Ab52128

Samples were completely decalcified in 15% EDTA pH 7.0 in ddH₂O and embedded in paraffin. Histological tissue sections (7 μm) were stained with Masson’s trichrome (Réactifs RAL, Martillac, France) as previously described [34 (link),35 (link)]. Expression of bone sialoprotein (BSP) was determined using immunohistochemistry. Binding of BSP primary antibody (ab52128, Abcam, Cambridge, UK) was visualized using Vectastain Fast Red kit (Vector Laboratories, Glostrup, Denmark) according to the manufacturer’s instructions. Images were taken with a BX61 microscope (Olympus, Tokyo, Japan). Quantification of BSP staining area per scaffold area was performed using ImageJ (National Institutes of Health, Bethesda, MD, USA).

Seven days after osteoinduction, Western blotting was conducted as described in our previous study,²⁸ using primary antibodies against DMP 1 (ab103203, Abcam), DSPP (sc‐73632, Santa Cruz Biotechnology, Dallas, TX), BSP (ab52128, Abcam), OSX (ab94744, Abcam), and VEGF (sc‐57496, Santa Cruz Biotechnology), with GAPDH as an internal control. Membranes were visualized using a chemiluminescent reagent (Sigma‐Aldrich) under a Western blotting imaging system (Bio‐Rad, Hercules, CA), and protein expression was quantified using ImageJ software.

Samples were fixed in 4% paraformaldehyde overnight, decalcified using EDTA (if necessary), embedded in paraffin and sliced at a thickness of 5 μm. The sections were stained with hematoxylin and eosin (H&E; Sigma‐Aldrich), Safranin‐O, Masson trichome, Alizarin red (both from Solarbio), or Movat's pentachrome staining (Abcam). Sections were also analyzed for tartrate‐resistant acid phosphatase (TRAP) activity using the leukocyte acid phosphatase kit (Sigma‐Aldrich).
For immunohistochemical staining after rehydration, the sections were blocked with 5% goat serum and incubated with the following primary antibodies: rabbit anti‐human collagen type I (Col I; ab138492), rabbit anti‐human collagen type II (Col II; ab34712), mouse anti‐human collagen type X (Col X; ab49945), rabbit anti‐human VEGF (ab46154), rabbit anti‐human bone sialoprotein (BSP; ab52128), rabbit anti‐human matrix metalloproteinase‐9 (MMP‐9; ab38898), rabbit anti‐human MMP‐13 (ab39021), rabbit anti‐human BMP‐2 (ab6285), rabbit anti‐human nuclear mitotic apparatus protein (NuMA; ab241470), and rabbit anti‐human and rat Osterix (ab209484; all from Abcam). After sequential incubation with a biotinylated secondary antibody and an ABC‐alkaline phosphatase complex, specific staining was revealed by using Fast Red (Dako).

After culturing for 7 and 14 days, cells were washed with PBS and lysed using a RIPA lysis buffer (Sigma-Aldrich). 30 μg of lysate protein samples were loaded to SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were blotted with primary antibodies overnight at 4 °C and subsequently with secondary antibodies (Beyotime Biotech) for 1 h at room temperature. The proteins were visualized using enhanced chemiluminescence reagents from Pierce Biotechnology (Rockford, IL, USA). Image J (softonic^®) software was used to qualify the protein expression. The primary antibodies used were as follows: anti-BSP (AB52128, Abcam, Cambridge, MA), anti- Runx2 (AB76956, Abcam), and anti-OCN (AB13420, Abcam).