Recombinant bmp 2

Co-IP assays were performed using conditioned media from hVSMCs cultured in the presence of 500 ng/ml of recombinant BMP-2 (Preprotech) for 24 h in M199 with 10% FBS, and the Dynabeads Co-immunoprecipitation Kit (Novex, Life Technologies), according to manufacturer’s recommendations. The BMP-2/4 monoclonal (Santa Cruz Biotechnology, sc-137087) and negative IgG antibodies were used as capture antibodies in the Co-IP reactions, and Western blot analysis of the eluted proteins were performed using CTerm-Ucma/GRP (GenoGla Diagnostics, Faro, Portugal) and BMP-2/4 antibodies as described above.

Anti-p-p65, anti-p65, anti-p-ERK1/2, anti-ERK1/2, anti-p-AKT, and anti-AKT were purchased from Cell Signaling Technology (Boston, MA, USA). Anti-GAPDH antibody was purchased from the ProteinTech Group (Chicago, IL, USA). Anti-BMP2, anti-BMP4, anti-BMPRIa, anti-BMPRII, anti-BMPRIb, and anti-occludin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant BMP2, BMP4, and noggin were purchased from Peprotech (NJ, USA). The inhibitor of NF-κB, PDTC, was purchased from Beyotime (Wuhan, China).

Collagen solution (collagen: BD, USA; HEPES: Gibco®, USA; 1 N NaOH: Gibco®, USA; HBSS: Gibco® USA) was mixed with recombinant BMP-2 (1 μg/ml, PeproTech, Rocky Hill, NJ, USA) or PBS solution. After cultivating in an incubator for 12 h at 37 °C, further incubation was done at − 80 °C for 12 h in a deep freezer. Thereafter, a collagen sheet was fabricated by using a lyophilizer. Collagen sheet (width 10 mm, thickness 0.5 mm) was inserted into the calvarial defect area. Control (PBS-loaded) or BMP-2-loaded collagen scaffolds were used in experiments on cranial bone regeneration models.
Scanning electron microscopy (SEM, JSM-6700F, Jeol, Tokyo, Japan) was performed on the external surface of the collagen scaffold. Photographs were taken at a magnification of ×1,000 and ×10,000.
In vitro BMP-2 release from the BMP-2-loaded collagen scaffold (BMP-2, 1 μg/ml) was evaluated using a commercially available ELISA kit (Quantikine®, R&D systems, Minneapolis, MN, USA). A 10 mm × 10 mm-size BMP-2-loaded collagen scaffold (BMP-2, 1 μg/ml) was cut and immersed in 1 ml PBS (pH 7.4) solution. The amount of release of BMP-2 protein into the media at indicated time points was measured (n = 4).