Df12303

mCOBs after 14 days of osteogenic induction were collected and lysed in RIPA buffer, as previously described (29 (link)). The protein concentrations in each sample were measured using Bradford assays with Coomassie brilliant blue G-250 (Bio-Rad, California, USA). A total of 20 μg protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). The SDS membranes were blocked with 5% nonfat milk for 1 hour and incubated with primary OCN antibodies overnight (DF12303, Affinity Biosciences, Beijing, China). ACTB (Abclonal, Wuhan, China) was examined as the housekeeping reference protein. After incubation for 1 hour with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody at a dilution of 1:5,000, band signals were detected using a Bio-Rad imaging system (Bio-Rad, USA) with NcmECL High (NCM Biotech, Jiangsu, China). The Image lab software program (Bio-Rad, USA) was utilized to perform relatively quantitative analysis of protein levels.

The protein expression of SD rat BMSCs was detected by Western Blotting. After 24 h, the medium was replaced with normal osteogenic induction medium and osteogenic induction medium containing 10% of different scaffold extracts. After culturing for 14 days, cell precipitation was collected; 100 µl of RIPAcell lysate (Beyotime, China) containing 1 mM PMSF, (Beyotime, China) was added to each 6-well plate, lysed, centrifuged, and collected the supernatant. Total cellular protein was quantified using a BCA protein assay kit. Then the samples were subjected to SDS electrophoresis and transferred to a polyvinylidene fluoride membrane. The membranes were first blocked with 5% skim milk for 1 h, and then incubated at 4 °C overnight with primary antibodies as follows: RUNX2 (AF5186, Affinity, 1:500), OCN (DF12303, Affinity, 1:500), ALP (DF6225, Affinity, 1:500), and Collagen I (AF7001, Affinity, 1:500). After being washed with TBST three times, the membranes were incubated with HRP-conjugated secondary antibodies (1:10,000). The antigen–antibody complex was visualized. Signal intensities were quantified using ImageJ software.

Paraffin sections were warmed at 65 °C for 1 h, dewaxed with xylene and ethanol, subjected to antigen retrieval by the dropwise addition of 0.25% trypsin and incubated at 37 °C for 30 min. Peroxidase blocking solution was added dropwise, and the sections were incubated for 15 min. Then, the goat serum working solution was added dropwise to the tissue for blocking, and the sections were incubated at 37 °C for 30 min. SDF-1α (ab9797, 1:100; Abcam), CD44 (ab189524, 1:50, Abcam), CD90 (DF6849, 1:50, Affinity), RUNX2 (ab23981, 1:50, Abcam), and OCN (DF12303, 1:50, Affinity) primary antibodies were added dropwise to the tissues until they were fully covered, and the sections were incubated at 4 °C for 12 h. The samples were heated at 37 °C for 30 min, incubated with DV-9001 for 30 min at 37 °C, and incubated with a secondary antibody for 60 min at 37 °C. DAB was added for color development, and then the sections were stained with hematoxylin for 5 min, differentiated with hydrochloric acid-alcohol for 5 s and rinsed with tap water until the tissue appeared blue. The sections were dehydrated in ethanol, cleared in xylene, mounted with neutral gum, allowed to dry naturally, observed and imaged.