In Vitro and In Vivo Studies of Antibacterial Coatings on Titanium Alloy Implants for Veterinary Application.

The aim of this work was the evaluation of biological properties of hybrid coatings modified with Ag, Cu, and Zn nanoparticles (NPs) applied on TPLO medical implants by the sol-gel process. The implant coatings enriched with various concentrations of metallic NPs were investigated in the in vitro bactericidal efficacy tests against Gram+ and Gram- bacteria and pathogenic yeast. Next, the designed materials were tested on human osteosarcoma cell lines. The cells adhesion, proliferation, viability, and differentiation were investigated. The cell growth wasevaluated using SEM, and the metallic ion release was measured. The results revealed that the NPs concentration in the hybrid layers decreased with the incubation time. In the last stage, the implants were tested in vivo on six canine patients. Three months after the operation, the radiological evaluation of the performed anastomosis was carried out as well as the histopathological evaluation of tissue regeneration. The strongest bactericidal efficacy was observed for the layers containing AgNPs. Along with an increased concentration of metallic additives, a growing toxic effect was clearly observed. The most pronounced toxic effect was especially evident with the AgNPs concentration exceeding 1 mol %. In all the operated patients, no deviations were found during the follow-up examinations in the postoperative period. The low dose of AgNPs in the hybrid layer facilitated the tissue healing process. It was proven that silver nanoparticles may accelerate the bone healing process. The correct tissue reparation was observed.

Antibacterial composite hybrid coatings of veterinary medical implants.

The aim of the work was to develop innovative antibacterial hybrid coatings applied on implants that are used for anastomoses of animals' long bones and to assess their physicochemical and biological properties. Plates made of the titanium alloy were covered with composite hybrid layers so as to protect the implant surface against corrosion and to enhance it with antibacterial properties.The hybrid coatings were obtained electrochemical oxidation and sol-gel. First, a layer of titanium nanotubes was applied to the implants surface through anodization. Next, the sol-gel method was used to create the second layer with silver nanoparticles. The microstructure examination of the materials was performed with the SEM. The phase composition analysis was carried out via the X-ray diffraction. The surface parameters (roughness, contact angle and free surface energy) were assessed. Biological studies of implants were conducted, including the analysis of degradation processes, cell response and bactericidal activity. The results confirmed that the hybrid antibacterial layers effectively protected the implant surface against scratches and corrosion and eliminated bacteria, which in turn would promote bone healing. The advantageous physicochemical and biological properties of metallic implants with hybrid composite layers raise hopes for their applicability in the veterinary treatment of bone fractures.

Substantial differences between human and ovine mesenchymal stem cells in response to osteogenic media: how to explain and how to manage?

It is expected that use of adult multipotential mesenchymal stem cells (MSCs) for bone tissue engineering (TE) will lead to improvement of TE products. Prior to clinical application, biocompatibility of bone TE products need to be tested in vitro and in vivo. In orthopedic research, sheep are a well-accepted model due to similarities with humans and are assumed to be predictive of human outcomes. In this study we uncover differences between human and ovine bone marrow-derived MSCs (BMSCs) and adipose tissue-derived MSCs (ADSCs) in response to osteogenic media. Osteogenic differentiation of BMSCs and ADSCs was monitored by alkaline phosphatase (ALP) activity and calcium deposition. Mineralization of ovine BMSC was achieved in medium containing NaH2PO4 as a source of phosphate ions (Pi), but not in medium containing ß-glycerophosphate (ß-GP), which is most often used. In a detailed study we found no induction of ALP activity in ovine BMSCs and ADSCs upon osteogenic stimulation, which makes ß-GP an unsuitable source of phosphate ions for ovine cells. Moreover, mineralization of human ADSCs was more efficient in osteogenic medium containing NaH2PO4. These results indicate major differences between ovine and human MSCs and suggest that standard in vitro osteogenic differentiation techniques may not be suitable for all types of cells used in cell-based therapies. Since mineralization is a widely accepted marker of the osteogenic differentiation and maturation of cells in culture, it may lead to potentially misleading results and should be taken into account at the stage of planning and interpreting preclinical observations performed in animal models. We also present a cell culture protocol for ovine ADSCs, which do not express ALP activity and do not mineralize under routine pro-osteogenic conditions in vitro. We plan to apply it in preclinical experiments of bone tissue-engineered products performed in an ovine model.