Dimethyl suberimidate dihydrochloride

Hyflo Super Cel (Diatomaceous earth), 3-aminopropyl(diethoxy)methylsilane (APDMS, 97%), dimethyl suberimidate dihydrochloride (DMS, 98%), lysozyme solution (50 mg/mL in distilled water), sodium hydroxide solution (50% in H₂O), N-Acetyl-L-cysteine (NALC, 99%), sodium citrate, and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tris-HCI (pH 8.0), distilled water (DNase/RNase-Free), and EDTA (pH 8.0) were purchased from Invitrogen (Waltham, MA, USA). Proteinase K solution (>600 mAU/mL) was purchased from Qiagen (Hilden, Germany). Absolute ethanol was purchased from Merck (Whitehouse Station, NJ, USA). Phosphate-buffered saline (PBS; 10×, pH 7.4) was purchased from Gibco (Grand Island, NY, USA).

The ACC preparation protocol was simplified from (Saltel et al., 2004 (link)). Briefly, 6-well plates or 13 mm diameter glass coverslips were coated with 50 μg/mL calf skin type I collagen (Sigma, #C9791) in 20 mM acetic acid, incubated for 1 h at 37°C and dried overnight. Then supports were successively incubated in (1) 200 mM Tris-buffered saline (TBS) pH 9 containing 0.13 mg/mL egg yolk phosvitin (Sigma, #P1253), 0.13 mg/mL alkaline phosphatase (Sigma, #P764) and 1 mg/mL dimethyl suberimidate dihydrochloride (Sigma, #179523) as a cross-linking reagent during 24 h at 37°C, (2) 6 mM calcium β-glycerophosphate (Sigma, #G6626) for 48 h at 37°C and (3) washed with 200 mM Tris pH 9. The last 3 steps were repeated 3–4 times depending on the amount of precipitated calcium phosphate. Next, the supports were rinsed with distilled water and air dried. Osteoclasts at day 3 of differentiation were detached with Accutase (Sigma, #A6964), scrapped, seeded and grown for 2 more days onto ACC.