Biological evaluation of acellular bovine bone matrix treated with NaOH

We mainly proceed from the view of biological effect to study the acellular bovine bone matrix (ABBM) by the low concentration of hydrogen oxidation. After cleaning the bovine bone routinely, it was cleaned with different concentrations of NaOH and stained with hematoxylin-eosin (HE) to observe the effect of decellulization. The effect of bovine bone matrix treated with NaOH were observed by optical microscopy and scanning electron microscopy (SEM), and compared by DNA residue detection. Cell toxicity was also evaluated in MC3T3-E1 cells by CCK-8. For the in vitro osteogenesis detection, alkaline phosphatase (ALP) staining and alizarin red (AR) staining were performed in MC3T3-E1 cells. And the in vivo experiment, Micro CT, HE and Masson staining were used to observe whether the osteogenic effect of the materials treated with 1% NaOH solution was affected at 6 and 12 weeks. After the bovine bone was decellularized with different concentrations of NaOH solution, HE staining showed that ultrasonic cleaning with 1% NaOH solution for 30 min had the best effect of decellularization. The SEM showed that ABBM treated with 1% NaOH solution had few residual cells on the surface of the three-dimensional porous compared to ABBM treated with conventional chemical reagents. DNA residues and cytotoxicity of ABBM treated with 1% NaOH were both reduced. The results of ALP staining and AR staining showed that ABBM treated with 1% NaOH solution had no effect on the osteogenesis effect. The results of micro-CT, HE staining and Masson staining in animal experiments also showed that ABBM treated with 1% NaOH solution had no effect on the osteogenesis ability. The decellularization treatment of ABBM with the low concentration of NaOH can be more cost-effective, effectively remove the residual cellular components, without affecting the osteogenic ability. Our work may provide a novelty thought and a modified method to applicate the acellular bovine bone matrix clinically better. Graphical abstract.