ИСТИНА

The objective of this study is to evaluate acute toxicity of the novel recombinant Bone Morphogenetic Protein 2 with an additional s-tag domain (s-tag-BMP-2) and EPO-HBD fusion protein consisting of sequences of human erythropoietin (EPO) and the heparin-binding protein domain (HBD) from Bone Morphogenetic Protein 2 that were immobilized on the demineralized bone matrix (DBM). Both EPO-HBD and s-tag-BMP-2 were expressed in Escherichia coli and extracted from the inclusion bodies. Following purifiation and refolding proteins were bound to DBM disks of 1.5 (thickness) x 4 (diameter) mm and sterilized. Study of toxic effcts was carried out in inbred CBA mice line (4-6 weeks old, average weight18.7±1.3 g, n=40). For this study, there were four experimental groups each consisting of five male and five female mice. The fist group of 10 intact animals served as the control. The second and third groups received DBM with s-tagBMP-2 (10 mkg) and with EPO-HBD (10 mkg) respectively. The fourth group was administered by DBM with both recombinant proteins with the same dosages. Composite materials were administered by single subcutaneous implantation.Following the two-weeks healing interval, toxic effects were assessed using clinical and laboratory diagnostic methods: clinical examination and observation, assessment of body weight dynamics, hematological and biochemical blood analysis, and pathologic anatomical research with the determination of mass coeffients of internal organs. Compared to the control group none of the experimental groups registered induced changes in body weight, food intake, clinical signs, and zoosocial behavior. None of the composite materials caused any death. There were no changes in the level of enzymes, bilirubin, and the protein and carbohydrate metabolism in the body. There were single statistically signifiant changes in the clinical blood parameters of the third and fourth experimental groups which are specifi to the activity of erythropoietin. The pathoanatomical analysis showed no abnormalities in the anatomical structure of organs, and there were no diffrences in mass coeffients. The results of this study show that s-tag-BMP-2 and EPO-HBD immobilized on the demineralized bone matrix did not induce any remarkable toxic response in mice and are biologically safe. This work was supported by the Russian Science Foundation (Grant No. 16-15-00133).