Ammonium hydroxide

LC-MS grade ACN was purchased from VWR Chemicals BDH (Radnor, Pennsylvania, USA). HPLC grade >98% FA and >99% TFA, 37% hydrochloric acid (HCl), 70–72% PCA, Trizma base, DL-dithiothreitol (DTT), NEM, NMM, 25% ammonium hydroxide, Tween, and ammonium bicarbonate (AMBIC) were purchased from Merck (Darmstadt, Germany). Ethanol (99.9%, EtOH) was purchased from Antibac (Asker, Norway). Glycine >99%, iodoacetamide (IAA), and CaCl₂ were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Crystalline porcine trypsin (4500 K) was obtained from Novozymes A/S (Bagsværd, Denmark). Recombinant HMGB1 for biochemistry and dsHMGB1 were obtained from HMGBiotech S.r.l. (Milano, Italy). Anti-HMGB1 antibodies (Ab18256, polyclonal, rabbit) were purchased from Abcam (Cambridge, UK). Antihuman haptoglobin antibodies from immunoturbidimetry clinical chemistry reagents (Roche Diagnostics, Rotkreuz, Switzerland) were purified to remove the tris-buffer by three rounds of dialysis with 0.1% sodium chloride, using cellulose membrane dialysis tubing of approximately 14k MWCO (Merck, Darmstadt, Germany).

The silicon preceramic polymer used in this study is Loctite 5248
(AG & Co., Germany, Henkel), a UV-curable alkoxy silicone with
thixotropic behavior and a viscosity range of 50–80 cP at 25
°C. The Si/O/C ratio calculated by XPS spectra was 19:27:54 (see
Figure S1 in the Supporting Information), while the number-average molecular weight was = 55 kDa, as measured from static light scattering analysis (SLS,
Zetasizer Nano ZEN3600, Malvern Instruments). Loctite 5248, which
is a straw-colored one-component liquid solution, was directly dissolved
in ethyl acetate (vapor pressure = 73 mmHg at 20 °C; Sigma-Aldrich)
to prepare BFTSA precursor solutions at 2 and 5% w/v (mg/mL). The
solution was stirred overnight at room temperature and wrapped into
an aluminum foil to prevent curing from ambient light. Double-side
mechanically polished Ti6Al4V discs of a diameter of 10 mm and a thickness
of 2 mm (provided by Eurocoating SpA, Italy) were used as substrates.
Calcium nitrate, Ca(NO₃)₂ (Sigma-Aldrich, USA);
diammonium phosphate, (NH₄)₂HPO₄ (Sigma-Aldrich,
USA); gelatin from cold water fish skin (Sigma-Aldrich, USA); deionized
(DI) water; and ammonium hydroxide (28%, Millipore, USA) were used
to synthesize HAp NPs.

Ammonium persulfate (APS), aniline hydrochloride (ANI-HCl), acetone, hydrochloric acid, glycerol, and 30% ammonium hydroxide were purchased from Sigma Aldrich (St. Louis, MO, USA), while ethanol, methanol, toluene, and cassava starch were purchased from R&M Chemicals (Petaling Jaya, Malaysia), Systerm (Shah Alam, Malaysia), ChemSoln (Shah Alam, Malaysia), and Cap Kapal ABC (Georgetown, Malaysia), respectively.

Silane-PEG-COOH (average Mw 5000) were purchased from Biochempeg Scientific Inc. Ethanol and 30% ammonium hydroxide were obtained from Sigma-Aldrich. MilliQ-grade water was used in the preparation of buffers and aqueous solutions.

After 28 days, osteogenic cultures were washed three times with PBS and fixed with 4% (wt/vol) PFA and washed thrice with deionized water. Mineralized matrices were stained with 2% (wt/vol) alizarin red solution and quantified using an earlier protocol 16. Briefly, the stained wells were washed three times with PBS, and 200 μl of 10% (vol/vol) acetic acid (Sigma, Irvine, U.K.) was added to each well (24 well plate) and incubated for 30 minutes in a shaker to elute the stain. The eluted stain was heated to 85°C for 10 minutes, cooled, and neutralized with 10% ammonium hydroxide (Sigma, Irvine, U.K.) read at 405 nm using a spectrophotometer. Fold increase in the absorbance value was calculated by comparing with un‐induced cells in osteo‐permissive medium.