules were dissolved in hexadecylpyridinium chloride monohydrate (10%; Sigma Aldrich, St. Louis, MO, USA), and the absorbance was read at 570 nm (SpectraMax iD3, Molecular Devices, LLC, San Jose, CA, USA). Hydrogels without cells were used as a background control.