Sodium tartrate

Sections were deplastified prior to staining. To show TRAP activity, sections were incubated in Sodium Acetate buffer, Naphthol‐AS‐TR phosphate (N6125‐1G, Sigma, Germany) and Sodium Tartrate (Merck, Germany) at 37°C for 60 min.
IHC was done using the following primary antibodies (Abcam Company, Cambridge, UK): rabbit monoclonal (EPR53) to α‐SMA, rabbit polyclonal (OAA100188) to osteopontin, mouse monoclonal (LS‐C83497‐100) to osteocalcin and rabbit monoclonal (EPR7785) to collagen type I.
To study the blood vessel formation, α‐SMA, osteopontin, osteocalcin, and collagen type‐I were diluted in DAKO‐Diluent (S 0809), 1:400, 1:400, 1:1200, and 1:1200, respectively. Collagen type‐I, α‐SMA, osteocalcin, and osteopontin staining were quantified as described for histomorphometry, using the same ROIs. The number of osteoblasts was counted manually in TRAP‐stained sections at the bone interface of ROItot. Blood vessels were classified and quantified as circular, intermediate, or irregular by α‐SMA in ROI400 μm.

Medium bran oat flour (i.d. 112-001) with a particle size percentage distribution of 2.00 mm (0.8%), 0.841 mm (61.5%), 0.595 mm (32.1%), 0.420 mm (5.0%), and Pan (0.6%) was donated by Richardson Milling (Portage La Prairie, MB, Canada). The enzymes (α-amylase, Flavourzyme^®, Alcalase^®, and Papain, sodium tartrate, sodium dodecyl sulfate, cupric sulfate pentahydrate, Trolox, 1,10-phenanthroline, iron(II) sulfate heptahydrate, hydrogen peroxide (H₂O₂), Tris-HCl, Tris-Base, potassium bromide, pyrogallol, reduced glutathione, L-serine, 3,5-dinitrosalicylic acid, sodium potassium phosphate tartrate, starch, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Ltd. (Oakville, Ontario, Canada). The solvents, including concentrated hydrochloric acid, methanol and Folin-Ciocalteau Phenol reagent, fluorescein, and 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH), were purchased from Fisher Scientific Co. (Nepean, Ontario, Canada).

Experiments were carried out with Milli-Q water (18.2 MΩ·cm). Inorganic salts, hydrogen peroxide (30% solution), glucose, potassium lactate (60% solution), fructose, mannose, sodium gluconate, sodium tartrate, (3-aminopropyl)triethoxysilane (99%), and organic solvents were obtained from Sigma-Aldrich (Burlington, MA, USA) or Reachim (Moscow, Russia) at the highest purity. Perfluorosulfonated ionomer (PFSI) (10% solution in isopropyl alcohol), a structural analogue of Nafion, was obtained from Plastpolimer (St. Peterburg, Russia). Hydrochloric acid solutions were prepared from fixanals manufactured by Germed (Dresden, Germany).
Lactate oxidase (LOx, EC1.1.3.2) from Pediococcus species (Sorachim, Lausanne, Switzerland) was used in the form of a lyophilized protein with a declared activity of 32.8 U/mg. glucose oxidase (GOx, EC 1.1.3.4) from Aspergillus niger (Sigma-Aldrich, Burlington, MA, USA) was used in the form of a lyophilized protein with a declared activity of 246.6 U/mg. Standard samples of blood serum were obtained from Spinreact (Girona, Spain).

Suramin (Cat. S2671), ATP (Cat. A6419), TRAP staining (387A-1KT), sodium tartrate (Cat. S4797), pNPP (Cat. N2765), LPS (Cat. L2630) and apyrase (Cat. A6535) were from Sigma-Aldrich (St Louis, MO, USA). Antibodies used in immunoblots to NFATc1 (Cat. sc-7294), c-Src (Cat. Sc-19) and IL-1β (Cat. sc-7884), and for the P2X5 blocking experiment (Cat. sc-15192) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), to DC-STAMP (Cat. MABF39) was from EMD Millipore (Bedford, MA, USA), to caspase-1 (Cat. AG-20B-0042-c100) was from Adipogen (San Diego, CA, USA), to P2X5 (Cat. ARP35511_P050) was from Aviva systems biology (San Diego, CA, USA). Unless indicated otherwise, all other reagents were from Sigma-Aldrich.

Samples were washed with PBS and fixed with 4 % formaldehyde in PBS for 15 min at room temperature. Fixation solution was removed by washing with PBS. TRAP staining was performed for 30 min at 37 °C with 0.3 mg/mL Fast Red Violet LB (Sigma-Aldrich) dissolved in an aqueous staining buffer containing 0.05 M sodium acetate (Sigma-Aldrich), 0.05 M acetic acid (Sigma-Aldrich), 0.03 M sodium tartrate (Roth), 0.1 mg/mL naphthol AS-MX phosphate disodium salt (Sigma-Aldrich) and 0.1 % Triton X-100 (Sigma-Aldrich). Afterwards, samples were washed with PBS and cell nuclei were stained for 10 min using Mayers Haemalaun solution (AppliChem) followed by rinsing in tap water. Bright field images of stained samples were taken using a Keyence BZ-X810 microscope.

Isopropyl-β-D-thiogalactopyranoside (IPTG), dithiothreitol, hemin, oxidized glutathione, veratryl alcohol (VA), manganese(II) sulphate and sodium tartrate were from Sigma-Aldrich; urea and H₂O₂ from Merck; and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) from Roche.

CCl₄, reduced glutathione (GSH), glutathione reductase, γ-glutamyl p-nitroanilide, bovine serum albumin (BSA), 1,2-dithio-bis nitro benzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), 2,6-dichlorophenolindophenol, thiobarbituric acid (TBA), picric acid, potassium iodide, sodium tartrate, copper sulphate, bromo cresol green, hydrogen peroxide solution, phenazine methosulphate, glycylglycine, magnesium chloride, guaiacol, sulfosalicylic acid, sodium azide, reduced nicotinamide adenine dinucleotide (NADH), sodium hydroxide, and trichloroacetic acid (TCA) were purchased from Sigma Chemicals Co. USA.