Hybrid Implants Based on Calcium-Magnesium Silicate Ceramic Diopside as a Carrier of Recombinant BMP-2 and Demineralized Bone Matrix as a Scaffold: Ectopic Osteogenesis in Intramuscular Implantation in Mice.

High efficiency of hybrid implants based on calcium-magnesium silicate ceramic, diopside, as a carrier of recombinant BMP-2 and xenogenic demineralized bone matrix (DBM) as a scaffold for bone tissue regeneration was demonstrated previously using the model of critical size cranial defects in mice. In order to investigate the possibility of using these implants for growing autologous bone tissue using in vivo bioreactor principle in the patient's own body, effectiveness of ectopic osteogenesis induced by them in intramuscular implantation in mice was studied. At the dose of 7 µg of BMP-2 per implant, dense agglomeration of cells, probably skeletal muscle satellite precursor cells, was observed one week after implantation with areas of intense chondrogenesis, initial stage of indirect osteogenesis, around the implants. After 12 weeks, a dense bone capsule of trabecular structure was formed covered with periosteum and mature bone marrow located in the spaces between the trabeculae. The capsule volume was about 8-10 times the volume of the original implant. There were practically no signs of inflammation and foreign body reaction. Microcomputed tomography data showed significant increase of the relative bone volume, number of trabeculae, and bone tissue density in the group of mice with BMP-2-containing implant in comparison with the group without BMP-2. Considering that DBM can be obtained in practically unlimited quantities with required size and shape, and that BMP-2 is obtained by synthesis in E. coli cells and is relatively inexpensive, further development of the in vivo bioreactor model based on the hybrid implants constructed from BMP-2, diopside, and xenogenic DBM seems promising.

Hybrid Proteins with Short Conformational Epitopes of the Receptor-Binding Domain of SARS-CoV-2 Spike Protein Promote Production of Virus-Neutralizing Antibodies When Used for Immunization.

Based on the previously developed approach, hybrid recombinant proteins containing short conformational epitopes (a.a. 144-153, 337-346, 414-425, 496-507) of the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein (S protein) were synthesized in Escherichia coli cells as potential components of epitope vaccines. Selected epitopes are involved in protein-protein interactions in the S protein complexes with neutralizing antibodies and ACE2 (angiotensin-converting enzyme 2). The recombinant proteins were used for immunization of mice (three doses with 2-week intervals), and the immunogenicity of protein antigens and ability of the resulting sera to interact with inactivated SARS-CoV-2 and RBD produced in eukaryotic cells were examined. All recombinant proteins showed high immunogenicity; the highest titer in the RBD binding assay was demonstrated by the serum obtained after immunization with the protein containing epitope 414-425. At the same time, the titers of sera obtained against other proteins in the RBD and inactivated virus binding assays were significantly lower than the titers of sera obtained with the previously produced four proteins containing the loop-like epitopes 452-494 and 470-491, the conformation of which was fixed with a disulfide bond. We also studied activation of cell-mediated immunity by the recombinant proteins that was monitored as changes in the levels of cytokines in the splenocytes of immunized mice. The most pronounced increase in the cytokine synthesis was observed in response to the proteins containing epitopes with disulfide bonds (452-494, 470-491), as well as epitopes 414-425 and 496-507. For some recombinant proteins with short conformational epitopes, adjuvant optimization allowed to obtained mouse sera displaying virus-neutralizing activity in the microneutralization assay with live SARS-CoV-2 (hCoV-19/Russia/StPetersburg-3524/2020 EPI_ISL_415710 GISAID). The results obtained can be used to develop epitope vaccines for prevention of COVID-19 and other viral infections.

Development of a Platform for Producing Recombinant Protein Components of Epitope Vaccines for the Prevention of COVID-19.

A new platform for creating anti-coronavirus epitope vaccines has been developed. Two loop-like epitopes with lengths of 22 and 42 amino acid residues were selected from the receptor-binding motif of the Spike protein from the SARS-CoV-2 virus that participate in a large number of protein-protein interactions in the complexes with ACE2 and neutralizing antibodies. Two types of hybrid proteins, including one of the two selected epitopes, were constructed. To fix conformation of the selected epitopes, an approach using protein scaffolds was used. The homologue of Rop protein from the Escherichia coli ColE1 plasmid containing helix-turn-helix motif was used as an epitope scaffold for the convergence of C- and N-termini of the loop-like epitopes. Loop epitopes were inserted into the turn region. The conformation was additionally fixed by a disulfide bond formed between the cysteine residues present within the epitopes. For the purpose of multimerization, either aldolase from Thermotoga maritima, which forms a trimer in solution, or alpha-helical trimerizer of the Spike protein from SARS-CoV-2, was attached to the epitopes incorporated into the Rop-like protein. To enable purification on the heparin-containing sorbents, a short fragment from the heparin-binding hemagglutinin of Mycobacterium tuberculosis was inserted at the C-terminus of the hybrid proteins. All the obtained proteins demonstrated high level of immunogenicity after triplicate parenteral administration to mice. Sera from the mice immunized with both aldolase-based hybrid proteins and the Spike protein SARS-CoV-2 trimerizer-based protein with a longer epitope interacted with both the inactivated SARS-CoV-2 virus and the Spike protein receptor-binding domain at high titers.

Dual-Functional Implant Based on Gellan-Xanthan Hydrogel with Diopside, BMP-2 and Lysostaphin for Bone Defect Repair and Control of Staphylococcal Infection.

A new dual-functional implant based on gellan-xanthan hydrogel with calcium-magnesium silicate ceramic diopside and recombinant lysostaphin and bone morphogenetic protein 2 (BMP-2)-ray is developed. In this composite, BMP-2 is immobilized on microparticles of diopside while lysostaphin is mixed directly into the hydrogel, providing sustained release of BMP-2 to allow gradual bone formation and rapid release of lysostaphin to eliminate infection immediately after implantation. Introduction of diopside of up to 3% (w/v) has a negligible effect on the mechanical properties of the hydrogel but provides a high sorption capacity for BMP-2. The hydrogels show good biocompatibility and antibacterial activity. Lysostaphin released from the implants over a 3 h period efficiently kills planktonic cells and completely destroys 24 h pre-formed biofilms of Staphylococcus aureus. Furthermore, in vivo experiments in a mouse model of critically-sized cranial defects infected with S. aureus show a complete lack of osteogenesis when implants contain only BMP-2, whereas, in the presence of lysostaphin, complete closure of the defect with newly formed mineralized bone tissue is observed. Thus, the new implantable gellan-xanthan hydrogel with diopside and recombinant lysostaphin and BMP-2 shows both osteogenic and antibacterial properties and represents a promising material for the treatment and/or prevention of osteomyelitis after bone trauma.

Osteoconductive, Osteogenic, and Antipathogenic Plasma Electrolytic Oxidation Coatings on Titanium Implants with BMP-2.

We report a one-pot plasma electrolytic oxidation (PEO) strategy for forming a multi-element oxide layer on the titanium surface using complex electrolytes containing Na2HPO4, Ca(OH)2, (NH2)2CO, Na2SiO3, CuSO4, and KOH compounds. For even better bone implant ingrowth, PEO coatings were additionally loaded with bone morphogenetic protein-2 (BMP-2). The samples were tested in vivo in a mouse craniotomy model. Tests for bactericidal and fungicidal activity were carried out using clinically isolated multi-drug-resistant Escherichia coli (E. coli) K261, E. coli U20, methicillin-resistant Staphylococcus aureus (S. aureus) CSA154 bacterial strains, and Neurospora crassa (N. crassa) and Candida albicans (C. albicans) D2528/20 fungi. The PEO-Cu coating effectively inactivated both Gram-positive and Gram-negative bacteria at low concentrations of Cu2+ ions: minimal bactericidal concentration for E. coli and N. crassa (99.9999%) and minimal inhibitory concentration (99.0%) for S. aureus were 5 ppm. For all studied bacterial and fungal strains, PEO-Cu coating completely prevented the formation of bacterial and fungal biofilms. PEO and PEO-Cu coatings demonstrated bone remodeling and moderate osteoconductivity in vivo, while BMP-2 significantly enhanced osteoconduction and osteogenesis. The obtained results are encouraging and indicate that Ti-based materials with PEO coatings loaded with BMP-2 can be widely used in customized medicine as implants for orthopedics and cranio-maxillofacial surgery.