Infection in orthopedics is a challenge, since it has high incidence (rates can be up to 15-20%, also depending on the surgical procedure and on comorbidities), interferes with osseointegration and brings severe complications to the patients and high societal burden. In particular, infection rates are high in oncologic surgery, when biomedical devices are used to fill bone gaps created to remove tumors. To increase osseointegration, calcium phosphates coatings are used. To prevent infection, metal- and mainly silver-based coatings are the most diffused option. However, traditional techniques present some drawbacks, including scarce adhesion to the substrate, detachments, and/or poor control over metal ions release, all leading to cytotoxicity and/or interfering with osteointegration. Since important cross-relations exist among infection, osseointegration and tumors, solutions capable of addressing all would be a breakthrough innovation in the field and could improve clinical practice.

Here, for the first time, we propose the use antimicrobial silver-based nanostructured thin films to simultaneously discourage infection and bone metastases. Coatings are obtained by Ionized Jet Deposition, a plasma-assisted technique that permits to manufacture films of submicrometric thickness having a nanostructured surface texture. These characteristics, in turn, allow tuning silver release and avoid delamination, thus preventing toxicity. In addition, to mitigate interference with osseointegration, here silver composites with bone apatite are explored. Indeed, capability of bone apatite coatings to promote osseointegration had been previously demonstrated in vitro and in vivo. Here, antibacterial efficacy and biocompatibility of silver-based films are tested in vitro and in vivo. Finally, for the first time, a proof-of-concept of antitumor efficacy of the silver-based films is shown in vitro.

Coatings are obtained by silver and silver-bone apatite composite targets. Both standard and custom-made (porous) vertebral titanium alloy prostheses are used as substrates.

Films composition and morphology depending on the deposition parameters are investigated and optimized. Antibacterial efficacy of silver films is tested in vitro against gram+ and gram- species (E. coli, P. aeruginosa, S. aureus, E. faecalis), to determine the optimal coatings characteristics, by assessing reduction of bacterial viability, adhesion to substrate and biofilm formation. Biocompatibility is tested in vitro on fibroblasts and MSCs and, in vivo on rat models. Efficacy is also tested in an in vivo rabbit model, using a multidrug resistant strain of S. aureus (MRSA, S. aureus USA 300). Absence of nanotoxicity is assessed in vivo by measuring possible presence of Ag in the blood or in target organs (ICP-MS). Then, possible antitumor effect of the films is preliminary assessed in vitro using MDA-MB-231 cells, live/dead assay and scanning electron microscopy (FEG-SEM). Statistical analysis is performed and data are reported as Mean ± standard Deviation at a significance level of p <0.05. Silver and silver-bone apatite films show high efficacy in vitro against all the tested strains (complete inhibition of planktonic growth, reduction of biofilm formation > 50%), without causing cytotoxicity. Biocompatibility is also confirmed in vivo.

In vivo, Ag and Ag-bone apatite films can inhibit the MRSA strain (>99% and >86% reduction against ctr, respectively). Residual antibacterial activity is retained after explant (at 1 month). These studies indicate that IJD films are highly tunable and can be a promising route to overcome the main challenges in orthopedic prostheses.

Intraosseous Transcutaneous Amputation Prosthesis (ITAP) is a new generation of limb replacements that can provide to amputees, an alternative solution to the main problems caused by the most common used external prosthesis such as pressure sores, infections and unnatural gait. ITAP is designed as one pylon osteointegrated into the bone and protruding through the skin, allowing both the mechanical forces to be directly transferred to the skeleton and the external skin being free from frictions and infections. The skin attachment to the implant is fundamental for the success of the ITAP, as it prevents the implant to move and consequently fail.

In this study we wanted to test if cell viability and attachment was improved using TiO2 nanotubes.

Human keratinocytes and human dermal fibroblasts were seeded for three days on TiO2 nanotubes with different sizes (18–30nm, 40–60nm and 60–110nm), compared with controls (smooth titanium) and tested for viability and attachment. A Mann-Whitney U test was used to compare groups where p values < 0.05 were considered significant. The results showed that the viability and cell attachment for keratinocytes were significantly higher after three days on controls comparing with all nanotubes (p=0.02), while attachment was higher on bigger nanotubes and controls. Cell viability for fibroblasts was significantly higher on nanotubes between 40 and 110nm comparing with smaller size and controls (p=0.03), while investigation of cell attachment is ongoing.

From these early results, we can say that TiO2 nanotubes can improve the soft tissue attachment on ITAP. Further in-vitro and ex-vivo experiments on cell attachment will be carried out.