Oxygen vacancy boosting Fenton reaction in bone scaffold towards fighting bacterial infection

Bacterial infection is a major issue after artificial bone transplantation due to the absence of antibacterial function of bone scaffold, which seriously causes the transplant failure and even amputation in severe cases. In this study, oxygen vacancy (OV) defects Fe-doped TiO2 (OV-FeTiO2) nanoparticles were synthesized by nano TiO2 and Fe3O4 via high-energy ball milling, which was then incorporated into polycaprolactone/polyglycolic acid (PCLGA) biodegradable polymer matrix to construct composite bone scaffold with good antibacterial activities by selective laser sintering. The results indicated that OV defects were introduced into the core/shell-structured OV-FeTiO2 nanoparticles through multiple welding and breaking during the high-energy ball milling, which facilitated the adsorption of hydrogen peroxide (H2O2) in the bacterial infection microenvironment at the bone transplant site. The accumulated H2O2 could amplify the Fenton reaction efficiency to induce more hydroxyl radicals (·OH), thereby resulting in more bacterial deaths through ·OH-mediated oxidative damage. This antibacterial strategy had more effective broad-spectrum antibacterial properties against Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus). In addition, the PCLGA/OV-FeTiO2 scaffold possessed mechanical properties that match those of human cancellous bone and good biocompatibility including cell attachment, proliferation and osteogenic differentiation.

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Introduction
Bacterial infection after artificial bone transplantation is the most common and serious complication due to the fact that bone defect is often accompanied by infectant and open wounds, and artificial bone scaffold provides stronghold and microenvironment for bacterial adhesion and growth [1,2].Postoperative infection may hamper the reconstruction of bone tissue and cause the failure of bone transplantation or even amputation, which has turned into an issue beyond neglect [3].Currently, antibiotic combined therapy is the preferred and direct approach for postoperative infection, yet the abuse of antibiotic has shown drug toxicity (hepatotoxicity, nephrotoxicity, etc) in other parts of the human body, and even spurred the production of superbugs [4].Besides, researchers have developed antibacterial functional coating by loading antibacterial substances onto the surface of bone scaffold through solvent soaking or polydopamine modification, and so on [5,6].The approach exhibits effective antibacterial properties in the early stage of bone implantation but cannot achieve sustained antibacterial activity.Moreover, to achieve long-term antibacterial activity, researchers have also incorporated antibacterial substances such as metal ions or their oxides into bone scaffold to continuously inhibit bacterial growth or kill bacteria [1,7,8].Although studies on antibacterial strategies have obtained promising effect, the biocompatibility of heavy metals like Ag, Cu, and Zn with human is in dispute [9].In this regard, it is imperative to propose more effective and safer strategies for the functionalization of bone scaffold with antibacterial properties.
Iron (Fe), an essential nutrient element in human body, is known to perform routine duties in DNA synthesis, erythropoiesis, energy metabolism and other life activities.Fe 3 O 4 nanoparticles have attracted extensive attention in bone transplantation due to their excellent biocompatibility and safety [10,11].It is known that Fe 2+ /Fe 3+ of Fe 3 O 4 can participate in the Fenton reaction, which decomposes hydrogen peroxide (H 2 O 2 ) into highly toxic hydroxyl radical (•OH), which is the most toxic reactive oxygen species (ROS) with high broad-spectrum antibacterial activity [12][13][14][15].The specific reaction formula is as follows: (1) Fe 3+ + H 2 O 2 → Fe 2+ + OOH − + H + ; (2) Fe 2+ + H 2 O 2 → Fe 3+ + OH − + •OH.Researchers indicated that H 2 O 2 is not just produced by cell normal metabolism, but also provided from the oxidative burst of immune cells, thus leading to the higher concentration of H 2 O 2 in the bacterial infection microenvironment [16,17].Nevertheless, it is still not enough for Fe 2+ /Fe 3+ to generate adequate •OH to achieve satisfactory antibacterial effect.In order to improve the Fenton reaction efficiency of Fe 2+ /Fe 3+ , Huang et al designed superparamagnetic iron oxide nanoparticles system, which enhanced Fenton reaction with higher •OH, owing to the continuous generation of H 2 O 2 in β-lap through futile redox [18].In another study, Li et al encapsulated Fe 3 O 4 nanoparticles and H 2 O 2 into poly lactic-co-glycolic acid polymer, and the collapsed polymer released H 2 O 2 at higher concentration could improve the Fenton reaction efficiency [19].However, the above methods require additional supplements of H 2 O 2 , which might cause other side effects to normal cells.
Taking into consideration the characteristic of increased H 2 O 2 concentration in the bacterial infection microenvironment, it is expected to enrich H 2 O 2 from microenvironment and amplify the ability of Fenton reaction to produce •OH.Oxygen vacancy (OV), as a representative defect type, is acknowledged as an active site for redox reaction and capable of adsorbing small molecules such as H 2 O 2 , NO, CO and others [20][21][22].Titanium dioxide (TiO 2 ) is a representative metal oxide semiconductor, and the oxygen (O) atoms in the crystal lattice are easily broken off and cause oxygen loss under the specific process conditions (temperatureassisted reduction, ion-doping or high-energy ball milling), resulting in the formation of OV [23][24][25].Besides, TiO 2 nanoparticles are biocompatible, non-toxic and widely utilized in biomedical research, which is approved by the Food and Drug Administration of United States [26].Therefore, it is feasible and significant to introduce OV into TiO 2 that is able to realize the aggregation of inherent H 2 O 2 from microenvironment for enhancing the Fenton reaction and the extensive generation of •OH in the bacterial infection site.
In this study, nano TiO 2 and Fe 3 O 4 powders were designed to synthesize Fe-doped TiO 2 nanoparticles rich in OV defects (denoted as OV-FeTiO 2 nanoparticles) by high-energy ball milling.Then, the OV-FeTiO 2 nanoparticles were incorporated into polycaprolactone/polyglycolic acid (PCL/PGA, denoted as PCLGA) scaffold fabricated via selective laser sintering (SLS) to achieve antibacterial functionalization.The macro/microstructure, mechanical properties, Fenton oxidation/catalytic properties and antibacterial properties of bone scaffold were comprehensively studied.Likewise, the evolution mechanism of defect structure of the OV-FeTiO 2 nanoparticles during high-energy ball milling, and the antibacterial mechanism of the PCLGA/OV-FeTiO 2 scaffold were emphatically studied and discussed.In addition, the cell culture assays were performed to evaluate the in vitro biocompatibility of the scaffold.

Materials
Nano TiO 2 powders (RT150, (20 ± 5) nm, ⩾99% purity) were purchased from Tangshan Caofeidian Taihongshengda New Material Co., Ltd (China).Fe 3 O 4 powders (100-200 nm, 97% purity) were supplied by Shanghai Aladdin Reagent Co., Ltd (China).And they were used as starting materials for highenergy ball milling.PCL (with a molecular weight of 100 kDa) and PGA (with a molecular weight of 100 kDa) powders were purchased from Shenzhen Polymtek Biomaterials Co., Ltd (China).Phosphate buffer solution (PBS) and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd (China).PCL and PGA were used as matrix materials for the construction of bone scaffold by SLS.

Synthesis of OV-FeTiO 2 nanoparticles
OV-FeTiO 2 nanoparticles were synthesized via highenergy ball milling (also known as mechanochemistry).Mechanochemistry was carried out in a single tank planetary ball mill (Fritsch Pulverisette 6, Germany).Briefly, TiO 2 and Fe 3 O 4 powders (mass ratio of 3:1) were mixed by high-energy ball milling with 400 rpm for 8 h using tungsten carbide (WC) balls as the milling medium, and then pure argon gas was repeatedly filled into the ball milling tank to remove air [27].The ball to powder mass ratio was fixed at 20:1 (20 g grinding balls per 1 g powders), and absolute ethanol was added before ball milling as process control agent.

Structural characterizations of the OV-FeTiO 2
The morphologies and microstructures of the OV-FeTiO 2 nanoparticles were characterized by the scanning electron microscope (SEM, TESCAN MIRA, China) and transmission electron microscopy (TEM, FEI Tecnai G2 F30, USA).For TEM analyses, the OV-FeTiO 2 nanoparticles were studied by the high-resolution TEM (HRTEM), conventional brightfield images and selected area electron diffraction (SAED).The formation of defects was monitored using the x-ray diffractometer (XRD, Bruker D8 Advance, Germany), Fourier transform infrared spectrum (FTIR, Nicolet iS50, USA), x-ray photoelectron spectrometer (XPS, Thermo Scientific Nexsa, USA), and electron paramagnetic resonance (EPR, Bruker A300, Germany).Specifically, the phase structure, grain size and lattice distortion of the OV-FeTiO 2 nanoparticles were analyzed by XRD using Cu-Kα radiation (λ = 0.154 nm) with scanning a speed of 5 • •min −1 .FTIR spectrometer was used to characterize the changes in chemical bonds or functional groups with KBr pellet method.Chemical elements were analyzed by XPS, and Ti 2p and O 1s peak positions were corrected to take the C 1s binding energy (284.6 eV) as the internal reference.The EPR spectra were recorded at 100 K to analyze the concentration of OV.

Preparation of bone scaffold
PCL was blended with PGA as the matrix materials of bone scaffold to make up for the shortcomings of the slow degradation rate and insufficient mechanical properties of PCL, while having better biological activity [28,29].PCLGA powders were mixed with TiO 2 , Fe 3 O 4 , and OV-FeTiO 2 powders to prepare the PCLGA/TiO 2 , PCLGA/Fe 3 O 4 , and PCLGA/OV-FeTiO 2 composite bone scaffolds by SLS, respectively, while the PCLGA scaffold was used as control.Take the PCLGA/OV-FeTiO 2 scaffold for example, the specific preparation process was illustrated.Firstly, the PCLGA/OV-FeTiO 2 composite powders were prepared.PCL and PGA powders with the mass ratio of 3:1 were dispersed into ethanol solution and then treated with magnetic stirring for 1 h at room temperature until the polymers were well-diversified and mixed evenly.At the same time, the OV-FeTiO 2 powders of 5 wt% were added to the ethanol solution, ultrasonically dispersed for 1 h, then mixed with PCLGA suspensions and ultrasound again for 30 min.Subsequently, the PCLGA/OV-FeTiO 2 composite powders could be obtained by the process of filtrating, drying and grinding.Eventually, upon attaining the above composite powders, composite bone scaffold was constructed layer-by-layer by a home-made SLS platform equipped with computer control system, CO 2 laser, option scanner system, powder delivery platform and other parts in figure 2(a) [30,31].The following was a brief description of a typical SLS processing cycle: firstly, a thin layer of powder was spread on the build platform by the roller, and then the powder was selectively sintered by the laser beam according to the slice data of the three-dimensional model input to the computer control system.Next, the build platform was dropped the height of one layer of powder after sintering was completed, and a new layer of powder was covered by the roller on the surface of the previous sintered layer.After that, the cycle was repeated until the scaffold was sintered.Ultimately, the scaffold was obtained by removing the unsintered powder [30].The primary sintering parameters were as follows: laser power of 2 W (duty cycle of 1.8%), laser spot diameter of 50 µm, sweep speed of 200 mm•s −1 , and a layer of powder with a height of 0.1 mm.During the laser sintering process, the laser energy density (ED, J•mm −3 ) could be calculated according to the formula 'ED = P/(V × h × H)', where P (W) was the laser power, V (mm•s −1 ) was the scanning speed, h (mm) was the hatching space, approximately the line width (laser spot diameter), and H (mm) was the layer thickness [32,33].Therefore, in this paper, the laser ED was 2 J•mm −3 .

Characterizations of bone scaffold
The macrostructure and microstructure of different composite bone scaffolds were studied with digital camera and SEM, and all of the scaffolds needed to be sprayed with gold to improve their conductivity.Chemical composition and phase analysis of the scaffolds were characterized by FTIR and XRD, respectively.Mechanical properties of the scaffolds were carried out by tensile and compression tests for five times with universal mechanics testing machine (Ji'nan Zhongluchang Testing Machine Manufacturing Co., Ltd, China), thereby obtaining some mechanical properties parameters such as tensile strength and modulus, breaking elongation, compressive strength and modulus [34].

Detection of H 2 O 2 adsorption and •OH produced by Fenton reaction
The adsorption properties of the PCLGA/OV-FeTiO 2 sample to H 2 O 2 were measured by three-electrode system built by an electrochemical workstation (CHI 760E, China).Briefly, the current-time (i-t) curve was recorded in PBS buffer solution (0.01 mol•l −1 , pH = 7.2-7.4)with the working glass carbon electrode (GCE, φ = 3 mm) as the working electrode (WE), saturated calomel electrode and platinum wire electrode as the reference electrode and the counter electrode (CE).The concentration of H 2 O 2 in solution was monitored by the change of current, thereby reflecting the adsorption of the sample to H 2 O 2 .Specifically, the PCLGA/OV-FeTiO 2 sample from the ground scaffold was ultrasonically dispersed in the mixed solution of deionized water and ethanol with Nafion as the binder for 30 min, and then added dropwise to clean GCE.After drying at room temperature, the sample was added twice to evenly load onto the WE.The three-electrode system was connected to the potentiostat with the voltage set at +0.5 V, and the test was carried out at room temperature.
Gram-negative E. coli and Gram-positive S. aureus were obtained to evaluate the antibacterial activity of scaffolds.In vitro antibacterial assays, 30% H 2 O 2 was used as Fenton reagent, which was diluted to 1 mm, and the concentration of bacterial suspension was 1 × 10 6 CFU•ml −1 .Both E. coli and S. aureus were resuscitated and pre-cultured in Luria-Bertani medium.While the PCLGA, PCLGA/TiO 2 , PCLGA/Fe 3 O 4 , and PCLGA/OV-FeTiO 2 scaffold samples (Φ8 mm × 1 mm) were irradiated to sterilize under UV light for 1 h.The antibacterial properties of the scaffolds were evaluated by the plate counting method, live/dead staining with double fluorescent dye and SEM [36].
The antibacterial rates of the scaffolds were also measured using plate counting method, and the effect of scaffolds on the cellular morphology of E. coli and S. aureus was analyzed by SEM.The scaffolds were immersed in corresponding test tubes containing 0.8 ml of E. coli and S. aureus suspensions (1 × 10 6 CFU•ml −1 , pH = 7.4) and 0.2 ml of H 2 O 2 (1 mm).The PCLGA scaffold was the control group, and the other scaffolds were the experimental groups.After all test tubes were allowed to incubate for 24 h at 37 • C, the scaffolds were removed, and 100 µl of residual suspension was coated on the agar plate for 24 h.Then, the growth of colonies was observed through the digital camera, and the antibacterial rate (%) of different scaffolds was calculated according to (N 0 − N 1 )/N 0 × 100%, where N 0 and N 1 represented the number of colonies in the control group and experimental group, respectively.At the same time, the scaffolds were fixed with 2.5% glutaraldehyde solution for 2 h at 4 • C, and then gradually dehydrated with ethanol solution (30%, 50%, 70%, 90% and 100% v/v).After the scaffolds were fully dried and gold-sprayed, the morphological characterizations of bacteria on scaffolds were observed by SEM, which further investigated the antibacterial mechanism.
In addition, the antibacterial properties of the scaffolds were also evaluated by LIVE/DEAD BacLight Bacterial Viability Kit (Thermo Fisher Scientific L7007, USA).Briefly, each scaffold sample was placed in a 24-well plate with 0.4 ml of bacterial suspension and 0.1 ml of H 2 O 2 per well and cultured for 24 h.Then, the bacteria were stained with prepared 4 ′ ,6-diamidino-2-phenylin dole dihydrochloride (SYTO9) and propidium iodide dual fluorescent dye for 15 min in dark and 37 • C, and the remaining dye was gently rinsed three times with PBS solution.Finally, the fluorescence images were observed under the fluorescence microscope (Olympus IX73, Japan).

In vitro cell culture assays
To evaluate the vitro cytocompatibility of the PCLGA and PCLGA/OV-FeTiO 2 scaffolds, the cell culture assays of human bone marrow mesenchymal stromal cells (hBMSCs) were performed to observe the adhesion and proliferation of cells.Briefly, hBMSCs were seeded at a density of 1 × 10 5 cells per well, and they cultured in Dulbecco's modified Eagle's medium added with 10% fetal bovine serum and 1% penicillin/streptomycin in an incubator at 37 • C under 5% CO 2 atmosphere, and the culture medium was changed every 2 d for the entire cultivation.Before the cell assays, the scaffold samples (7 mm × 7 mm × 1 mm) were sterilized with ultraviolet lamp for 1 h and then placed into a 24-well culture dish.After hBMSCs were seeded for 1, 3 and 7 d, the scaffolds were removed from the medium, washed with PBS, fixed with 4% glutaraldehyde for 30 min, then progressively dehydrated with gradient ethanol solutions and completely dried.Finally, the adhesion morphologies of the cells were observed by SEM.Also, the diffusion area of cells on scaffolds was measured through Image J software.
In order to evaluate cell proliferation of the scaffolds, the probe was implemented to stain and recognize the cell health.At each evaluation period, the hBMSCs were stained with 2 µM calcein acetoxymethyl ester (Calcein AM, Beyotime) and 4 µM Ethidium Homodimer 1 (EthD-1, Beyotime) for 30 min, and the fluorescence microscope was photographed with fluorescence microscopy.The Cell Counting Kit-8 (CCK-8, Beyotime) test was carried out to quantitatively detect the proliferation of hBMSCs on the scaffolds.Briefly, at each evaluation period, the medium was removed, the scaffolds were washed with PBS and transferred to a fresh medium added 10% CCK-8, and then incubated for another 2 h.Subsequently, the optical density (OD) value of the supernate in each well was measured at λ = 450 nm via microplate reader (Thermo Multiskan FC).Alkaline phosphatase (ALP) activity, as an important marker for early osteoblastic differentiation, was employed to qualitatively and semiquantitative investigate hBMSCs differentiation on the scaffold.On 1, 3 and 7 d of cell seeding, the cells were removed with trypsin and fixed with 4% glutaraldehyde.After that, ALP reagent (CTCC-JD002) was performed to stain and observe with microscopy.Also, the relative activity of ALP on scaffold was analyzed by calculating the grey values of the stained images via Image J software with the control group as 1.

Statistical analysis
All the quantitative data were presented as mean value ± standard deviation for each group of samples.The level of statistical significance was determined by one-way analysis of variance.The difference of * p < 0.05 was recognized as statistically significant, and * * p < 0.01 was highly significant.

OV defects structure of OV-FeTiO 2
Core/shell-structured OV-FeTiO 2 nanoparticles were synthesized via high-energy ball milling process as displayed in figure 1(a).Fe 3 O 4 particles were basically wrapped in TiO 2 nanoparticles through mechanical impaction, and the titanium (Ti) atoms in TiO 2 were partially replaced by the Fe atoms, that is, the Fe atoms were solidified in TiO 2 .Simultaneously, while the escape of the O atoms from the oxide surface lattice resulted in the creation of OV defects [37].It has been found that the OV-FeTiO 2 nanoparticles exhibited the typical core/shell structure, with TiO 2 nanoparticles basically enfolded on the surface of Fe 3 O 4 in the SEM and TEM images (figures 1(b) and (c-i)).As observed from the HRTEM images (figure 1(cii)), the lattice spacings of the OV-FeTiO 2 were measured to be 0.34 nm and 0.26 nm corresponding to the (101) plane of TiO 2 and the (311) plane of Fe 3 O 4 , respectively.What's more, the presence of a large number of lattice disorders and dislocations originated from the absence of O atoms from the lattice and the formation of OV [27,38].The polycrystalline nature of OV-FeTiO 2 nanoparticles was shown in the SAED pattern (figure 1(c-ii) inset).The elemental mappings clearly demonstrated that the elements for Ti, O and Fe were distributed homogeneously over the OV-FeTiO 2 nanoparticles in figure 1(c-iii).Therefore, it was evident that the oxide came through multiple welding and breaking under the impact of the grinding balls during high-energy ball milling, generating a large number of lattice distortions and consequently inducing OV defects.
The Bragg reflection peaks of all samples in the XRD pattern were identified according to standard JCPDS cards.As observed in figure 1 According to Bragg's law, it could be seen that the corresponding interplanar crystal spacing of the OV-FeTiO 2 decreased relatively.This was consistent with the above result of partial replacement of the Ti atoms (r Ti = 2 Å) in TiO 2 by the Fe atoms (r Fe = 1.72 Å) with relatively smaller atomic radius to create OV, and force the lattice contraction in the vicinity of the Fe atoms [39].Additionally, the diffraction peaks of the OV-FeTiO 2 were significantly broadened.It was well-known that high-energy ball milling induced fracture and deformation of the powder materials and produced lattice strain, resulting in a decrease in crystallinity.The FTIR signal of the TiO 2 and OV-FeTiO 2 had similar pattern of spectrum (figure 1(e)).The large absorption band at 3440 cm −1 was caused by the stretching vibrations of the hydration water and −OH groups on the surface of the samples, and the peak at 1635 cm −1 was the bending vibration of the −OH groups.Also, it was ascribed that the Ti-O-Ti stretching mode of TiO 2 was assigned to the intense bands around 500-750 cm −1 and Fe-O stretching mode of Fe 3 O 4 was related to the signals at 580 cm −1 around.On the other hand, the absorption peak of Fe-O stretching vibration was not clearly shown in the OV-FeTiO 2 spectrum, and the peak of Ti-O-Ti was shifted towards lower wavenumber.Overall, it was believed that TiO 2 wrapped on the surface of the OV-FeTiO 2 , and the Fe atoms partially replaced the Ti atoms of the Ti-O-Ti bond to form the Ti-O-Fe bond.
Moreover, the XPS analysis was performed to study the chemical state and quantitative information before and after ball milling (figure 1(f-i)).The four signals of different intensities were observed corresponding to C, Ti, O, and Fe, among which the characteristic peak of Fe 2p was weak.And this was because the mechanical ball milling inevitably wrapped incompletely and unevenly, leading to the surface of Fe 3 O 4 being partially exposed, which was confirmed by the above SEM and TEM results.The removed O atoms left behind two excess electrons in each OV, which were harvested by the neighboring Ti atoms and induced the formation of Ti 3+ ions near the surface area.This was confirmed by high resolution XPS spectra of the Ti 2p level in figure 1(f-ii).As for OV-FeTiO 2 , the spectrum of Ti 2p shifted to lower energies compared to TiO 2 , suggesting the changes of chemical state for the Ti atom.The binding energies of 457.9 eV and 464.0 eV were identified as the Ti 2p 3/2 and Ti 2p 1/2 spin-orbit splitting peaks of Ti 4+ ions.At the same time, the peaks located at 457.4 eV and 463.0 eV were assigned to Ti 2p 3/2 and Ti 2p 1/2 spin-orbit splitting of Ti 3+ ions, respectively.These all indicated the partial reduction of Ti 4+ ions and the presence of Ti 3+ ions, i.e., the existence of ligand-unsaturated Ti atoms, further verifying the production of OV indirectly.Moreover, the fine spectra of O 1s as well as peak-differentiated and imitated results could be seen in figure 1(f-iii).The peak of O 1s was asymmetric and broad, indicating the presence of more than one chemical state for oxygen in the OV-FeTiO 2 , which might contain the lattice oxygen in metal oxides, defective oxygen source of oxygen vacancies, adsorbed oxygen or water from surface hydroxyl groups, etc.Based on the characteristic of peak and the analysis of the elemental binding energy, it could be seen that the blinding energies of peaks at 529.2 eV, 530.8 eV and 532.0 eV of the OV-FeTiO 2 , assigned to the lattice oxygen in metal oxides (labeled as O1), defective oxygen source of OV (labeled as O2) and adsorbed oxygen from surface hydroxyl groups (labeled as O3), respectively [40].The peak position of the OV-FeTiO 2 slightly shifted to low binding energy in comparison to the TiO 2 on account of the existence of an extra electron from OV. Apart from that, the area of the O2 peak in the OV-FeTiO 2 increased from 27.8% to 34.2%, relatively.This further indicated the conclusion that OV defects were introduced during the high-energy ball milling.
EPR is a highly sensitive and immediate way to visually monitor OV defects.To further confirm the existence and properties of OV defects, EPR analysis of the TiO 2 and OV-FeTiO 2 was performed.The peak of g = 2.001-2.004could be credited to OV as reported in past studies.In figure 1(g), a strong axisymmetric signal of the OV-FeTiO 2 at g = 2.002 was observed, which was associated with OV defects of the samples [25,41].Furthermore, the signal intensity of the OV-FeTiO 2 was stronger than the TiO 2 , thus it was inferred that a large amount of OV was presented in the OV-FeTiO 2 , which was also confirmed by the XPS analysis above-mentioned.

Microstructure and mechanical properties
The three-dimensional structure of bone scaffold was designed based on the internal structure of natural bone.Histologically, natural bone is composed of cortical bone as the outer layer, which contains longitudinal Haversian canals for the repair and growth of tissues such as blood vessels and nerves, and cancellous bone in the interior, which is a meshwork consisting of rod-like or plate-like structure and is the site of 80% bone tissue remodeling process [42,43].It could be observed that all the scaffolds were equipped with uniformly connected and interlaced porous structure from the macroscopic photographs and optical images of the PCLGA, PCLGA/TiO 2 , PCLGA/Fe 3 O 4 and PCLGA/OV-FeTiO 2 composite bone scaffolds (figures 2(b) and (c)).The longitudinal channels in the outer layer of scaffolds were uniformly annular arrays of circular holes (φ ≈ 1.4 mm).Likewise, the internal hollow channels were constituted by intersecting meshes (triangular pores of ≈1 mm) at the angle of 120 • with spaces of 1 mm longitudinally.It has been suggested that the interconnected porous structure of bone scaffold contributes to the inward growth of bone tissue as well as blood vessels and nerves, which can provide nutrient delivery for cellular behavior.Besides, some studies also confirmed that porous scaffold with an appropriate range of pore sizes (400-1600 µm) has no significant differences in terms of bone growth [44][45][46].Based on this result, the designed scaffold was by and large adequate for the growth and migration of cells and new tissue.
PCLGA has better mechanical properties and biocompatibility as tissue engineering material [28,47].The dispersion of the nanofillers in the polymer matrix plays a crucial role in the comprehensive properties of biopolymer scaffold.Hence, the microscopic morphologies of bone scaffolds were observed by SEM.It could be observed from figure 2(d) that the TiO 2 , Fe 3 O 4 and OV-FeTiO 2 nanoparticles were uniformly dispersed and embedded in the PCLGA matrix, and the surface of scaffolds was relatively flat without any cracks and pores.In consequence, it showed that the dispersion of nanoparticles and the sintering quality of scaffolds both performed well.The chemical composition of scaffolds was identified by FTIR spectrum in figure 2(e).The absorption peak located at 2952 cm −1 (C-H stretching vibration) was assigned to the typical characteristic peak of PCL, and the peaks at 1162 cm −1 (C-O-C stretching vibration) and 1 732 cm −1 (C=O stretching vibration) corresponded to the common characteristic peaks of both PCL and PGA, which were well detected in all scaffolds [48,49].Remarkably, the absorption peak of PGA about 584 cm −1 overlapped with the stretching vibration peaks of TiO 2 at 500-700 cm −1 and Fe 3 O 4 about 580 cm −1 .Therefore, the crystal structure of scaffolds was further explored with XRD patterns (figure 2(f)).The diffraction peaks at 21.7 • , 24.1 • were ascribed to the (110) and (200) crystal planes of PCL, and the peaks at 22.4 • and 29.0 • were the (110) and (020) reflection phases of PGA, respectively.Compared to the PCLGA scaffold, the XRD patterns of the PCLGA/TiO 2 and PCLGA/Fe 3 O 4 composite scaffolds exhibited some new diffraction peaks that perfectly matched the (101) phase of TiO 2 and the (220) and (311) phases of Fe 3 O 4 .Furthermore, the typical diffraction peaks of TiO 2 and Fe 3 O 4 were not clearly shown in the XRD pattern of the PCLGA/OV-FeTiO 2 scaffold.This was probably due to the result of lattice distortion and reduced crystallinity during the high-energy ball milling process.
As far as bone transplantation was concerned, the most basic mechanical properties were sufficiently desirable for bone scaffold [50,51].Therefore, the stress-strain curves of different scaffolds were tested by universal testing machine to calculate their tensile strength and modulus, elongation at break, compressive strength and modulus.As shown in figure 2(g), it was apparent that the mechanical properties of the PCLGA/TiO 2 , PCLGA/Fe 3 O 4 and PCLGA/OV-FeTiO 2 composite scaffolds with different nanoparticles were improved in various degrees.Compared with the pure PCLGA scaffold, the tensile strength and modulus of other three composite scaffolds were remarkably increased by 159%-164% and 128%-147%, respectively.And the breaking elongation was increased by 112%-124%, the compressive strength and modulus were improved by 121%-130% and 120%-131%.These results were mainly due to the strong interaction between the nanoparticles and the polymer matrix, thus enabling the effect of strengthening and toughening [52].For another, from the perspective that the mechanical properties of the preparation of composite bone scaffolds were further improved compared with polymer matrix, which were within the range of human cancellous bone strength requirements [53].

H 2 O 2 adsorption and •OH generation
To get some insights into the interaction process of the PCLGA/OV-FeTiO 2 and H 2 O 2 , an amperometry experiment was designed to monitor the H 2 O 2 concentration based on the changes of current [54].The representations illustrated the experimental setup and provided the operating conditions of the electrodes (figures 3(a-i) and (a-ii)).In simple terms, the working glass carbon electrode, the reference and CEs were partially placed inside the PBS buffer and connected to the potentiostat.The PCLGA/OV-FeTiO 2 sample was uniformly loaded on the GCE beforehand that the potential of 0.5 V was applied.Until the current was stabilized, H 2 O 2 solution (10 mm) of 10 µl was added to the previous PBS solution at 60 s intervals, and the changes of current were recorded.It was observed that the current response was followed by an irregular step and rapid decrease after gradual addition of H 2 O 2 in figure 3(b), and this could be attributed to the consecutive decrease of the H 2 O 2 concentration caused by the adsorption and activation of H 2 O 2 on the surface of the PCLGA/OV-FeTiO 2 (figure 3(c)) [23,55].From the above experiment, the PCLGA/OV-FeTiO 2 still had a better interaction with H 2 O 2 in low concentration.The inference from the analysis was that the OV-FeTiO 2 owned more OV defects could provide more active sites, thus making it easier for the O atoms of H 2 O 2 to interact with metal atoms (Ti atoms or Fe atoms) exposed at the surface of the PCLGA/OV-FeTiO 2 , which facilitated the adsorption of H 2 O 2 .
The generation of •OH via Fenton reaction is critical for antibacterial therapy.TMB and MB were utilized to identify the production of •OH, among which the color of TMB could be transformed and oxidized from colorless to blue-green, and MB could be degraded and weaken the absorption at 663 nm (figures 3(d) and (e)) [56].As shown in figures 3(d-ii) and (diii), the obvious absorption peak at λ = 653 nm, and the bluegreen reaction were showed of TMB in the presence of the PCLGA/OV-FeTiO 2 sample, indicating the successful formation of •OH.And the absorbance gradually increased in the wake of the increase of H 2 O 2 concentration, indicating that the increase of H 2 O 2 concentration had a positive strengthening effect on Fenton reaction.Similarly, the similar results could be presented in the absorption spectra of MB (figures 3(e-ii) and (e-iii)).In comparison without the PCLGA/OV-FeTiO 2 sample, the absorption of MB decreased significantly in the presence of the PCLGA/OV-FeTiO 2 sample.This was because MB was degraded through the reaction product •OH of the PCLGA/OV-FeTiO 2 with H 2 O 2 .And the amount of •OH gradually increased with the increase of H 2 O 2 concentration, suggesting that the PCLGA/OV-FeTiO 2 effectively performed Fenton reaction and produced •OH.The above results indicated that the generation of •OH by the PCLGA/OV-FeTiO 2 was H 2 O 2 concentration-dependent, which was consistent with the characteristics of Fenton reaction.Further, as shown in figure 3(e-iv), the impact of pH environments on •OH was evaluated by the degradation degree of MB [57].It could be seen that under the conditions of weak acidity (pH = 5.5, 6.5) and weak alkalinity (pH = 7.4), the absorbance of three groups of experimental at 665 nm decreased by about 48%, 35% and 16%, respectively.These results indicated that the generation of •OH by the PCLGA/OV-FeTiO 2 was pH-dependent.The Fenton reaction had stronger ability to generate •OH in acidic environment, which was also consistent with the characteristics of Fenton reaction.At the same time, this also demonstrated the non-toxic side effects of the PCLGA/OV-FeTiO 2 on normal cells and tissues.
Additionally, the generation of •OH was also confirmed by EPR spectroscopy (figure 3(f)).The PCLGA/OV-FeTiO 2 group exhibited a typical 1:2:2:1 characteristic signal, which was the classical map after the addition of •OH and DMPO [58].While the pure H 2 O 2 group did not have related signal, as a consequence for H 2 O 2 activation to produce •OH on the surface of the PCLGA/OV-FeTiO 2 .This result was consistent with the degradation and oxidation of MB and TMB in the PCLGA/OV-FeTiO 2 system discussed above.According to the above discussion, the specific reaction mechanism was illustrated in figure 3(g).OV was expected to serve as the preferentially active site for H 2 O 2 activation, benefiting H 2 O 2 adsorption on the PCLGA/OV-FeTiO 2 and further promoting the rapid generation of •OH in situ via Fenton reaction.

Antibacterial activities
The antibacterial activities of scaffolds were evaluated.The results of the bacterial plate counting and the antibacterial rate in each group of scaffolds with E. coli and S. aureus were shown in figures 4(a) and (b).Compared with the PCLGA scaffold, the number of colonies in the PCLGA/TiO 2 and PCLGA/Fe 3 O 4 scaffolds showed slight and limited reduction, and the antibacterial rates were 14% (10%) and 32% (31%) with weak antibacterial effect, respectively.Under the same conditions, the number of colonies in the PCLGA/OV-FeTiO 2 scaffold decreased more in a certain degree, and the antibacterial rate reached 64% (60%), indicating that the antibacterial efficiency of the OV-FeTiO 2 was better than the pure TiO 2 and Fe 3 O 4 .The analysis was mainly attributed to the higher efficiency of Fenton reaction to generate •OH by enhancing H 2 O 2 adsorption of the OV-FeTiO 2 .
Similarly, the bacteria cultivated on different scaffolds were examined by live/dead fluorescence staining.According to the fluorescence images and quantitative results, only the PCLGA/OV-FeTiO 2 scaffold processed the relatively better antibacterial effect against E. coli and S. aureus, as distinct red fluorescence (61% of E. coli, 57% of S. aureus) and comparatively little green fluorescence were detected (figures 4(c) and (d)).These results were consistent with the above experiment, indicating that the PCLGA/OV-FeTiO 2 scaffold had a stronger destructive effect on bacteria, and its high antibacterial potential from outstanding Fenton reaction efficiency in bone transplantation.However, compared to the ideal antibacterial effect (antibacterial rate ⩾90%), there was still more room to improve the antibacterial ability of the PCLGA/OV-FeTiO 2 scaffold.The analysis suggested that the reasons for limited antibacterial capacity might include the lower concentration of H 2 O 2 and the less exposure of antibacterial substances of the scaffold, along with the small porosity of scaffold itself, thus not fully utilizing its antibacterial effect.
In order to investigate the potential antibacterial mechanism, the bacterial morphologies on the scaffolds were further observed.As shown in figure 4(e), the adhered E. coli on the PCLGA scaffold maintained typical rod-like structure with intact and smooth membranes, while the bacteria cultivated on the PCLGA/TiO 2 and PCLGA/Fe 3 O 4 scaffolds occurred slight wrinkles and membrane damage.In contrast, those on the PCLGA/OV-FeTiO 2 scaffold showed irregular shapes, severely ruptured and atrophic membranes.These results indicated that the PCLGA/OV-FeTiO 2 scaffold had a more destructive effect on bacterial structure.Elevated levels of ROS have been demonstrated to be a crucial factor in bacterial apoptosis.The analysis considered that OV as the active site could accumulate H 2 O 2 at the bacterial infection and enhance Fenton reaction to produce •OH, which could be utilized to fight bacteria [25,59].
In conclusion, the antibacterial mechanism of the PCLGA/OV-FeTiO 2 scaffold was proposed as seen in figure 5.In this system, more H 2 O 2 in bacterial infection microenvironment was accumulated at the bacterial infection with the assistance of OV-FeTiO 2 adsorption, which amplified the Fenton reaction of Fe 2+ /Fe 3+ to produce •OH.The central modes of action were: (1) direct interaction of Fe 2+ /Fe 3+ released from gradual degradation of the scaffold with the negatively charged bacterial cell membrane by electrostatic interaction, giving rise to membrane disruption and entocyte leakage; and (2) the OV-FeTiO  of oxidative stress, thus significantly inducing bacterial apoptosis [8,60].
Photothermal therapy is a promising antibacterial therapy that is gradually becoming a research hotspot in the field of bone transplantation by its characteristics of low invasiveness, low side effects, and faster recovery time [61].It has been reported that TiO 2−x nanoparticles with a large number of OV defects have a high photothermal conversion efficiency (39.8%) in the near infrared ray bio-window, and the photothermal conversion efficiency of Fe 3 O 4 nanoparticles can also reach 39% [36,59].And it was found that photothermal conditions may promote the release of Fe 2+ /Fe 3+ , thus strengthening the Fenton reaction.Therefore, it is expected that the antibacterial properties of scaffolds can be further reinforced by the photothermal effect in combination with the enhanced Fenton reaction, which can achieve rapid and efficient antibacterial treatment at sites of bacterial infection for scaffold implants.

Biocompatibility
The biocompatibility of bone scaffold is crucial for the adhesion, proliferation and differentiation of cells and the growth of new bone tissue [62].Based on the above experimental results, the PCLGA/OV-FeTiO 2 scaffold with favorable mechanical and antibacterial properties was the current candidate material, and thus the PCLGA scaffold (as the control group) and PCLGA/OV-FeTiO 2 scaffold were selected for further cell culture assays.The adhesion and morphologies of hBMSCs cultured on different scaffolds for 1, 3 and 7 d were shown in figure 6(a).After 1 d of culture, they were spindle-shaped on the PCLGA and PCLGA/OV-FeTiO 2 scaffolds.It was worth noting that the cells expanded in a multilateral shape and appeared obvious filopodia on the PCLGA/OV-FeTiO 2 scaffold after 3 d.After 7 d, the number of cells in both groups increased, but the cells adhered to the PCLGA/OV-FeTiO 2 scaffold exhibited more spreading, filopodia extension and intercellular fusion than did the cells in the control group.Similar results were obtained by cell relative area calculation, and there were significant differences in cell diffusion area between the two groups (figure 6(b)).
The regulation of scaffold on hBMSCs activity was studied via fluorescence staining.Obviously, there was no obvious red fluorescence in the image, and the number of cells was positively correlated with the culture time.This result demonstrated that all the scaffolds had almost no cytotoxicity (figures 6(c) and (d)).After 3 and 7 d of cell culture, the number of cells on the PCLGA/OV-FeTiO 2 scaffold was significantly higher than that in the control group.Further, the cell counting kit-8 (CCK-8) test was used to quantitatively evaluate the proliferation of hBMSCs, and the results were shown in figure 6(e).In general, the OD value was proportional to the number of living cells.More importantly, it was evident that after 3 and 7 d of cell culture, the OD value of the PCLGA/OV-FeTiO 2 scaffold was significantly higher than that of the control group, and much higher cell density was generated.Hence, in this study, it was believed that the OV-FeTiO 2 nanoparticles introduced into the PCLGA scaffold might be beneficial to cell adhesion and conducive to cell proliferation.It could be due to the rough bulges of scaffold caused by nanofillers or the particular magnetocaloric effect of Fe 3 O 4 in the earth's magnetic field [53,63].As a biochemical marker for osteoblast activity, ALP activity was widely accepted to assess the early differentiation of hBMSCs [7,64].The ALP staining images and relative activity of hBMSCs cultured on different scaffolds after culturing for 1, 3 and 7 d were presented in figures 6(f) and (g).The ALP activity level on scaffold gradually increased over time, and the cells on the PCLGA/OV-FeTiO 2 scaffold exhibited higher ALP positive areas (blue areas) at 3 and 7 d of incubation.Thus, it was considered that the osteogenic potential was superior to that of PCLGA scaffold.Based on the above analysis, the PCLGA/OV-FeTiO 2 scaffold had better biocompatibility, which might facilitate the cell adhesion, proliferation and osteogenic differentiation.

Conclusion
In summary, the PCLGA/OV-FeTiO 2 composite bone scaffold with favorable mechanical properties, more effective antibacterial properties and better biocompatibility was successfully constructed by SLS in this study, of which the PCLGA biodegradable aliphatic polymers were used as the matrix material, and the OV-FeTiO 2 nanoparticles rich in OV defects were used as reinforced phase and antibacterial substance.Mechanistically, the OV defects of the OV-FeTiO 2 nanoparticles could adsorb and enrich H 2 O 2 at the bacterial infection site, which could directly enhance the interaction between Fe 2+ /Fe 3+ and H 2 O 2 to amplify Fenton reaction efficiency and generate more •OH, incurring more bacterial death.The results of mechanical tests and cell culture assays demonstrated that the PCLGA/OV-FeTiO 2 scaffold featured favorable mechanical properties and better biocompatibility.Overall, this study enabled Fenton antibacterial activity by enriching H 2 O 2 within the bacterial infection of bone defect through a simple and economical technique, without additional delivery of toxic drugs.Accordingly, this study proposed a new, effective and safe strategy against bacterial infection, which will broaden more new avenues for scientific research in bone implantation.

Figure 2 .
Figure 2. Fabrication and characterizations of different scaffolds.(a) Schematic illustration of preparation process for scaffold by selective laser sintering.(b)-(d) Photographs, SEM images and microscopic morphologies of the PCLGA, PCLGA/TiO 2 , PCLGA/Fe 3 O 4 and PCLGA/OV-FeTiO 2 scaffolds.(e) and (f) FTIR spectra and XRD pattern of scaffolds.(g) Mechanical properties include tensile strength and modulus, compressive strength and modulus and elongation at break of scaffolds.

Figure 3 .
Figure 3. OV boosting Fenton reaction with H 2 O 2 activation and •OH generation.(a) Experimental set-up used for the amperometric monitoring of H 2 O 2 .(b) Current responses upon H 2 O 2 (10 µl) additions.(c) Schematic representation of H 2 O 2 adsorption at the surface of PCLGA/OV-FeTiO 2 .(d) Oxidation of TMB by PCLGA/OV-FeTiO 2 .(i) and (ii) Illustrative diagram of chemical reaction in oxidation of TMB, and photos as well as UV-vis absorption spectra of TMB for •OH detection.(iii) H 2 O 2 concentration-dependent oxidation of TMB due to generated •OH by PCLGA/OV-FeTiO 2 .(e) Degradation of MB by PCLGA/OV-FeTiO 2 .(i) and (ii) Illustrative diagram of chemical reaction in degradation of MB, and degradation of MB due to generated •OH.(iii) and (iv) H 2 O 2 concentration-dependent and pH-dependent degradation of MB due to generated •OH by PCLGA/OV-FeTiO 2 .(f) EPR spectra of DMPO trapped for PCLGA/OV-FeTiO 2 suspension in the presence of H 2 O 2 .(g) Schematic illustration of H 2 O 2 activation and •OH generation of PCLGA/OV-FeTiO 2 via Fenton reaction.

Figure 4 .
Figure 4. Antibacterial properties of different scaffolds.(a) Photographs of the bacterial colonies formed by E. coli and S. aureus after co-culture with PCLGA, PCLGA/TiO 2 , PCLGA/Fe 3 O 4 and PCLGA/OV-FeTiO 2 scaffolds.(b) Antibacterial rates of the scaffolds corresponding to (a).(c) Fluorescence images showing the live/dead bacteria of E. coli and S. aureus on the scaffolds.(d) Quantitative analysis of live/dead staining.(e) SEM images on the scaffolds.E. coli is cyan, and S. aureus has a golden color.
2 nanoparticles exposed to scaffold amplified the efficiency of Fenton reaction to generate abundant •OH by adsorbing H 2 O 2 in bacterial infection microenvironment; and (3) formation of •OH could destroy membrane, damage DNA, destruct proteins and the ATP synthesis enzymes by accumulation

Figure 5 .
Figure 5. Schematic of oxygen vacancy boosting Fenton reaction in PCLGA/OV-FeTiO 2 bone scaffold towards fighting bacterial infection.More H 2 O 2 in bacterial infection microenvironment were accumulated at the bacterial infection with the assistance of OV-FeTiO 2 adsorption, which amplified Fenton reaction of Fe 2+ /Fe 3+ .

Figure 6 .
Figure 6.In vitro biocompatibility of hBMSCs on the PCLGA and PCLGA/OV-FeTiO 2 scaffolds after 1, 3 and 7 d of culture.(a) Pseudo-colored SEM images of hBMSCs after culturing for different time.(b) Cell relative area through image analysis.(c) Fluorescence microscopy images of the scaffolds after culturing for different time.(d) Cell density through image analysis.(e) Cell proliferation on the control, PCLGA and PCLGA/OV-FeTiO 2 scaffolds after culturing for different time determined by CCK-8 assay.(f) The ALP staining images of the scaffolds after culturing for different time.(g) Relative activity of ALP through image analysis.* denotes p < 0.05 and * * denotes p < 0.01, compared with the control group.