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.

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.

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.

Biocompatibility and Physico-Chemical Properties of Highly Porous PLA/HA Scaffolds for Bone Reconstruction.

The major problem in bone tissue engineering is the development of scaffolds which can simultaneously meet the requirements of porous structure, as well as have the ability to guide the regeneration of damaged tissue by biological fixation. Composites containing biodegradable matrix and bioactive filler are the new hope in this research field. Herein we employed a simple and facile solvent casting particulate-leaching method for producing polylactide acid/hydroxyapatite (PLA/HA) composites at room temperature. FT-IR analysis confirmed the existence of necessary functional groups associated with the PLA/HA composite, whereas energy-dispersive X-ray (EDX) spectra indicated the uniform distribution of hydroxyapatite particles in the polymer matrix. The beehive-like surface morphology of the composites revealed the presence of macropores, ranged from 300 to 400 µm, whereas the thickness of the pores was noticed to be 1-2 µm. The total porosity of the scaffolds, calculated by hydrostatic weighing, was found to be 79%. The water contact angle of pure PLA was decreased from 83.6 ± 1.91° to 62.4 ± 4.17° due to the addition of hydroxyapatite in the polymer matrix. Thus, the wettability of the polymeric biomaterial could be increased by preparing their composites with hydroxyapatite. The adhesion of multipotent mesenchymal stromal cells over the surface of PLA/HA scaffolds was 3.2 times (p = 0.03) higher than the pure PLA sample. Subcutaneous implantation in mice demonstrated a good tolerance of all tested porous scaffolds and widespread ingrowth of tissue into the implant pores. HA-containing scaffolds showed a less pronounced inflammatory response after two weeks of implantation compared to pure PLA. These observations suggest that PLA/HA composites have enormous potential for hard tissue engineering and restoring maxillofacial defects.

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.