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 Implants Based on Calcium-Magnesium Silicate Ceramics Diopside as a Carrier of Recombinant BMP-2 and Demineralized Bone Matrix as a Scaffold: Dynamics of Reparative Osteogenesis in a Mouse Craniotomy Model.

Calcium-magnesium silicate ceramics, diopside, is a promising material for use in bone plastics, but until now the possibility of its use as a carrier of recombinant bone morphogenetic protein-2 (BMP-2) has not been studied, as well as the features of reparative osteogenesis mediated by the materials based on diopside with BMP-2. Powder of calcium-magnesium silicate ceramics was obtained by solid-state synthesis using biowaste - rice husks and egg shells - as source components. Main phase of the obtained ceramics was diopside. The obtained particles were irregularly shaped with an average size of about 2.3 µm and ~20% porosity; average pore size was about 24 nm, which allowed the material to be classified as mesoporous. Diopside powder adsorbs more than 150 µg of recombinant BMP-2 per milligram, which exceeds binding capacity of hydroxyapatite, a calcium-phosphate ceramic often used in hybrid implants, by more than 3 times. In vitro release kinetics of BMP-2 was characterized by a burst release in the first 2 days and a sustained release of approximately 0.4 to 0.5% of the loaded protein over the following 7 days. In vivo experiments were performed with a mouse model of cranial defects of critical size with implantation of a suspension of diopside powder with/without BMP-2 in hyaluronic acid incorporated into the disks of demineralized bone matrix with 73-90% volume porosity and macropore size from 50 to 650 µm. Dynamics of neoosteogenesis and bone tissue remodeling was investigated histologically at the time points of 12, 21, 48, and 63 days. Diopside particles were evenly spread in the matrix and caused minimal foreign body reaction. In the presence of BMP-2 by the day 63 significant foci of newly formed bone tissue were formed in the implant pores with bone marrow areas, moreover, large areas of demineralized bone matrix in the implant center and maternal bone at the edges were involved in the remodeling. Diopside could be considered as a promising material for introduction into hybrid implants as an effective carrier of BMP-2.

Effect of recombinant BMP-2 and erythropoietin on osteogenic properties of biomimetic PLA/PCL/HA and PHB/HA scaffolds in critical-size cranial defects model.

Osteoplastic materials PLA/PCL/HA and PHB/HA and scaffolds with a highly porous structure based on them with potential applications in regenerative medicine have been obtained by solvent casting with thermopressing and salt leaching for PLA-based samples and solid-state mixing with subsequent thermopressing and salt leaching for PHB-based samples. The scaffolds were characterized by SEM-EDX, DSC, FTIR spectroscopy, mechanical tests in compression, measurement of the contact angle, in vitro studies, including loading by recombinant BMP-2 and EPO and their release kinetics, and in vivo studies on a model of regeneration of critical-sized cranial defects in mice. Biomimetic scaffolds with micropores sizes ranged from 300 to 500 µm and volume porosity of 70% imitate trabecular bone's structure and have increased hydrophilicity to achieve osteoconductive properties. Mechanical characteristics correspond to native trabecular bone. Elastic modulus - key mechanical characteristics of bone implants - showed the values of 0.15 ± 0.04 and 0.18 ± 0.08 GPa for PLA/PCL/HA and PHB/HA scaffolds, respectively. Both materials have high biocompatibility and can be used together with recombinant proteins BMP-2 and EPO. Introduction of BMP-2 leads to induction of new bone formation, introduction of EPO results in increased angiogenesis in the implantation area. The obtained scaffolds with recombinant proteins can be used as bone implants for reconstruction of defects of lightly or non-loaded bones.

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.

Biomimetic UHMWPE/HA scaffolds with rhBMP-2 and erythropoietin for reconstructive surgery.

A promising direction for the replacement of expanded bone defects is the development of bioimplants based on synthetic biocompatible materials impregnated with growth factors that stimulate bone remodeling. Novel biomimetic highly porous ultra-high molecular weight polyethylene (UHMWPE)/40% hydroxyapatite (HA) scaffold for reconstructive surgery with the porosity of 85 ± 1% vol. and a diameter of pores in the range of 50-800 µm was developed. The manufacturing process allowed the formation of trabecular-like architecture without additional solvents and thermo-oxidative degradation. Biomimetic UHMWPE/HA scaffold was biocompatible and provided effective tissue ingrowth on a model of critical-sized cranial defects in mice. The combined use of UHMWPE/HA with Bone Morphogenetic Protein-2 (BMP-2) demonstrated intensive mineralized bone formation as early as 3 weeks after surgery. The addition of erythropoietin (EPO) significantly enhanced angiogenesis in newly formed tissues. The effect of EPO of bacterial origin on bone tissue defect healing was demonstrated for the first time. The developed biomimetic highly porous UHMWPE/HA scaffold can be used separately or in combination with rhBMP-2 and EPO for reconstructive surgery to solve the problems associated with difference between implant architecture and trabecular bone, low osteointegration and bioinertness.

Recombinant domains III of Tick-Borne Encephalitis Virus envelope protein in combination with dextran and CpGs induce immune response and partial protectiveness against TBE virus infection in mice.

BACKGROUND: E protein of tick-borne encephalitis virus (TBEV) and other flaviviruses is located on the surface of the viral particle. Domain III of this protein seems to be a promising component of subunit vaccines for prophylaxis of TBE and kits for diagnostics of TBEV. METHODS: Three variants of recombinant TBEV E protein domain III of European, Siberian and Far Eastern subtypes fused with dextran-binding domain of Leuconostoc citreum KM20 were expressed in E. coli and purified. The native structure of domain III was confirmed by ELISA antibody kit and sera of patients with tick-borne encephalitis. Immunogenic and protective properties of the preparation comprising these recombinant proteins immobilized on a dextran carrier with CpG oligonucleotides as an adjuvant were investigated on the mice model. RESULTS: All 3 variants of recombinant proteins immobilized on dextran demonstrate specific interaction with antibodies from the sera of TBE patients. Thus, constructed recombinant proteins seem to be promising for TBE diagnostics. The formulation comprising the 3 variants of recombinant antigens immobilized on dextran and CpG oligonucleotides, induces the production of neutralizing antibodies against TBEV of different subtypes and demonstrates partial protectivity against TBEV infection. CONCLUSIONS: Studied proteins interact with the sera of TBE patients, and, in combination with dextran and CPGs, demonstrate immunogenicity and limited protectivity on mice compared with reference "Tick-E-Vac" vaccine.