Dual-Functional Nano-Functionalized Titanium Scaffolds to Inhibit Bacterial Growth and Enhance Osteointegration

Implantable biomaterials play a key role for the success of orthopedic surgery procedures. However, infections remain one of the most damaging post-operative complications that lead to the implant failure. Recently, several approaches have been proposed to avoid or manage implant-associated infections. Among these, an appropriate surface functionalization to confer intrinsic antibacterial properties preserving the osteo-integration ability represents an appealing strategy for the development of innovative implant materials. Titanium and its alloys are the most used materials for manufacturing of both articular and bone skull prostheses as well as dental implants. However, to date there is still a significant clinical need to improve their bioactivity, osseointegration and antibacterial activity. In this study, titanium biomimetic scaffolds are prepared by nano-functionalization with TiO2 (Ti_TiO2) and γFe2O3 (Ti_γFe2O3). Both cytocompatibility and antibacterial activity have been evaluated. Data show that both nano-functionalized scaffolds exhibit a good antibacterial activity towards Staphylococcus aureus, reducing colony number to 99.4% (Ti_TiO2) and 99.9% (Ti_γFe2O3), respectively. In addition, an increase of both human adipose-derived mesenchymal stem cells (hADSCs) cell proliferation (up to 4.3-fold for Ti_TiO2 and 3.7-fold for Ti_γFe2O3) and differentiation has been observed. These data suggest that these nano-functionalized titanium substrates represent promising prototypes for new antimicrobial and osteoconductive biomaterials to be used in the orthopedic field to reconstruct significant bone defect.


Introduction
In recent years, the number of people undergoing surgery for osteoarticular problems, such as osteoarthritis and osteoporotic fractures, is greatly grown due to an overall increase in life expectancy. Biomaterials play a key role for the success of orthopedic surgery procedures. However, despite significant advances in the patient's quality of life, implantassociated infections remain one of the most damaging post-operative complications [1]. Actually, the prosthesis insertion in the body is associated to the risk of microbial infection and osteomyelitis occurrence causing implant failure [2,3] inducing surgery procedures to replace the infected prosthesis and, in the worst cases, limb amputation. Some evidences showed that the infection rate in patients with open fractures is about 20%, but it can be higher than 50% in much more severe cases [4]. Bacterial contamination during the surgery includes several sources such as operating environment, surgical equipment, clothing worn by medical and paramedical personnel and resident bacteria on the skin and mucous membranes of the patient himself [5]. Most contaminations belong to the Gram-positive Staphylococci family such as Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis, and Gram-negative bacilli including Pseudomonas aeruginosa and Escherichia coli [6]. The infections are caused by bacteria adhesion on the implant surface and subsequent biofilms formation [7]. This leads to many complications including chronic infections, resistance to treatment with antibiotics and chronic inflammatory response at the site of the biofilm itself [7,8]. Therefore, the inhibition of both bacterial surface adhesion and proliferation are the main points to be considered to design antibacterial materials. The elements influencing bacterial adhesion to the implant surface are the physical properties such us the material microstructure, hydrophobicity, superficial charge and surface roughness [9].
Titanium (Ti) and its alloys are the most used materials for orthopedic implants due to their exceptional mechanical and chemical properties, good biocompatibility, corrosion resistance, good bone affinity and osteoconductivity [10,11]. Several approaches are reported in the literature to enhance the bone tissue regeneration of Ti-based devices by the development of appropriate trabecular topography, bio-coating [12] or specific pore structure [13]. The internal pore structure also affects the mechanical stiffness of the material [14][15][16] thereby being instrumental for the design of novel long-lasting implants.
However, although Ti shows these excellent properties, to date there is still a significant clinical need to improve its antibacterial properties preserving osteo-integration ability. Therefore, the development of new biomaterials with bactericidal/osteoinductive function is still a challenge. Several approaches have been proposed in the literature for Ti substrates mainly involving: (1) the use of materials with intrinsic antibacterial properties (Ag, Cu, Zn and polymers) [17][18][19] and (2) surface functionalization by specific coating [20,21] or surface structuring [22,23]. In this context, nanotechnology is a powerful tool allowing the design of innovative biomaterials by playing on chemistry of materials and size. Using nanostructured materials, free-living or surface-associated bacteria (biofilm) can be inhibited by an ad-hoc modulation of the biomaterial chemical-physical properties (size, surface morphology, charge and zeta potential) [24]. Antimicrobial activity of nano-materials mainly involves both oxidative stress induction and metal ion release. The first derived from free radical's generation leading to damage on cellular wall and nucleic acids. The release of metal ions (positively charged) to the surface of bacteria (negatively charged) enhances antimicrobial activity by destabilization of cell membrane and, inside cells, by proteins denaturation (especially ribosome proteins) [25]. However, often antibacterial effects are associated to cytotoxicity with a consequent increase of health risk [26][27][28].
In particular, metal or metal oxide nanoparticles (NPs) are increasing their use as alternative to antibiotics due to their broad spectrum of action against both gram +(ve) and gram −(ve) bacteria [15]. Among these, TiO 2 NPs are among the most studied due to their efficiency and versatility based on photo-catalytic mechanism upon light irradiation depending on crystal structure, doping metals and irradiation wavelength [29,30]. The bactericidal mechanism of TiO 2 is mainly due to Reactive Oxygen Species (ROS) generation and lipid oxidation of bacterial cell wall. The ROS generation has been also found in the absence of light in presence of species undergoing catalytic decomposition (like H 2 O 2 ) [31,32]. Among the form of nanostructures, titania nanotubes have been extensively studied for osteogenesis highlighting that a combination of nano-and microscale roughness at the implant surface of these nano-systems can promote relevant bioactivity [33].
Iron (III) oxide NPs are also appealing to nano-materials for tissue regeneration, since iron is an essential micro nutrient for cell growth and can also exhibit magnetic properties promoting cell proliferation [34,35]. Additionally, they can show antibacterial effects both as Fe 2 O 3 and as Fe 3 O 4 due to multiple mechanisms including ROS species, superoxide radical's generation and singlet oxygen formation [36,37]. However, despite the advantages described above, chemically synthesized iron oxide nanoparticles agglomerate and possess reduced stability [38], requiring post-synthesis modification [39]. Therefore, the integration of nano-materials with new properties into Ti biomimetic scaffolds can be an effective strategy to develop innovative biomaterials for tissue engineering.
In this paper we proposed the preparation of new dual functional material with antibacterial and osteo-integration ability based on surface nano-functionalization of Ti alloy with TiO 2 and γFe 2 O 3 NPs (Ti_TiO 2 and Ti_γFe 2 O 3 ) by wet chemistry. Both the antibacterial properties towards S. aureus and the cell growth and differentiation using hADSCs have been assessed by in vitro studies and discussed.

Chemicals and Materials
All chemicals were obtained from commercial sources at the highest possible purity and were used as received. All solvents used were spectrophotometric grade. Milli-Qgrade water was used in all preparations. Ti supports were provided by Istituto Ortopedico Rizzoli (Bologna, Italy). The supports are made of trabecular Ti 6 Al 4 V alloy and circular shaped with diameter of 8 mm and height of 4 mm.

Titanium Scaffolds Nano-Functionalization Procedure
(a) Nano-functionalization with TiO 2 (Ti_TiO 2 ). Ti supports were dipped in a titanium isopropoxide (0.1 M) isopropyl alcohol solution for 5 min and heated in air at 600 • C for three times. After that, the Ti_TiO 2 scaffolds were rinsed with isopropyl alcohol and dried under nitrogen. (b) Nano-functionalization with γFe 2 O 3 NPs (Ti_γFe 2 O 3 ). Ti supports were cleaned in ethanolic ultrasound bath for 10 min and after, they were placed in a quartz tube containing ethyl alcohol, iron (III) acetylacetonate and acetone as photosensitizers. After 15 min of degassing with N 2 , the solution was irradiated with four lamps (254 nm 16 W) for 90 min. NaOH 1 M was added to the resulting yellow solution and stirred in air for 60 min. Finally, the red-brown Ti_γFe 2 O 3 substrates were rinsed three times with deionized water and dried with nitrogen flow.

Physical-Chemical Characterization of Titanium Scaffolds
The morphological and microstructural properties of nano-derivatised Ti scaffold surfaces were evaluated by Scanning Electron Microscopy (SEM-EDX) performed on a SEM-LEO 438 VP (Carl Zeiss AG, Oberkochen, Germany) and a Zeiss EVO MA10 (Carl Zeiss AG, Oberkochen, Germany) equipped with a LaB6 electron gun. Transmission electron microscopy (TEM) analysis was performed using the bright field in conventional parallel beam (CTEM) mode (BF). An ATEMJEOL JEM-2010 (Pleasanton, CA, USA) equipped with a 30-mm 2 window energy-dispersive X-rays spectrometer was used. Atomic force measurements (AFM) were performed using a Solver P47 (NT-MDT instrument, Moscow, Russia) in contact mode. We used standard silicon AFM probes (NT-MDT instrument, Russia) having cantilever force constant in the 0.01-0.5 Nm range. The zeta potential was evaluated using a ZetaSizer NanoZS90 (Malvern Panalytical Ltd., Cambridge, UK), equipped with a 633 nm laser, at the scattering angle of 90 • C and 25 • C temperature. Z-potential measurements were performed in aqueous dispersion at pH 7.2. Each measurement was performed three times.

In-Vitro Tests
(a) Cell culture and proliferation analysis. hADSCs were acquired from Lonza (Lonza Group Ltd., Basel, Switzerland) and cultured as previously reported [40] until 80-90% of confluence.
Trypan blue test. 1 × 10 6 cell were plated on Ti scaffolds (Ti_CTRL (not-treated Titanium substrate), Ti_TiO 2 , Ti_γFe 2 O 3 ) in culture medium and incubated in a humidified atmosphere containing 5% CO 2 at 37 • C for 1, 3 and 7 days. After, hADSCs were detached from Ti-derivatized supports with trypsin (Sigma-Aldrich, Milan, Italy), stained with trypan blue (Termo Fisher Scientific, NYSE: TMO) and counted by using a Leica DMI 4000B fluorescence microscope (Leica Microsystems Srl, Milano, Italy). 4 ,6-Diamidino-2-Phenylindole (DAPI, Thermo Fischer Scientific, NYSE: TMO, Waltham, MA, USA) staining. 1 × 10 6 hADSCs were cultured on each scaffold for 1, 3 and 7 days, fixed in 4% PFA (paraformaldehyde) and washed three times in PBS (Sigma-Aldrich, Milan, Italy). After, each scaffold was permeabilized in 0.3% Triton X-100 for 10 min, washed in PBS and the nuclei stained with DAPI (1:5000) in PBS for 5 min. Finally, the images (20 for each scaffold) were acquired using a Leica DMI 4000B fluorescence microscope and the nuclei counted by Fiji Image J recognition software. The proliferation rates differences were assessed using One-way ANOVA test with Holm test as post-hoc for multiple comparisons. Each biological test was conducted in triplicate for each time points.
Alizarin Red S (ARS) staining quantification assay. ARS staining (Sciencell, Italy) was performed to evaluate calcium deposits in cells culture according to the manufacturer's instructions. Specifically, 1 × 10 6 hADSCs were cultured on each scaffold for 1, 3 and 7 days in 6-well plates. After that, the cells on the scaffolds were washed with PBS and fixed in 4% PFA at room temperature for 15 min and then washed twice with excess dH 2 O prior to addition of 1 mL of 40 mM ARS (pH 4.1) per well. The scaffolds were incubated at room temperature for 20 min under shaking. After aspiration of the unincorporated dye, they were washed four times with 4 mL dH 2 O while shaking for 5 min, re-aspirated and stored at −20 • C prior to dye extraction. For the quantification of ARS, 800 µL 10% (v/v) acetic acid was added to each well, and the plate was incubated at room temperature for 30 min with shaking. The cells were then detached from the scaffolds, transferred with 10% (v/v) acetic acid to a 1.5-mL microcentrifuge tube and vortexed for 30 s. After, the samples were sealed with parafilm, heated to 85 • C for 10 min and transferred to ice for 5 min. They were then centrifuged at 20,000× g for 15 min and 500 µL of the supernatant was removed to a new 1.5-mL microcentrifuge tube. Then 200 µL of 10% (v/v) ammonium hydroxide was added to neutralize the acid. In total, 150 µL of the supernatant for each sample were read in triplicate at 405 nm by a Microplate Reader (Biotek, Waltham, MA, USA).
Alkaline phosphatase (ALP) activity evaluation. ALP activity was evaluated by colorimetric assay performed on cell culture supernatants. Specifically, 1 × 10 6 hADSCs were cultured on each scaffold for 1, 3 and 7 days and the culture media collected and used to measure ALP activity. This was enzymatically determined using an alkaline phosphatase assay kit (ab83369, Abcam, Cambridge, UK) according to the manufacturer's instructions. The ALP level was measured by reading the absorbance at 405 nm by a Microplate Reader (Biotek, USA). All experiments were performed in triplicate.
(c) Bacteria strain and viability assay.
S. aureus (ATCC 29213) was purchased from American Type Culture Collection (LGC Promochem, Milan, Italy) and cultured in Tryptone Soya Broth (TSB, Sigma-Aldrich, Milan, Italy). Ti scaffolds were sterilized in autoclave at 121 ± 1 • C for 20 min and placed in 24-well plates (one scaffold per well). Bactericidal activity was evaluated according to Hu et al. [41] with some modifications. Briefly, a colony of S. aureus cultured overnight in Tryptone Soya Agar (TSA, Sigma-Aldrich, Milan, Italy), was inoculated in Mueller-Hinton Broth (MHB, Sigma-Aldrich, Milan, Italy) and incubated at 150 rpm on a rotary shaker for 6-8 h at 37 • C. After incubation period, the bacteria suspension (10 7 cells/mL) was placed onto the titanium scaffold surfaces with a 60 µL/cm 2 density, and incubated in a humidified atmosphere at 37 • C for 24 h. Then, the scaffolds were placed in tubes containing phosphate-buffered saline (PBS) solution and, vigorously stirred for 60 s to detach the bacteria from the coating surface. Finally, a Colony Forming Units (CFU) assay was performed by using an untreated titanium scaffold as control, as following. Specifically, 100 µL of bacterial suspension were serially diluted in 900 µL of PBS, 100 µL of each dilution Nanomaterials 2021, 11, 2634 5 of 14 spread on solid medium and incubated overnight at 37 • C. After incubation, colonies in the range of 30-300 were counted to determine the number of CFU: The bacterial adhesion on the surface TiO 2 e γFe 2 O 3 nano-functionalized scaffolds were evaluated by live/dead cell staining kit (BacLight™ Bacterial Viability Kit, ThermoFischer Scientific, NYSE: TMO). Briefly, after bacteria incubation, the scaffolds were stained with a mixture of SYTO 9 (green-fluorescent staining viable cells) and propidium iodide (red-fluorescent staining dead cells) for 15 min. After, they were visualized under fluorescence microscopy by using Leica DMRE epifluorescence microscope (Leica Microsystems, Heerbrugg, Switzerland) with Leica C Plan 63xobjective and BP 515-560 nm excitation filter in combination with a LP 590 nm suppression filter.
For ROS evaluation, bacteria were detached, washed twice with PBS, stained with 2 , 7'-dichlorodihydrofluorescein diacetate (H 2 DCFDA, Sigma-Aldrich, Milan, Italy) at 10 µM final concentration and incubated at 37 • C for 20 min. After the incubation time, bacteria were washed twice with PBS, to remove the excess of dye, and finally images were acquired by using a Leica DMRE epifluorescence microscope with Leica C Plan 100x objective and BP 515-560 nm excitation filter in combination with a LP 590 nm suppression filter.
Data were analyzed either as raw data or as mean ± standard error (SE), as appropriate. Differences between the two time points and the different types of scaffolds were evaluated by using one-and two-way ANOVA with post-hoc Holm test, where appropriate. The values p < 0.05 were considered to be significant.

Morphological and Microstructural Analyses of Titanium Scaffolds
Both Ti_TiO 2 and Ti_γFe 2 O 3 scaffolds were prepared by surface in-situ growth with wet chemistry. Ti-isopropoxide precursor were converted in nanosized TiO 2 on scaffold surface trough thermal growth (Figure 1a). Similarly, Ti_γFe 2 O 3 scaffold were obtained with a two-steps approach based on the photochemical reaction of Fe(acac) 3 precursor in the presence of photosensitizers, followed by pH-basic oxidation to obtain nanosized γFe 2 O 3 on the scaffold surface ( Figure 1b).   Both proposed synthetic methods were based on chemical reactions converting specific precursors (Ti(isopropoxide) 4 and FeIII(acac) 3  surface. The nano-derivatized biomimetic scaffolds were morphologically and chemically characterized trough AFM, SEM, EDX and TEM inspections. Figure 2 reports SEM images of the biomimetic scaffolds before (Figure 2a,b) and after TiO 2 nano-functionalization (Figure 2c,d). The images clearly demonstrate the presence of TiO 2 nanostructures with dimensions between 300 nm and 800 nm in diameter (Figure 2c,d).  The EDX analysis indicates a significant increase of the O peak (0.55 KeV) compared to the untreated Ti_CTRL titanium scaffold (see Figure S1), confirming the presence of TiO2 nanostructures. In the case of Ti_γFe2O3 it was not possible to detect the presence of γFe2O3 NPs in the SEM images because of their nanosize (Figure 3a). The presence of iron was assessed through EDX analysis which showed the specific peaks for the iron species values for the maghemite (γFe2O3): 2.95 Å (28%), 2.54 Å (100%), 2.09 Å (20%), 1.73 Å (10%), 1.64 Å (25%) and 1.47 Å (42%) (see Figure S2). AFM (Figure 3c), performed on samples obtained on flat Si(100) substrates, indicate the presence of γFe2O3 NPs with an average size of about 14 ± 3 nm (Figure 3d). The average value of the size obtained from the AFM is higher than the typical TEM sizes likely due to the slight NPs aggregation occurring during NPs deposition. These values are in accordance with the data reported in literature and definitively confirm the presence of γFe2O3 NPs. In addition, we performed Z-potential investigation on TiO2 and γFe2O3 NPs in solution. Our results indicate the presence of negative charged nanoparticles with Z-potential values of −11.8 ± 1.4 mV for TiO2 and −39.2 ± 0.9 mV for γFe2O3. Surface negative charges that can promote the adsorption of hydrophilic proteins (vitronectin, fibronectin, etc.) leading to increased bone cell adhesion [42].

hADSCs Proliferation and Differentiation Evaluation on Titanium Scaffolds.
To gain insight on the osteo-integration ability of both Ti_TiO2 and Ti_γFe2O3, cell proliferation was investigated by observing the viability of hADSCs for 1, 3 and 7 days on nano-functionalized scaffolds (cultured at the same density in parallel cultures) compared In addition, we performed Z-potential investigation on TiO 2 and γFe 2 O 3 NPs in solution. Our results indicate the presence of negative charged nanoparticles with Zpotential values of −11.8 ± 1.4 mV for TiO 2 and −39.2 ± 0.9 mV for γFe 2 O 3 . Surface negative charges that can promote the adsorption of hydrophilic proteins (vitronectin, fibronectin, etc.) leading to increased bone cell adhesion [42].
In addition to the osteo-integration, we also evaluated another important aspect for implantable-devices, the osteogenic differentiation. To this aim, ARS staining quantification and ALP activities after 1, 3, and 7 days of culture on both Ti_TiO2 and Ti_γFe2O3 were measured. Data obtained from ARS staining quantification show a higher calcium depos- These data were also confirmed with DAPI staining which, after 24 h of cell growth (D1), showed no significant difference in the cell number in the nano-functionalized scaffolds compared to Ti_CTRL. After 3 and 7 days of culture in both Ti_TiO 2 and Ti_γFe 2 O 3 scaffolds a considerable cell number increasing of for 3.7-fold Ti_TiO 2 and 4.3-fold for Ti_γFe 2 O 3 , compared to the Ti_CTRL control was found (see Figure S3). Contrary, the cell number difference between the two nano-functionalized scaffolds was not significant.
In addition to the osteo-integration, we also evaluated another important aspect for implantable-devices, the osteogenic differentiation. To this aim, ARS staining quantification and ALP activities after 1, 3, and 7 days of culture on both Ti_TiO 2 and Ti_γFe 2 O 3 were measured. Data obtained from ARS staining quantification show a higher calcium deposits content in both Ti_TiO 2 and Ti_γFe 2 O 3 scaffolds than Ti_CTRL control at the same incubation times ( Figure 4B). In detail, after 3 days (D3) of culture on scaffolds the ARS value increases of about 7% for Ti_TiO 2 and 11% for Ti_γFe 2 O 3 respect to the Ti_CTRL control; while, at D7 these values correspond to about 15% for Ti_TiO 2 and 19% for Ti_γFe 2 O 3 , respectively. On the contrary, not significant difference was found between Ti_TiO 2 and Ti_γFe 2 O 3 scaffolds at all times analyzed (p > 0.05).
In accordance with ARS staining quantification data, ALP results show values significantly higher in both Ti_TiO 2 and Ti_γFe 2 O 3 scaffolds than Ti_CTRL control at the same incubation times ( Figure 4C). Specifically, after 3 days (D3) the ALP activities increase of about 9% for Ti_TiO 2 and 11% for Ti_γFe 2 O 3 respect to the Ti_CTRL control; while, at D7 these values correspond to about 6% for Ti_TiO 2 and 8% for Ti_γFe 2 O 3 , respectively. Additionally, in this case, no significant difference was found between Ti_TiO 2 and Ti_γFe 2 O 3 scaffolds at all times analyzed (p > 0.05). These data together indicate that both nano-functionalizations are able to induce osteogenic differentiation.
After cell culture, the nano-derivatized scaffolds were inspected by SEM to characterize the surface topographical features upon cellular interaction. Figure 5 reports representative SEM images of Ti_CTRL (Figure 5a,b), Ti_TiO 2 (Figure 4c,d) and Ti_γFe 2 O 3 (Figure 5e,f) after 7 days of culture. The images visibly show that the cells are able to adhere and proliferate in nano-functionalized titanium scaffolds and that at 7 days after cell seeding, the cells on all scaffolds became even more stretched maintaining the typical morphology of mesenchymal stem cells, characterized by long and thin cellular processes.
these values correspond to about 6% for Ti_TiO2 and 8% for Ti_γFe2O3, respectively. Additionally, in this case, no significant difference was found between Ti_TiO2 and Ti_γFe2O3 scaffolds at all times analyzed (p > 0.05). These data together indicate that both nano-functionalizations are able to induce osteogenic differentiation.
After cell culture, the nano-derivatized scaffolds were inspected by SEM to characterize the surface topographical features upon cellular interaction. Figure 5 reports representative SEM images of Ti_CTRL (Figure 5a,b), Ti_TiO2 (Figure 4c,d) and Ti_γFe2O3 (Figure 5e,f) after 7 days of culture. The images visibly show that the cells are able to adhere and proliferate in nano-functionalized titanium scaffolds and that at 7 days after cell seeding, the cells on all scaffolds became even more stretched maintaining the typical morphology of mesenchymal stem cells, characterized by long and thin cellular processes.  The above results highlight that both Ti_TiO 2 and Ti_γFe 2 O 3 biomimetic scaffolds are able to promote hADSCs adhesion, proliferation, grow and differentiation, indicating their effective osteo-integrative/inductive ability. This finding is corroborated by several literature evidences showing that the nanostructing of the scaffold combined with surface properties can improve the cell adhesion and proliferation achieving better osseointegration [42,43]. In our case, several factors can be considered to comment the obtained results. Firstly, the biocompatibility of the nano-materials. Actually, both TiO 2 and γFe 2 O 3 have been proved to be biocompatible for bone regenerative application [33,34]. Then, the scaffolds surface topography whose biomimetic feature is an important parameter affecting both cell adhesion and proliferation specially during the phase of osseointegration. It has been really observed that even if nanotopography by itself promotes bone cell functions, however the combination of nano-and micro-scale roughness can enhance bioactivity [33]. Our finding shows that both TiO 2 and γFe 2 O 3 NPs form nanostructured coatings (300-800 nm for TiO 2 NPs and 10-20 nm for γFe 2 O 3 NPs) spread onto the micrometric trabecular microstructure of the pristine Ti substrate. These results, therefore, evidence that a combined micro-nanotopography can promote and induce the cellular integration.
Another important factor for cell-biomaterial interaction is the surface hydrophilicity, that improve adhesion and spread of cells, osteoblastic differentiation and maturation [44]. Both TiO 2 and γFe 2 O 3 nano-systems exhibit negative charges at the surface (see above the Z-potential data) indicating the possibility to improve the adsorption of hydrophilic proteins (vitronectin, fibronectin, etc.) leading to increased bone cell adhesion [41].

In Vitro Antibacterial Activity Evaluation
In order to assess the antibacterial properties of both Ti_TiO 2 and Ti_γFe 2 O 3 scaffolds, S. aureus bacteria were incubated on them for 24 h, and after CFU assay and live/dead staining were performed. Results are reported in Figure 6. Data highlight a good antibacterial activity for both for Ti_TiO 2 and Ti_γFe 2 O 3 scaffolds compared to the Ti_CTRL scaffold. Specifically, the CFU assay reveals that TiO 2 and γFe 2 O 3 NPs incorporated on the titanium scaffold surfaces are able to reduce the colony number of about 99.4% for Ti_TiO 2 (3.2 ± 0.7 × 10 5 CFU/mL) and 99.9% for Ti_γFe 2 O 3 (4.3 ± 0.9 × 10 2 CFU/mL) compared to the Ti_CTRL (3.7 ± 1.4 × 10 7 CFU/mL) ( Figure 6A). In addition, Ti_γFe 2 O 3 scaffold exhibited a stronger statistically significant antibacterial activity compared to the Ti_TiO 2 scaffold. These results were in agreement with those of the Live/Dead staining showing the presence of very few living (green fluorescent) and many dead (red fluorescent) cells for Ti_TiO 2 , while almost all of them were red for Ti_γFe 2 O 3 compared to the Ti_CTRL, demonstrating their strong biocidal activity ( Figure 6B). Consequently, it's possible to deduce that bactericidal effect of γFe 2 O 3 is both higher and faster compared to TiO 2 . These findings could be related to the different nature of NPs, as well as to the size (TiO 2 NPs size are higher than γFe 2 O 3 NPs). In fact, bactericidal effect is increased in nanoparticles with smaller size, due to cell uptake, intracellular distribution and interaction with biological macromolecules (in particular with microbial membranes) [45]. In order to verify the presence of intracellular ROS induced by the nano-functionalized scaffolds, 2′, 7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining was performed after 1, 2 and 4 h post bacteria incubation and fluorescence images detected. Specifically, ROS signal was evaluated in the early hours (1, 2 and 4 h), because ROS-positive cells decrease due to the reduction of viable cells [51]. Results are reported in Figure 7. It can be noticed that green fluorescence clearly appears in both Ti_TiO2, and Ti_γFe2O3 scaffolds after 2 h (Figure 7e,h) and increases after 4 h (Figure 7f,i). On the contrary, the Ti_CTRL does not show any ROS evidence (Figure 7a-c). This finding indicates the ROS The antibacterial activity of both TiO 2 and γFe 2 O 3 is reported to mainly due to generation of free radicals and liberation of metal ion [46,47]. About γFe 2 O 3 , it has been found that ROS generation induces membrane depolarization, lipid peroxidation and DNA damage, while ion release negatively affects metabolic activities, cell homeostasis and protein functions [48,49]. Further, iron nanoparticles are able to penetrate into biofilms [50]. In terms of TiO 2 , its antibacterial effect is closely related to generation of ROS under UVradiation or in dark condition in presence of oxidizing species [29][30][31]. It has also been reported that UV-independent antibacterial activity increases by the use of transition metal ions as dopants [47,51,52].
In order to verify the presence of intracellular ROS induced by the nano-functionalized scaffolds, 2 , 7'-dichlorodihydrofluorescein diacetate (H 2 DCFDA) staining was performed after 1, 2 and 4 h post bacteria incubation and fluorescence images detected. Specifically, ROS signal was evaluated in the early hours (1, 2 and 4 h), because ROS-positive cells decrease due to the reduction of viable cells [51]. Results are reported in Figure 7. It can be noticed that green fluorescence clearly appears in both Ti_TiO 2 , and Ti_γFe 2 O 3 scaffolds after 2 h (Figure 7e,h) and increases after 4 h (Figure 7f,i). On the contrary, the Ti_CTRL does not show any ROS evidence (Figure 7a-c). This finding indicates the ROS presence in agreement with the literature data above mentioned.

Conclusions
In this study, we prepared Ti scaffold nano-functionalized with TiO2 and γFe2O3 NPs by surface in situ growing method and their osteo-integrative/inductive capability and antimicrobial activity were assessed. Morphological characterization of both Ti_TiO2 and Ti_γFe2O3 scaffolds showed the presence of TiO2 NPs with dimensions between 300 nm and 800 nm in diameter spread onto Ti surface, while in the case of γFe2O3 both TEM and AFM measurements highlight the presence of NPs with a diameter in the range of 10-20 nm. Both Ti_TiO2 and Ti_γFe2O3 scaffolds exhibited good osteo-integration ability. In particular, a considerable increase of hADSCs cell growth after 7 days of culture equal to 2.3fold for Ti_TiO2 and 2.6-fold for Ti_γFe2O3, compared to the control, was found. Further, an improved osteogenic differentiation was observed with an ALP activity increasing up to 17%. SEM analyses highlighted good cell adhesion and colonization over the scaffold The above results highlight that both Ti_TiO 2 and Ti_γFe 2 O 3 are able to match the double function to promote osteo-integration and inhibit bacterial invasion. To further investigate these aspects, we plan further studies focusing on the optimization of the nanofunctionalization (in terms of NPs densities, surface charges) and to gain more insight on the regeneration and bactericidal mechanisms with in vitro studies to evaluate the specific genes and cellular pathways to be finally validated by in vivo study.

Conclusions
In this study, we prepared Ti scaffold nano-functionalized with TiO 2 and γFe 2 O 3 NPs by surface in situ growing method and their osteo-integrative/inductive capability and antimicrobial activity were assessed. Morphological characterization of both Ti_TiO 2 and Ti_γFe 2 O 3 scaffolds showed the presence of TiO 2 NPs with dimensions between 300 nm and 800 nm in diameter spread onto Ti surface, while in the case of γFe 2 O 3 both TEM and AFM measurements highlight the presence of NPs with a diameter in the range of 10-20 nm. Both Ti_TiO 2 and Ti_γFe 2 O 3 scaffolds exhibited good osteo-integration ability. In particular, a considerable increase of hADSCs cell growth after 7 days of culture equal to 2.3-fold for Ti_TiO 2 and 2.6-fold for Ti_γFe 2 O 3 , compared to the control, was found. Further, an improved osteogenic differentiation was observed with an ALP activity increasing up to 17%. SEM analyses highlighted good cell adhesion and colonization over the scaffold porosities.
Good antibacterial activities were exhibited from both Ti_TiO 2 and Ti_γFe 2 O 3 scaffolds with a reduction of 99.4% and 99.9% of S. aureus colony number, respectively, compared to the not-treated sample. H 2 DCFDA staining investigation reveals the presence of ROS occurring in presence of both TiO 2 and γFe 2 O 3 nano-functionalization. These data prove that both Ti_TiO 2 and Ti_γFe 2 O 3 scaffolds are able to match the double function to promote osteo-integration and deplete bacterial invasion. Further in vitro investigations are planned to optimize the nano-functionalized coating (in terms of NPs densities, surface charges, etc.) and gain more insight on mechanisms inducing bone regeneration and antibacterial activity, before final validation by in vivo studies. Results here presented pave the way for future use of Ti_TiO 2 and Ti_γFe 2 O 3 biomimetic scaffolds to reconstruct large bone defect in orthopedic fields.