Osteodiff

The mineralization or calcification ability of the hBMMSCs in contact with the tested NPs was analyzed by alizarin red S Staining (ARS) after 21 d of culture. The hBMMSCs were seeded onto 24-well plates at 1 × 10⁴ cells/well concentrations and allowed for attachment. The cells were then transferred into the NPs-conditioned medium and cultured for 21 d. After the culture period, the cells were fixed in 95% ethanol for 30 min at room temperature (RT), rinsed three times with double-distilled water, stained with 5% of alizarin red (pH = 4.2, Sigma Aldrich, St. Louis, MO, USA) for 5–10 min, washed repeatedly with double distilled water, and then dried at RT. The dried plate was observed under a stereomicroscope (Leica Microsystems GmbH, Wetzlar, Germany) to acquire relevant images. For quantification of the calcified nodules, the alizarin red was dissolved in 10% cetylpyridinium chloride (Sigma-Aldrich, MO, USA). After that, the plate was read at an absorbance of 405 nm by the spectrophotometric microplate reader (Thermo Fisher, USA). For this assay, both a negative control (hBMMSCs cultured in unconditioned growth medium, DMEM; Gibco, USA) and a positive control (hBMMSCs cultured in osteoinductive media (OsteoDiff^®, Miltenyi Biotec, Germany) were used for reference.

An Alizarin Red S Staining (ARS) assay was performed to assess hPDLSC calcified nodule formation in contact with the tested sealers (AHPbcs, AHP and ESbcs), as a measurement of their biomineralization potential. Twenty‐thousand hPDLSCs per well were seeded onto 12‐well plates (n = 3) and left to proliferate until confluency was reached. The cells were then transferred into undiluted (1:1) sealer‐conditioned medium and cultured for 21 days. After the culture period, the samples were rinsed with foetal bovine serum and fixed with 70% ethanol for 1 h. Then, samples were stained with 2% Alizarin Red solution (Sigma Aldrich) for 30 min in controlled conditions (dark ambient and room temperature) and solubilized using 10% cetylpyridinium chloride monohydrate solution (Sigma‐Aldrich). Lastly, absorbance values of the samples were measured using Synergy H1 multi‐mode microplate reader (BioTek) at 570 nm. For this assay, both a negative control (hDPSCs cultured in unconditioned growth medium [DMEM; Gibco]) and a positive control (hDPSCs cultured in osteogenic medium (OsteoDiff; Miltenyi Biotec) were used for reference.

The actual MSC preparations used on each day for the sheep treatments were tested to ensure that they retained tri‐lineage differentiation capacity consistently with other MSC preparations.
Chondrogenesis: 250 000 MSCs were pelleted by centrifugation at 500 g in chondrogenic selection media (ChondroDiff, Miltenyi Biotec, Mcquarie Park, NSW Australia) and cultured for 21 days with media changes every 2 to 3 days; cell pellets were washed in PBS, fixed in 10% neutral buffered formalin for 4 hours and stored in 70% ethanol, then dehydrated in sequential ethanol solutions and xylene then embedded in paraffin. Microtome sections (4 μm) were stained with toluidine blue‐fast green to visualize tissue proteoglycans.
Osteogenesis: 10 000 MSCs/well seeded in 24 well plates were cultured for 10 days in osteogenic selection media (OsteoDiff, Miltenyi Biotec), which was changed every 3 to 4 days; calcium deposition was detected by Alizarin Red S staining of monolayers using 0.37% w/v Alizarin red S, pH 4.2.
Adipogenesis: 50000 MSCs/well seeded in 12 well plates were cultured in αMEM containing 15% FCS, 10 mM L‐glutamine, 0.5 μM dexamethasone, 0.5 μM IBMS and 50 μM indomethacin for 2 weeks, media was changed every 3 to 4 days; Adipogenesis was demonstrated by triglyceride staining on formalin fixed monolayers using Oil Red O (5 g/mL in 70% v/v isopropanol) at 37°C.

ChondroDiff^® (cat no. 130-091-679), OsteoDiff^® (cat no. 130-091-678) selection medias were purchased from Miltenyi Biotec (North Ryde, New South Wales, Australia). Chondroitinase-ABC, dexamethasone, indomethacin and 3-isobutyl-1-methylxanthine were obtained from Sigma-Aldrich (New South Wales, Australia). Menzel and Glaser SuperFrost ultraPlus, positively charged microscope slides were obtained from Fisher Scientific, GmbH, Braunschweig, Germany. NovaRED substrate was obtained from Vector Laboratories (CA, USA). FGF-2 (cat no. 100-18C) and FGF-18 (cat no. 100-28) were purchased from PeproTech (Sapphire Biosciences, New South Wales, Australia).

At P3-P4, part of ASC expanded in complete Alpha-MEM medium added with 5% (vol/vol) SRGF—batch A or SRGF—batch B were detached by trypsinization. Cells were seeded at 10,000 cells/cm² and after complete adhesion (24 h) adipogenic, chondrogenic and osteogenic differentiation was induced utilizing StemMACS AdipoDiff, ChondroDiff and OsteoDiff media (Miltenyi Biotec GmbH, Bergisch Gladbach; Germany). After 21 days, differentiated osteocytes, adipocytes and chondrocytes were stained by Alizarin Red, Oil Red-O and Safranin-O (Sigma), respectively.