Ab8448

After sacrifice, LV tissue samples were collected and immediately cryo-preserved in liquid nitrogen and stored at −80 °C. Total protein lysates were prepared as described before⁵⁹ . Equal amounts of proteins were separated on a 4–12% Bis-Tri gradient gel (Invitrogen) and transferred onto polyvinylidene difluoride membrane (Millipore). The membrane was blocked with Odyssey blocking buffer (Li-cor) and probed for lysyl oxidase (LOX), (sc-373995 Santa Cruz), Periostin (ab79946, Abcam), TGF-β1 (ab92486, Abcam), Osteopontin (ab8448, Abcam), Fibroblast activation protein-α (orb97039, Biorbyt) and GAPDH (sc-25778, Santa Cruz). Immunofluorescence detection was performed with matching secondary antibodies (Li-cor 800CW or Alexa Fluor 680, ThermoFisher Scientific) using an Infrared imaging system (Li-cor, CLx).

After 21 days of osteogenic differentiation of DPSCs, PDLSCs, DFPCs, and ABMMSCs, the total cellular protein was extracted using the RIPA lysis buffer (Sigma, USA), and concentrations were measured by Bradford protein assay kit (Sangon Biotech, China). MSCs that were cultured in MEM-α medium containing 10% FBS were used as a control. The samples were heated for 8 min at 100°C. Equal amount of proteins was separated by 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, USA). The membranes were blocked with 5% skim milk for 2 hours at room temperature and then incubated with primary antibodies RUNX2 (1 : 2000, ab236639, Abcam), ALP (1 : 2000, ab83259, Abcam), OSX (1 : 2000, ab94744, Abcam), OPN (1 : 2000, ab8448, Abcam), and β-actin (1 : 2000, ab8227, Abcam) overnight at 4°C. After washing with TBST buffer, the blots were incubated with HRP goat anti-rabbit (1 : 1000, Abcam) for 1 hour at room temperature. The proteins were visualized using a ChemiDoc imaging system (Bio-Rad, USA).

After micro-CT scanning, specimens were decalcified using 10% EDTA buffered with Tris-EDTA solution (pH 7.2–7.4, to 700 mL of PBS, add 100 g EDTA and adjust pH as needed to 7.2–7.4 by cautiously adding drops of 10 N NaOH) for 2 weeks. Before being embedded into a paraffin block, the plastic tube was removed. Those blocks were sectioned at a thickness of 4 μm and then stained with hematoxylin/eosin (HE) and Masson trichrome (MT). Histological images were acquired with a digital slide scanner (Paranoramic 250 Flash III, 3d-Histech, Budapest, Hungary). The acquired 3D images were measured using an image analysis program (Image Pro Plus, Media Cybernetics, Inc., Rockville, MD, USA). For immunohistochemistry, the sections were incubated overnight in the primary antibody at 4 °C. The next day, the sections were stained with Alexa Fluor^® 488 goat anti-rabbit IgG (H + L) (Invitrogen, Waltham, MA, USA, A10034) for 1 h at room temperature. Primary antibodies used in staining were rabbit-polyclonal to osteocalcin (mouse-specific, 1:100, Abcam, Cambridge, UK, ab93876), rabbit-polyclonal to osteopontin (mouse/human-specific, 1:100, Abcam, Cambridge, UK, ab8448), and rabbit-polyclonal to CD31 (human/mouse/pig-specific, 1:50, Abcam, Cambridge, UK, ab28364), which were used to demonstrate new bone formation and angiogenesis.

Based on the in vitro release profiles, vehicle, L‐Pro and H‐Pro scaffolds (10 mm × 10 mm × 3 mm) were immersed in 5 mL of standard osteogenic induction media (Cyagen Biosciences, Inc, Santa Clara, CA, USA) for 24 h to prepare conditioned osteogenic media.
BMSCs were cultured in scaffold‐conditioned osteogenic media with or without isoproterenol (1 μM), and the Alkaline Phosphatase Color Development Kit (Beyotime Institute of Biotechnology) was used to measure ALP activity on day 7. Alizarin red S (ARS, Sigma‐Aldrich Co., Taufkirchen, Germany) staining was performed on day 14 to detect mineral nodules. Samples were processed for Western blotting using standard techniques and incubated with anti‐osterix (ab209484, Abcam) and anti‐osteopontin (ab8448, Abcam).

Cell lysates were obtained using RIPA lysis buffer (Beyotime, Shanghai, China) containing 10 mM phenylmethylsulphonylfluoride as a protease inhibitor (PMSF; Beyotime), and 50 μg of total protein was separated in a Bis‐Tris polyacrylamide gel and transferred onto a nitrocellulose membrane. The membrane was then incubated in 5% bovine serum albumin (BSA) containing primary rabbit‐anti‐human polyclonal antibodies at 4°C overnight. Next, samples were incubated with IRDye^® 800CW goat‐anti‐rabbit antibody at room temperature for 1 hr and visualized via chemiluminescence with an infrared laser scanning system (Odyssey Licor, Lincoln, NE, USA). The following primary rabbit‐anti‐human antibodies were used: anti‐JAG1 (1:1000, ab109536; Abcam); anti‐Runx2 (1:1000, ab23981; Abcam); anti‐Sp7/Osterix (1:2000, ab22552; Abcam); anti‐ALP (1:2000, ab95462; Abcam); anti‐OCN (1:500, ab93876; Abcam); anti‐OPN (1:1000, ab8448; Abcam); anti‐cleaved‐Notch 1 (V1754) (1:500, YC0067; Immunoway, Newark, DE, USA); anti‐cleaved‐Notch 2 (D1733) (1:500, YC0069; Immunoway); anti‐β‐Catenin (1:5000, ab32572; Abcam); anti‐ GSK‐3β (1:5000, ab32391; Abcam); and anti‐GAPDH (1:2500, ab9485; Abcam).

Total proteins were extracted using RIPA reagent (Beyotime Biotechnology, Shanghai, China) and the protein concertation was determined by BCA kit (Beyotime Biotechnology, Shanghai, China) as per the instructions. Then, 25 μg of protein was loaded on 10% SDS-PAGE and transferred on PVDF membranes (Millipore, Billerica, MA). After blocking with 5% skimmed milk for 1 h, PVDF membranes were incubated with primary antibodies (rabbit anti-RUNX2, 1:1000, ab236639; rabbit anti-OCN, 1:1000, ab133612; rabbit anti-OSX, 1:1000, ab209484; rabbit anti-OPN, 1:1000, ab8448; rabbit anti-GAPDH, 1:1000, ab9485; all purchased from Abcam, Shanghai, China) overnight at 4°C. The following day, membranes were washed 3 times with TBST buffer for 5 minutes each, and further incubated with secondary antibody (goat anti-rabbit H&L preadsorbed, 1:1000, ab7090; purchased from Abcam, Shanghai, China) for 2 h at room temperature. The protein bands were visualized using an enhanced chemiluminescence kit (Yeasen, Shanghai, China) and photographed on a gel imager system (Bio-Rad, Hercules, CA, United States). GAPDH functioned as the internal control.