
==== Front
Bioact Mater
Bioact Mater
Bioactive Materials
2452-199X
KeAi Publishing

S2452-199X(24)00318-9
10.1016/j.bioactmat.2024.07.035
Article
Vancomycin-encapsulated hydrogel loaded microarc-oxidized 3D-printed porous Ti6Al4V implant for infected bone defects: Reconstruction, anti-infection, and osseointegration
Zhang Teng a
Zhou Wenhao b
Yang Wanliang a
Bi Jingwei a
Li Hao a
Gao Xianlei a
Zhang Baoliang a
Shi Guidong a
Li Ka a
Wei Zhijian weizhijian2002@126.com
ac⁎⁎⁎
Pan Xin panxin0714@sina.com
a⁎⁎
Feng Shiqing shiqingfeng@sdu.edu.cn
a⁎
a Department of Orthopedics, Qilu Hospital of Shandong University, Jinan, 250012, China
b Shaanxi Key Laboratory of Biomedical Metallic Materials, Northwest Institute for Non-ferrous Metal Research, Xi'an, 710016, China
c International Cooperation Base of Spinal Cord Injury, Tianjin Key Laboratory of Spine and Spinal Cord Injury, Department of Orthopedics, Tianjin Medical University General Hospital, Tianjin, China
⁎ Corresponding author. Department of Orthopedics, Qilu Hospital of Shandong University, Jinan City, Shandong Province, China. shiqingfeng@sdu.edu.cn
⁎⁎ Corresponding author. panxin0714@sina.com
⁎⁎⁎ Corresponding author. Department of Orthopedics, Qilu Hospital of Shandong University, Jinan, 250012, China. weizhijian2002@126.com
21 8 2024
12 2024
21 8 2024
42 1831
6 6 2024
30 7 2024
30 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Infected bone defect is a formidable clinical challenge. Conventional approaches to prevention and treatment for infected bone defects are unsatisfactory. The key elements of the treatment are bone defect reconstruction, anti-infection, and osteogenesis. Conventional treatment methods remain unsatisfactory owing to the absence of composite integrating materials with anti-infective, and osteogenic activities as well as proper mechanical strength at the same time. In this study, we fabricated a vancomycin-encapsulated hydrogel with bacteria-responsive release properties combined with a shaved porous (submicron-micron) three-dimensional-printed Ti6Al4V implant. The implant surface, modified with submicron-sized pores through microarc oxidation (MAO), showed enhanced osteogenic activity and integrated well with the hydrogel drug release system, enabling sustained vancomycin release. In vitro experiments underscored the commendable antibacterial ability, biosafety, and osteoinductive potential. Effective antibacterial and osteogenic abilities of the implant were further demonstrated in vivo in infected rabbit bone defects. These results showed that the vancomycin-encapsulated hydrogel-loaded microarc-oxidized 3D-printed porous Ti6Al4V can repair the infected bone defects with satisfactory anti-infection and osseointegration effects.

Graphical abstract

Summary diagram of the preparation and surgical implantation of the MAO/hydrogel composite porous Ti6Al4V scaffolds designed in this study. MAO, microarc oxidation.Image 1

Highlights

• The TS-M/H/V can repair infected bone defects with satisfactory anti-infection and osseointegration effects as well as perfect mechanical properties.

• The TS-M/H/V can circumvent the drawbacks of traditional treatments and be assembly used intraoperatively, which is convenient for orthopaedic clinical application.

• We can precisely control the vancomycin release by regulating the degradation period of the hydrogel to realize accurate anti-bacteria.

Keywords

Porous Ti6Al4V implants
Infected bone defects
Anti-infection
Osseointegration
Abbreviations

ALP alkaline phosphatase

BI bone in-growth

BICR bone-implant contact ratio

CLSM confocal laser scanning microscopy

FT-IR Fourier transform infrared

hMSCs human mesenchymal stem cells

MRSA methicillin-resistant Staphylococcus aureus

PBWBCs peripheral blood white blood cells

PMMA polymethyl methacrylate

MAO microarc-oxidization (MAO)

Tetra- PEG-NH2 four-arm PEG amino

Tetra-PEG-SC four-arm PEG succinimide carbonate

Tetra-PEG-SG four-arm PEG succinimide glutarate

Tetra-PEG-SS four-arm PEG succinimide ester

TS-M Ti6Al4V implants without hydrogel

TS-M/H Ti6Al4V implants loaded with hydrogel

TS-M/H/V Ti6Al4V implants loaded with vancomycin-containing hydrogel
==== Body
pmc1 Introduction

Post-traumatic or post-tumor resection infection bone defects remain an intractable clinical challenge [1]. In the United States, the annual infection rates associated with joint and fracture surgeries are notably high, with approximately 1.2 million joint surgeries and 6 million fracture surgeries resulting in infection rates of up to 2 % and 5 %, respectively [2,3]. Traditional therapeutic methods for infected bone defects, such as autogenous bone grafts, the Ilizarov technique, and the Masquelet (induced-membrane) technique, along with systemic antibiotic administration [[4], [5], [6]], have several shortcomings. First, impaired local blood supply results in insufficient local antibiotic concentrations following systemic administration, and systemic antibiotic use fails to sufficiently increase antibiotic levels around the implant, hindering effective bacterial eradication [7]. Additionally, high doses of systemic antibiotics can induce systemic toxicity, including oto- and nephrotoxicity [8,9]. Second, autogenous bone grafts, restricted by size and anatomical shape, limit osteogenic potential and cannot entirely address diverse clinical bone defects. Third, the Ilizarov technique lacks immediate postoperative stable reconstruction and osteogenic ability, resulting in prolonged hospital stays and heavy social and economic burdens on patients [10]. Regarding the Masquelet technique, in addition to the absence of immediate postoperative stable reconstruction, residual monomers in the polymethyl methacrylate (PMMA) matrix can cause necrosis in the surrounding bone [11]. Antibiotic release from the PMMA matrix, characterized by an initial burst followed by a release at sub-lethal doses, cannot guarantee antibiotic effectiveness but rather promote resistance [12]. Consequently, additional surgery for removing the PMMA matrix after bone grafting is needed, adding further risks and trauma to the patients. Despite the recent emergence of methods based on antibacterial and osteogenic biomaterials, including hydroxyapatite [13], nanofiber [14], and bioactive glass [15] implants, the limited mechanical strength of these implants precludes their clinical application for large bone defects.

Three-dimensional (3D)-printed porous titanium alloy implants can be customized according to physiological anatomy and clinical treatment requirements, thereby reconstructing the original anatomy and restoring functionality [21]. Therefore, the utilization of 3D-printing microporous titanium alloy structures is important for practical implementation. Introducing a microporous structure can reduce the elastic modulus and other mechanical parameters of an implant, enhancing its biocompatibility with the surrounding bone tissue and effectively avoiding stress shielding and osteolysis [22]. Although 3D-printed implants fulfill the criteria for repairing the material morphology and microstructure of infected bone defects, these implants are still prone to infection because partially melted Ti6Al4V particles can enhance bacterial adhesion and impede the osteogenic potential of the implants [[23], [24]]. Antimicrobial hydrogels are simple to synthesize, and they can be prepared and stored for use before the fabrication of 3D-printed implants. To endow the 3D-printed porous Ti6Al4V implants with defect repair, anti-infection, and osteogenesis capacities, the antimicrobial hydrogel was immediately filled into the microarc-oxidized 3D-printed porous Ti6Al4V implants. This groundbreaking approach enables the delivery of higher antibiotic concentrations at the target site while ensuring immediate stability and long-term osseointegration for repairing infected bone defects.

2 Materials and methods

2.1 Preparation of vancomycin-containing hydrogel and rheological studies

We used a phosphate buffer solution (pH 7.4) to dissolve the gel precursors. First, a four-arm PEG amino (Tetra- PEG-NH2) was dissolved in a sample bottle to prepare gel precursor solution 1. Subsequently, gel precursor solution 2 was prepared by dissolving four-arm PEG succinimide carbonate (Tetra-PEG-SC), four-arm PEG succinimide glutarate (Tetra-PEG-SG), or four-arm PEG succinimide ester (Tetra-PEG-SS) in another sample bottle. The degradation time of the gel was regulated according to the proportions of PEG-SC, PEG-SG, and PEG-SS, and the release rate of vancomycin was generally controlled. Based on the characteristics of postoperative infections in orthopedics, a tetra-PEG-SC hydrogel with a 1-month degradation time was selected. Thereafter, vancomycin was reacted with one-arm PEG to form PEG-Van. After PEG-Van was incorporated into Tetra-PEG-NH2, it was mixed with gummy precursor solution 2 and injected into the porous implants to form a vancomycin-encapsulated hydrogel (10 %, 100 μg vancomycin per mg hydrogel) using a double-pass syringe till the porous implants were fully filled. In order to test the distribution of the vancomycin in the hydrogel, SEM（ThermoFisher Scientific, Helios 5 CX, USA）and EDS Mapping (Bruker, Quantax XFlash7, Germany) was employed. In addition, Rheometry was performed on a Thermo Haake (Thermo Fisher Scientific, Waltham, MA, USA) with a plate geometry (35 mm diameter) at 25 °C. The hydrogel discs (diameter = 35 mm, thickness = 1.2 mm) were tested at a gap distance of 1 mm. Before the tests, an amplitude sweep was first performed in order to define the linear viscoelastic region (LVR) in which the storage modulus is independent of the strain amplitude. Then an angular frequency ω of 15 rad s−1 and a deformation amplitude γ0 of 0.5 % were selected to perform the rheological studies.

2.2 Implant design and experimental groups

The 3D-printed macroporous Ti6Al4V implants (10-mm diameter, 5-mm height for in-vitro study; 5-mm diameter, 6-mm height for in-vivo study) were designed using the Magics software (Materialise, Leuven, Belgium) and prepared using electron beam melting. The implants were designed with parameters (pore size: 400–600 μm, strut diameter: 240–320 μm, porosity: 60%–80 %) conducive to bone and vessel ingrowth [21]. The MAO treatment was conducted employing a JH-10 pulsing power supply (Jinhu-lv-bao Co. Ltd, Beijing, China) and the electrolyte was an aqueous solution of 0.065 mol L−1 Ca-(CH3COO)2·H2O, 0.03 mol L−1 NaH2PO4, 0.065 mol L−1 EDTA-2Na, and 0.5 mol L−1 NaOH. After the MAO treatment, the nanoscale porous topography was further identified under scanning electron microscopy (SEM). For simplicity, hereafter the Ti6Al4V implants (scaffolds) are denoted as TS, the microarc-oxidized Ti6Al4V implants are denoted as TS-M, the microarc-oxidized Ti6Al4V implants loaded with hydrogel are denoted as TS-M/H, the microarc-oxidized Ti6Al4V implants loaded with vancomycin-encapsulated hydrogel are denoted as TS-M/H/V. Previous studies have already proved the osteogenesis-promoting effect of the MAO treatment on the 3D-printed porous Ti6Al4V implants, we set up the following experiment groups: TS-M, TS-M/H, TS-M/H/V.

2.3 In vitro drug release study

We examined drug release by placing the TS-M/H/V implants in buffer solutions of different pH values (7.4 and 5). Vancomycin concentration in the collected buffer solutions was assessed using high-performance liquid chromatography at 230 nm. The chromatographic conditions are described in Supplementary Information.

2.4 Elastic moduli study

Compression tests were conducted using an MTS electromechanical test system (E44.304; MTS Systems, Beijing, China) with a strain rate of 3 × 10−4 s−1. The strain was registered using a digital image correlation system (DIC, MatchID Stereo, SciTech Pty, Victoria, Australia). The elastic modulus was calculated based on the initial elastic loading stage.

2.5 Cell proliferation in implants

After cobalt-60 sterilization, the TS-M, TS-M/H, and TS-M/H/V implants (n = 4 each) were co-incubated with human mesenchymal stem cells (hMSCs) for 1, 3, and 5 days. The cell viability was assessed using Cell Counting Kit-8 (CCK-8; Dojindo, Tabaru, Japan). After 5 days, the number and morphology of the cells were analyzed using SEM (Hitachi, Tokyo, Japan), and the live and dead cells were stained using Calcein AM and propidium iodide (LIVE/DEAD cell viability kit, Dojindo). Subsequently, the stained samples were visualized using confocal laser scanning microscopy (CLSM; Leica TCS SP8; Leica, Wetzlar, Germany). ALP activity of hMSCs was determined using the Alkaline Phosphatase Assay Kit (Beyotime, Jiangsu, China) to detect their osteoblast differentiation ability after 7 and 14 days of co-culture.

2.6 Bacterial cultures

MRSA was separated from a human patient with bacteremia at Beijing Hospital. Ethical approval was obtained from the Ethics Committee and written consent was provided by the patient. Strains of fresh single colonies were diluted with normal saline until the turbidity reached 0.5 McFarland Standard (1 × 108 CFU/mL).

2.7 In vitro antibacterial function

For the antibacterial assay, all implants were co-cultured with bacterial suspensions (108 CFU/mL for 1 mL per well) for 4 and 24 h. The antibacterial rates of all implants for planktonic (Rap) and adhered (Raa) bacteria were evaluated using the Microbial Viability Assay Kit-WST (Dojindo) quantitatively, the detailed steps of which can be found in a previous study [25]. The morphology of bacteria adhered to the implants was observed using SEM. The viable and dead bacteria cells on implants at 24 h were distinguished using fluorescent staining with the Live/Dead Bac-Light™ Bacterial Viability Kit (Molecular Probes™, Invitrogen, Waltham, MA, USA). Further, the implants of each group were put into the 25 ml centrifuge tubes with S.aureus suspension (3 ml, 1 × 105 CFU mL−1) added to be incubated (37 °C, 220 rpm min−1). 100 μL bacterial culture medium was collected at specific time points (2h,4h,6h,8h,10h,12h) to be measured the OD value by enzyme-labeler at 600 nm.

2.8 Animal model and surgical procedure

All surgical procedures were approved by the Model Animal Research Centre of Shandong University, where the study animals were bred. All animal procedures were performed according to the principles of the Guide for the Care and Use of Laboratory Animals after obtaining approval from the Animal Ethics Committee of the Model Animal Research Centre of Shandong University (approval number, KYLL-2022(ZM)-588).

Eighteen specific pathogen-free male New Zealand white rabbits (average weight: 3.67 ± 0.22 kg) were employed in this study. The rabbits were randomly assigned to TS-M, TS-M/H, and TS-M/H/V groups (six rabbits per group). The capacity of the implants to cure infected bone defects was evaluated in an infected femoral condyle defect model. We used isoflurane inhalation to anesthetize the rabbits. Subsequently, we shaved the hair in the medial femur and sterilized the skin using iodophor. After opening the right medial femur condyles surgically, we created a 5-mm diameter defect in the femur condyles and injected 0.1 mL (108 CFU) MRSA into the defect. This dose can result in a 100 % infection rate [26]. Finally, implants sterilized with ethylene oxide were inserted into the bone defect, and the incision was closed using an absorbable thread (Fig. 4a–d).

2.9 Physical examination and hematological analysis

We recorded the body weight and temperature preoperatively and weekly for 6 weeks postoperatively. In addition, blood (0.5 mL) was collected to examine peripheral blood white blood cells (PBWBCs) using the hematology analyzer XS-800i (Sysmex Corporation, Kobe, Japan).

2.10 Radiographic analyses

At the day of euthanasia, lateral radiography was performed on the rabbits to identify the osteomyelitis grade in accordance with the modified osteomyelitis scoring system (Table S1). At 3 weeks postoperatively, all rabbits were anesthetized to obtain lateral femur radiographs.

2.11 Micro-computed tomography analysis

Ex vivo micro-computed tomography (CT) examination of the implants and surrounding area was conducted during the next 6 weeks. Micro-CT images were obtained using the Inveon MM system (Siemens, Munich, Germany) to analyze bone amount and distribution. Each specimen was analyzed using segmentation software (Inveon Research Workplace, Siemens). The osteomyelitic condition of rabbit femurs in each group was quantitatively studied using the osteomyelitis scoring system of micro-CT (Table S2). BVF (BV/TV) is the volume of mineralized bone. Illustrative 3D reconstructions of the implants were generated using a 3D visualization system (Inveon Research Workplace; Siemens).

2.12 Microbiological examination

Examination of the implant. After sacrifice, the implants and tibias were collected separately, and implant-adhering bacteria were detached from the implant into 1 mL phosphate-buffered saline (PBS) using an ultrasonic cleaning treatment (Sonorex Digital 10P; Bandelin, Berlin, Germany; 10 min at 80 % intensity). The tubes were then centrifuged at 4000 rpm for 10 min at 4 °C. The supernatants were collected and resuspended in 300 μL PBS. Ten-fold serial dilutions of samples were incubated on 5 % sheep blood agar plates (Thermo Fisher) at 37 °C for 48 h, and the culture dishes were then quantified for specific bacterial growth. Specifically, the inoculated viable cells in the culture dishes were counted and calculated.

Examination of peri-implant bone. The tibias of rabbits were mechanically homogenized (Omni Tissue Homogenizer and Hard Tissue Homogenizing tips; Omni International, GA, USA) to fully release the bacteria within them. Bacteria were then quantified using serial dilutions and viable counts on 5 % sheep blood agar plates.

2.13 Histological evaluation of osseointegration

The implants and femurs were obtained and fixed in 10 % formalin for 1 week, and dehydrated in a graded ethanol series (50 %, 70 %, 80 %, 90 %, 95 %, and 100 %) under vacuum for 2 days. Thereafter, we embedded the samples in methyl methacrylate, cut and ground them into slices (100–150 μm) using transverse saw cuts as well as a polishing machine (Exakt band saw; Exakt Apparatebau, Norderstedt, Germany). We used Masson–Goldner's trichrome method with NanoZoomer (Hamamatsu Photonics, Hamamatsu, Japan) to stain and photograph the samples. Infection signs were visualized and evaluated by three blinded independent observers using an osteomyelitis scoring system [29] (Table S3). Finally, the cross-sections of the implants were normalized to 100 %, and the percentage of BI and BICR were measured.

2.14 Histological evaluation of infection

The femurs were placed in 4 % paraformaldehyde for tissue fixation for 24 h. Subsequently, the specimens were decalcified with ethylenediaminetetraacetic acid for 4 weeks and embedded in paraffin. We then cut the specimens into 5-μm slices and stained them using hematoxylin-eosin before observation using a NanoZoomer Digital Pathology system. Pathological changes in bone tissue infection were quantitatively scored through the microscopic histological parameters and scoring system (Table S4).

2.15 Statistical analysis

All experiments were conducted in triplicate, and the results are expressed as the mean ± standard deviation or standard error of the mean. Statistical significance was analyzed using one-way variance analysis or Student's t-test, with significance defined as p < 0.01 or p < 0.05.

3 Results

3.1 Sample preparation and characterization

In this study, custom-designed porous Ti6Al4V implants with anti-infection function were prepared via a facile MAO reaction and vancomycin-loaded hydrogel (Fig. 1a). Scanning electron microscopy (SEM) images showed the porous structure of the implant surface treated with MAO and the hydrogel (Fig. 1b and c). No obvious particles were observed (Fig. 1d and e), indicating a homogeneous distribution of vancomycin in the hydrogel. As previous studies demonstrated the osteogenesis-promoting effect of MAO treatment on 3D-printed porous Ti6Al4V (scaffold) implants (TS) [25,26], we set up the following experimental groups: MAO Ti6Al4V implants without hydrogel (TS-M), those loaded with hydrogel (TS-M/H) and those loaded with vancomycin-containing hydrogel (TS-M/H/V). And the compression, tensile, shear strength and elastic modulus of the three groups was shown in Fig. 1f and g respectively, and the stress-strain curves under different loading modes were provided in the “Supplementary information” file (Fig. S2). Dynamic stress sweep rheological tests were conducted to reveal the mechanical strength of the polyethylene glycol (PEG) hydrogel; the yield stress was approximately 1600 Pa and storage modulus (G′) was approximately 400 Pa (Fig. 1h). The frequency sweep test showed that the G′ was conspicuously greater than the G″ over the entire frequency range, demonstrating that the PEG hydrogel possessed elastic dominant networks (Fig. 1i).Fig. 1 Three-dimensional-printed MAO-modified Ti6Al4V hydrogel characteristics. (a) Image of the final product (TS-M/H/V). (b) Representative SEM image of the MAO-modified Ti6Al4V implant. (c) Representative SEM image of the hydrogel loaded with vancomycin. (d) SEM image of the Vancomycin-encapsulated hydrogel. (e) Energy spectrum diagram of the surface vancomycin distribution, the blue spots indicated the vancomycin. (f) Strength of the implants in the TS-M, TS-M/H, and TS-M/H/V groups. (g) Elastic modulus of the hydrogels. Rheological analysis of hydrogel in the (h) stress sweep mode and (i) frequency sweep mode. (j) FT-IR spectra of the TS-M, TS-M/H, and TS-M/H/V implants. (k) Vancomycin release profiles from the TS-M/H/V implant in PBS at pH = 7.4, dotted line indicates the Day 3 and the vancomycin release quickly before Day 3 (blue arrow) and steadily after Day 3 (red arrow). (l) The hydrogel degradation curve of TS-M/H/V. (values are presented as the mean ± standard deviation, n = 6).

Fig. 1

In addition to SEM observations confirming the successful construction of functional composite coatings, Fourier transform infrared (FT-IR) spectroscopy was utilized to verify the successful crosslinking of vancomycin-loaded hydrogels. A comparison of the FT-IR spectra of TS-M, TS-M/H, and TS-M/H/V hydrogels revealed that the spectra of both TS-M/H and TS-M/H/V hydrogels had the absorption peak of the aldehyde carbonyl group C=O at 1728 cm−1, indicating that the aldehyde group was modified and the added vancomycin did not negatively affect the gel stability (Fig. 1j). Compared with that of TS-M/H/V hydrogel, the FT-IR spectrum of the vancomycin-free hydrogel showed the original absorption peak of the aldehyde carbonyl group C=O at 1728 cm−1 and disappearance of the absorption peak at 1079 cm−1. The disappearance of the C–O–C absorption peak revealed that the aldehyde groups in PEG participated in the reaction and affected the spatial structure of the gel.

The vancomycin release kinetics of TS-M/H/V hydrogel was also investigated (Fig. 1k). The release process comprises two phases. In the first 3 d, the vancomycin release rate was relatively fast and then gradually decreased and remained relatively stable over approximately 25 d. Quantitative calculations revealed that the total vancomycin releasing amount was almost the same (392.5 μg) and the hydrogel degradation curve was shown in Fig. 1l.

3.2 Osteocompatibility and osteogenic activities

An ideal implant surface should avoid the adhesion and colonization of bacteria while promoting the adherence, proliferation, and maturation of osteoblasts to realize its long-term stability. To test the biocompatibility of the implants, we evaluated cell morphology using SEM (Fig. 2a–i) and viability through live/dead staining analysis (Fig. 2j–l) of hMSCs co-cultured with the TS-M, TS-M/H, and TS-M/H/V implants for 7 days. Under SEM, the hMSCs in all groups presented a well-stretched filiform shape. Additionally, hMSCs survived on all implants (Fig. 2j–l). We then measured hMSC proliferation by testing cellular metabolic activity on day 1, 3, and 5. No significant difference was found these images indicate in proliferative activity between the three groups (p＞0.05) (Fig. 2m). Using the early-phase osteogenic marker alkaline phosphatase (ALP), we then tested the effect of the materials on osteogenic differentiation promotion. The TS-M/H group presented higher ALP activity compared to the other two groups on day 7 and 14, and no significant difference was observed between the TS-M and TS-M/H/V groups (Fig. 3), indicating that while the hydrogel favorably induced hMSC osteo-differentiation, adding vancomycin had the opposite effect.Fig. 2 Cell morphology, viability, and proliferation of hMSCs co-cultured with implants. SEM observations ( × 100, × 200, and × 1000 magnifications) of hMSCs on the TS-M (a–c), TS-M/H (d–f), and TS-M/H/V (g–i) implants after 5 days of co-culture (arrows indicate the cells). Live/dead staining results of the TS-M (j), TS-M/H (k), and TS-M/H/V (l) implants after 5 days of co-culture (live and dead cells appear green and red, respectively). (m) Cell proliferation on the TS-M, TS-M/H, and TS-M/H/V implants (*p < 0.05).

Fig. 2

Fig. 3 Osteogenic activity of hMSCs co-cultured with implants. ALP staining of the hMSCs in the TS-M (a, b), TS-M/H (c, d), and TS-M/H/V (e, f) groups after 7 and 14 days of co-culture; the bluish-violet zone (green arrows) indicates ALP expression. (g) ALP activity of cells on the TS-M, TS-M/H, and TS-M/H/V implants after 7 and 14 days of co-culture. (h).

Fig. 3

Fig. 4 Infected femoral condyle defect model and the antibacterial effects of the implants in rabbits. Surgical procedure: (a) skin incision; (b) drilling into the femur condyles medullary cavity and MRSA injection; (c) implant insertion; and (d) closing the incision. Physical and hematological changes in the TS-M, TS-M/H, and TS-M/H/V groups during the follow-up period: (e) changes in body weight; (f) changes in body temperature; and (g) changes in PBWBC count. (**p < 0.01, *p < 0.05, n = 6 per group). Specimen morphology of the TS-M (h), TS-M/H (i), and TS-M/H/V (j) groups at 6 weeks after injection of 0.1 mL (108 CFU) MRSA. Lateral radiographs of the TS-M (k), TS-M/H (l), and TS-M/H/V (m) groups at 6 weeks after injection of 0.1 mL (108 CFU) MRSA; the red arrows indicate infection signs in the TS-M and TS-M/H groups with subperiosteal calcification, whereas the blue arrows indicate infection signs in the TS-M and TS-M/H groups with osteolysis around the implants. (n) Osteomyelitis scores of the TS-M, TS-M/H, and TS-M/H/V groups (**p < 0.01) (n = 6 per group). MRSA, methicillin-resistant Staphylococcus aureus; TS-M, MAO Ti6Al4V implants without hydrogel; TS-M/H, MAO Ti6Al4V implants loaded with hydrogel; TS-M/H/V, MAO Ti6Al4V implants loaded with vancomycin-containing hydrogel.

Fig. 4

3.3 Surgery and general follow-up

An infected femoral condyle defect rabbit model was used to determine the capacity of the implants to cure infected bone defects (Fig. 4a–d). In total, 18 rabbits were subjected to implantation surgery. Rabbits in the TS-M and TS-M/H groups exhibited significantly greater weight loss compared to those in the TS-M/H/V group (p < 0.05; Fig. 4e). The body temperature of rabbits in the TS-M/H/V group (38.4°C-40.2 °C) was significantly lower than that of rabbits in the TS-M (38.8°C-42.3 °C) and TS-M/H (39°C-42 °C) groups (p < 0.05; Fig. 4f). All rabbits showed a distinct increase in PBWBC count postoperatively (Fig. 4g), and all groups showed a relative decrease in PBWBCs during follow-up. However, the PBWBC count of the TS-M/H/V group was significantly lower than those in the other groups at almost all postoperative time points (p < 0.01). Rabbits in the TS-M/H/V group showed no signs of osteomyelitis or abnormal bone morphology (Fig. 4j), whereas those in the other groups showed signs of osteomyelitis, including abnormal bone morphology (Fig. 4h and i) and periosteal reactivity with subperiosteal calcification (Fig. 4k and l). Metaphyseal osteolysis was also observed in the TS-M and TS-M/H groups (Fig. 4k and l). The modified osteomyelitis score of the TS-M/H/V group was significantly lower than that of the other groups (p < 0.01; Fig. 4n).

3.4 Micro-CT analysis

Micro-CT analysis of the excised femurs was performed at 6 weeks to observe the implants and surrounding area, focusing on signs of bone remodeling. Illustrative 3D reconstructions were generated based on the findings (Fig. 5a–i). Femurs of rabbits from the TS-M/H/V group showed clear mineralized cortical bone apposition (Fig. 5i) and no signs of infection; however, the TS-M and TS-M/H groups showed distinctive osteolysis (Fig. 5a, b, d, e, red arrows). Furthermore, the intraporous bone volume fraction (BVF) of the TS-M/H/V group (49.2 % ± 3.3 %) was significantly higher than that of the other two groups (TS-M 14.6 % ± 2.2 %, TS-M/H 16.4 % ± 3.2 %; p < 0.01; Fig. 5j). The peri-implant BVF of the TS-M/H/V group (50.3 % ± 3.6 %) was also significantly higher than that of the other two groups (8.3 % ± 1.06 %, 12.8 % ± 1.5 %; p < 0.05). The osteomyelitis score of the TS-M/H/V group (7.5 % ± 0.35 %) was significantly lower than that of the other two groups (6.1 % ± 0.46 %, 3 % ± 0.37 %; p < 0.05). The 3D images are presented in Fig. 5c–f, i. Collectively, these images indicated that the osteogenic ability of the implants was better in the TS-M/H/V group.Fig. 5 Quantitative micro-CT ex vivo imaging and BVF of the femurs of rabbits from the TS-M, TS-M/H, and TS-M/H/V groups after the 6-week follow-up period. TS-M group: side view (a) and top view (b). TS-M/H group: side view (d) and top view (e). TS-M/H/V group: side view (g) and top view (h). The red arrow indicates osteolysis. 3D reconstructions of the TS-M (c), TS-M/H, (f) and TS-M/H/V (i) groups after 6 weeks. Ti, bones in the peri-implant region or within the implants are labeled in gray and green, respectively; a larger green area indicates more bone growth. (j) BVF differentiation among the three groups based on quantification of the micro-CT scans. Error bars represent standard errors of the mean. (k) Osteomyelitis scores of the TS-M, TS-M/H, and TS-M/H/V groups. (*p < 0.05; **p < 0.01) (n = 6 per group). BVF, bone volume fraction; CT, computed tomography; TS-M, MAO Ti6Al4V implants without hydrogel; TS-M/H, MAO Ti6Al4V implants loaded with hydrogel; TS-M/H/V, MAO Ti6Al4V implants loaded with vancomycin-containing hydrogel.

Fig. 5

3.5 Microbiological examination

The in vitro antibacterial function was evaluated by incubating the implants with S. aureus for 4 h. Subsequently, the antibacterial effect of the TS-M/H/V implants against planktonic bacteria was found to be significantly greater than that of the TS-M/H and TS-M implants (Fig. 6a). However, the antibacterial effect of the TS-M/H/V implant against planktonic bacteria was similar to that of the TS-M/H implant, and the effect of both these implants was significantly higher than that of the TS-M implant (Fig. 6b). This finding suggested the susceptibility of the porous structure to bacterial colonization, as well as significant antibacterial and anti-adhesive efficiency of TS-M/H/V against S. aureus. Using SEM, we found that typical spherical S. aureus cells with intact membranes adhered to the TS-M and TS-M/H implants and formed aggregates at 4 h (Fig. 6c and d), whereas no visible cells were observed in the TS-M/H/V group, except for debris of disrupted bacterial membranes (Fig. 6e).Fig. 6 Quantification of planktonic and adhered bacteria after co-culture with the TS-M, TS-M/H, and TS-M/H/V implants for 4 h. (a) Planktonic bacteria. (b) Adhered bacteria. SEM images ( × 3000 and × 25000 magnifications) of samples with adhered S. aureus: TS-M (c, c’), TS-M/H (d, d’), and TS-M/H/V (e, e’). Red arrows indicate S. aureus colonies, whereas blue arrows indicate the debris of disrupted S. aureus membranes. CFU values of the femurs (f) and implants (g) in the TS-M, TS-M/H, and TS-M/H/V groups after 6 weeks. (h) The bacterial growth curves of TS-M, TS-M/H, and TS-M/H/V groups. All values are presented as the mean ± standard deviation (**p < 0.01) (n = 6 per group). SEM, scanning electron microscopy; TS-M/H, MAO Ti6Al4V implants loaded with hydrogel; TS-M/H/V, MAO Ti6Al4V implants loaded with vancomycin-containing hydrogel.

Fig. 6

The average bacterial counts in the rabbit femurs of the TS-M/H/V group were significantly lower than those of the TS-M and TS-M/H groups (p < 0.01; Fig. 6f). Bacterial growth was sparsely detected in the femurs of rabbits in the TS-M/H/V group. The average bacterial counts of the TS-M/H/V implants were also significantly lower than those of the TS-M and TS-M/H implants (p < 0.01; Fig. 6g).

3.6 Histological evaluation

Representative histological pictures of implant osseointegration are shown in Fig. 7a–c. Histological slices from infected sites in the TS-M and TS-M/H groups showed typical signs of chronic bone infection (Fig. 7d and e), including inflammatory cell (mononuclear cell and granulocyte) infiltrate, bone necrosis, and bone erosion. These features were notably diminished in the TS-M/H/V group (Fig. 7f). Fig. 7g shows the quantitative test results of bone in-growth (BI) and bone-implant contact ratio (BICR) in implants from the three groups. The BI and BICR of the TS-M/H/V group were higher than those of the TS-M and TS-M/H groups (p < 0.05), indicating that the eradication of infection is beneficial to improve bone ingrowth. Finally, the histopathological score of the TS-M and TS-M/H groups was remarkably higher than that of the TS-M/H/V group (Fig. 7h), demonstrating the milder infection state of the TS-M/H/V group compared with that of the other groups.Fig. 7 Histological images, bone growth, and osteomyelitis scores of the TS-M, TS-M/H, and TS-M/H/V groups at 6 weeks postoperatively. Goldner's trichrome staining (representative images) of the implants of the TS-M (a), TS-M/H (b), and TS-M/H/V (c) groups. Overview (H&E staining) of epi-metaphysis of the implanted femur condyles of the TS-M (d), TS-M/H (e), and TS-M/H/V (f) groups; black arrow indicates inflammatory infiltrate, red arrow indicates bone necrosis and blue arrow indicates bone erosion. (g) Quantitative analysis of BI and BICR of the TS-M, TS-M/H, and TS-M/H/V implant groups (n = 10 per group). (h) Histopathological score of the TS-M, TS-M/H, and TS-M/H/V implant groups (n = 10 per group). All values are presented as mean ± standard deviation (*p < 0.05; **p < 0.01).

Fig. 7

4 Discussion

4.1 Defect reconstruction in infected bone defects

Traditional therapeutic methods for infectious bone defects, such as the Ilizarov and Masquelet techniques, together with systemic antibiotic administration, have several drawbacks, the most significant one of which is the absence of immediate stable reconstruction after surgery, resulting in excessively long treatment cycles. One of the significant advantages of 3D-printed technology is it can fabricate personalized implants to realize precise reconstruction for patients with complex bone defects, which provides the major advantage of immediate stable reconstruction.

Another important factor for realizing immediate stable reconstruction for bone defects is the ideal mechanical strength of the 3D-printed porous Ti6Al4V. A severe disadvantage of the existing therapeutic bioactive materials for infected bone defect reconstruction (such as nanofiber, bioglass scaffolds loaded with biological factors, stem cells with bone promoting effect [[27], [28], [29]], chitosan-grafted PLGA/hydroxyapatite scaffold, and hydroxyapatite scaffolds containing vancomycin, [[30], [31], [32]]) is their inadequate mechanical strength. The 3D-printed porous Ti6Al4V has been proven to have sufficient mechanical strength for complex anatomical defects in load-bearing bones [33,34]. In the present study, we further quantitatively evaluated the excellent mechanical strength of TS-M/H/V implants (Fig. 1d).

4.2 Anti-infective effects in infected bone defects

An ideal and efficacious biomaterial for osseous tissue engineering, especially for remediating infected bone, remains elusive. Infection rates in open fractures can be as high as 20–50 % [16,17], resulting in significantly longer hospitalization durations and increased financial burdens on patients. The region of impairment and most bone substitutes are susceptible to microbial colonization and subsequent biofilm formation [[18], [19], [20]], thereby promoting the progression of infection. Furthermore, bone defects that lack vascularization render the curative effects of systemic antibiotics unsatisfactory [7]. Hence, exploring alternative bioactive substances capable of inhibiting bacterial proliferation while reconstructing bone defects is of scientific and clinical importance. In a previous study, 3D-printed Ti6Al4V implants with anti-infection and osteogenic functions were constructed by immobilizing vancomycin on an MAO-modified surface using PD/Hep-assisted coating [25,26]. However, the drug-loading capacity of this approach was lower and the production process was more complicated than the method for constructing TS-M/H/V in the present study. The hydrogel in the present study is entirely synthetic without the risk of being inhibited by anticoagulation agents, rejection reactions, and transferring disease. Their cost is low due to their easily preservable, highly accessible components. Because of the intrinsic properties of the ammonolysis reaction, the resulting hydrogels can gel instantly just by injection and adhere to the porous Ti6Al4V tightly through chemical bonds [35]. According to a previous study, to enhance the formation of capillaries and transportation of metabolites, the pore diameter must be at least 300 μm [36]. As 400-μm micropores are favorable for promoting the proliferation of bone tissue cells, we used a porous scaffold size of 400 μm. An interpenetrating porous structure facilitates the penetration of osteoblasts, nutrient exchange, and new bone formation [37,38], but it also provides a favorable environment for bacterial colonization [39]. The uniform filling of a hydrogel can effectively obstruct bacteria from entering the pores of the porous stent, and this is conducive to the overall antibacterial efficacy of the composite system by eliminating local antibacterial “blind spots” Staphylococcus aureus is the predominant microorganism involved in bone infections [46,47], which is detected in 80%–90 % of patients with purulent osteomyelitis [48,49], and the incidence of bone infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has notably increased [50]. Long-term, low-concentration antibiotic administration can foster bacterial resistance [51,52], pose risks to bone cells, and potentially cause cancer [53,54]. Hence, we set the degradation time of the hydrogel at 25 days by optimizing the molecular chain structure design which can match the healing cycle of infected bone defects. Furthermore, a previous study showed that the critical time to prevent implant-associated infections is within 6 h postoperatively, when the pathogen remains metabolically inactive [55]. Hence, administering a sufficiently high concentration of vancomycin at the implant site at the beginning of infection is crucial. The vancomycin release kinetics demonstrated in the present study align with the 6-h antibacterial rule.

A previous study revealed that a porous structure can enhance the combination with hydrogels [56] and delay hydrogel degradation, which is crucial for effectively clearing bacterial colonies and promoting bone ingrowth at infected sites. Hydrogels are ideal antibiotic carriers that steadily and slowly release antibiotics at implantation sites as they degrade, but they are too soft to provide good mechanical support. Therefore, to make the most of their strengths whilst circumventing the weaknesses, we first treated TS using MAO in the electrolyte for osteointegrative coating, which created even micro/nanopore TiO2/CaP coatings in situ on the TS macroporous strut walls to improve implant osseointegration performance and biocompatibility, and then we added the vancomycin-encapsulated hydrogel with ideal vancomycin release kinetics to the inner pores of the 3D-printed porous Ti6Al4V implants. Vancomycin is effective in eliminating the most common S. aureus and MRSA infections in osteomyelitis and has low cytotoxicity [57]. The results revealed that the TS-M/H/V implants exhibited outstanding anti-MRSA activity as well as satisfactory cytocompatibility. Additionally, vancomycin is one of the few antibiotics that does not stimulate the osteogenic transformation of hMSCs [58], as illustrated by the in vitro osteogenic activities tests which showed no significant difference between the ALP activities of hMSCs in the TS-M and TS-M/H/V groups (p＞0.05). In summary, the carefully designed PEG-based vancomycin-encapsulated hydrogel coupled with an MAO-modified active layer, as described here, possesses a synergistic combination of properties.

4.3 Bone regeneration in infected bone defects

Cell adhesion, proliferation, and spreading on an implant surface play crucial roles in determining the osseointegration of the interface between the implant and human bone tissue. Using MAO to produce nanoscale porous Ti6Al4V dioxide bioactive coatings on the implant surface can help mimic the architecture of the natural bone extracellular matrix and provide better biological characteristics for the adhesion of bone cells [59]. Our osteogenesis experimental results showed that the TS-M/H/V implant had good osseointegration capacity, suggesting an appropriate in vivo degradation rate that aligns with the pace of bone ingrowth. Interestingly, the results of osteogenic activities tests in vitro showed that the ALP activity of hMSCs on the TS-M/H implants was greater than that in the other two groups (p＜0.05), suggesting that the present PEG-based hydrogel has a positive effect on the osteogenic activity.

However, the presence of bacteria can severely impair osteogenic activity. Besides controlling local infections, promoting bone regeneration is another important factor for the application of biomaterials in infected bone defect treatment [60]. In this study, 3D reconstruction of the micro-CT image of the distal femur samples collected after 6 weeks showed that more bone tissues were regenerated inside and outside of the porous implants in the TS-M/H/V group compared with those in the other groups. We also observed that the in-vivo osteogenic activities of the TS-M/H/V were significantly better than those in the other groups based on the results of the histological analyses, which is inconsistent with the results of the in vitro test of the present study. The reason for this may be attributed to the following: S. aureus can be internalized into osteoblasts through endocytosis and then inhibit osteoblast proliferation [61,62]. S.aureus and its protein A, lipoteinehoic acid, and peptidoglycan can also promote osteoclast differentiation and enhance bone resorption activity [63,64], while the TS-M/H/V can significantly eliminate S. aureus.

Compared to the other groups, the TS-M/H/V can significantly reverse poor bone formation, and a notable enhancement in bone formation was observed. Successful osseointegration between implants and adjacent bone tissue involves a series of factors. First, bone formation is exceedingly restricted under S. aureus infection. Second, bone formation in the bone defects relies heavily on the local mechanical conditions. A perfect implant should be integrated with the encompassing bone tissue and have robust mechanical binding force to prevent complications, such as loosening and displacement, and restore the function of the reconstructed bone or joint. Aseptic loosening is also a major cause of implant failure [[40], [41]], which can arise from stress-shielding effects and implant surface structures that are not conducive to bone ingrowth and new bone deposition [42]. It is evident that 3D printing technology holds promise in alleviating the stress-shielding effect arising from mechanical property mismatches to avoid aseptic loosening of the implant [[43], [44], [45]]. The elastic moduli of TS-M/H/V (2.572 ± 0.48 GPa) were considerably lower than those of the solid titanium alloys (∼90–115 GPa) [65], which significantly exceeds that of the surrounding tissues (cortical hard bone: ∼15–25 GPa; cancellous or trabecular soft bone with an open cellular structure: ∼0.1–4.5 GPa) [65], resulting in stress shielding and affecting bone healing [66]. The elastic modulus of the TS-M/H/V (2.572 ± 0.48 GPa) in the present study was similar to that of cancellous bone and more beneficial for bone regeneration. Our work demonstrates that the designed composite implantable scaffold can be used to treat infected bone defects, with potential for application in clinical practice. Third, osteogenic elements in the bone defects are another essential factor for bone formation, which can be achieved using the MAO process. This process can combine porous titanium alloy implants and functional hydrogels while introducing key trace elements, such as calcium and phosphorus, which play key roles in bone formation and osseointegration [67].

In this study, we used the MAO process to incorporate trace elements into 3D-printed metal microporous scaffolds; it was also used as a platform to combine functional hydrogels. This composite scaffold, which is beneficial for bone regeneration, significantly enhanced the interaction between bone and the Ti6Al4V surface. Furthermore, the results of our in vitro and in vivo studies demonstrated the inhibition of bacteria and induced osteogenic differentiation and mineralization, indicating that our complex TS-M/H/V holds promise in the application in infected bone defects treatment.

Our study had some limitations. Continuous assessment and monitoring of the degradation of synthetic hydrogels in vivo were lacking. Additionally, further validation of our findings in large animal models, such as sheep or pigs, is a prerequisite for future preclinical studies.

5 Conclusion

In this study, we constructed a microarc-oxidized, 3D-printed porous Ti6Al4V implant loaded with a vancomycin-encapsulated hydrogel. The system displayed numerous characteristics desirable in an implant for treating infected bone defects. The results revealed that the newly developed TS-M/H/V therapy system can effectively treat infected bone defects by providing adequate mechanical support and eradicating infection while enhancing osteogenic activity. The TS-M/H/V therapy system can intelligently circumvent the drawbacks of traditional treatment. Our findings provide a new direction for the treatment of bone defects caused by various pathogeneses.

Ethics approval and consent to participate

All animal experiments were undertaken following a robust ethical review and in accordance with the principles of the Guide for the Care and Use of Laboratory Animals after obtaining approval from the Animal Ethics Committee of the Model Animal Research Centre of Shandong University (approval number, KYLL-2022(ZM)-588).

Funding

This work was supported by the 10.13039/501100001809 National Natural Science Foundation of China [grant number 82202729 ], the 10.13039/501100007129 Natural Science Foundation of Shandong Province [grant number ZR2022QH261 ], the 10.13039/501100010040 Taishan Scholar Project of Shandong Province [grant number tsqn202306355 ], the 10.13039/501100001809 National Natural Science Foundation of China [grant number 32101087 ] and the 10.13039/501100019065 Tianjin Municipal Science and Technology Program (21JCZDJC01100 ).

Data and materials availability

Additional related data due to large size are available upon request to the corresponding author at shiqingfeng@sdu.edu.cn.

CRediT authorship contribution statement

Teng Zhang: Conceptualization, Funding acquisition, Writing – original draft. Wenhao Zhou: Data curation, Methodology. Wanliang Yang: Data curation, Formal analysis, Investigation. Jingwei Bi: Data curation, Formal analysis, Investigation. Hao Li: Data curation, Methodology. Xianlei Gao: Data curation, Methodology. Baoliang Zhang: Software. Guidong Shi: Validation, Visualization. Ka Li: Conceptualization, Software. Zhijian Wei: Formal analysis, Supervision. Xin Pan: Formal analysis, Funding acquisition, Investigation. Shiqing Feng: Project administration, Resources, Supervision.

Declaration of competing interest

None.

Appendix A Supplementary data

The following is the Supplementary data to this article:Multimedia component 1

Multimedia component 1

Peer review under responsibility of KeAi Communications Co., Ltd.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.bioactmat.2024.07.035.
==== Refs
References

1 Papakostidis C. Giannoudis P.V. Reconstruction of infected long bone defects: issues and Challenges Injury 54 2023 807 810 36828614
2 Berríos-Torres S.I. Umscheid C.A. Bratzler D.W. Leas B. Stone E.C. Kelz R.R. Reinke C.E. Morgan S. Solomkin J.S. Mazuski J.E. Dellinger E.P. Itani K.M.F. Berbari E.F. Segreti J. Parvizi J. Blanchard J. Allen G. Kluytmans J.A.J.W. Donlan R. Schecter W.P. Centers for disease control and prevention guideline for the prevention of surgical site infection JAMA. Surg. 152 2017 784 791 28467526
3 Yokoe D.S. Avery T.R. Platt R. Huang S.S. Reporting surgical site infections following total hip and knee arthroplasty: impact of limiting surveillance to the operative hospital Clin. Infect. Dis. 57 2013 1282 1288 23912846
4 Kawakami R. Konno S. Ejiri S. Hatashita S. Surgical treatment for infected long bone defects after limb-threatening trauma: application of locked plate and autogenous cancellous bone graft, Fukushima J. Med. Sci. 61 2015 141 148
5 Li J. Li M. Wang W. Li B. Liu L. Evolution and development of Ilizarov technique in the treatment of infected long bone nonunion with or without bone defects Orthop. Surg. 14 2022 824 830 35343060
6 Frese J. Schulz A.P. Kowald B. Gerlach U.-J. Frosch K.-H. Schoop R. Treatment outcome of the Masquelet technique in 195 infected bone defects-A single-center, retrospective case series Injury 54 2023 110923
7 Benahmed A.G. Gasmi A. Tippairote T. Mujawdiya P.K. Avdeev O. Shanaida Y. Bjørklund G. Metabolic conditions and peri-implantitis Antibiotics 12 2022 65 36671266
8 Samarei R. Comparison of local and systemic ciprofloxacin ototoxicity in the treatment of chronic media otitis Global J. Health Sci. 6 2014 144 149
9 Karimi Z. Pakfetrat Z. Roozbeh J. Janfeshan S. Toll-like receptor-2 mediates systemic inflammation in gentamicin-induced rat nephrotoxicity Clin. Exp. Pharmacol. Physiol. 47 2020 1584 1590 32384574
10 Siddiqui A.A. Siddiqui F. Bashar M. Adeel M. Rajput I.M. Katto M.S. Impact of Ilizarov fixation technique on the limb functionality and self-esteem of patients with unilateral tibial fractures Cureus 11 10 2019 e5923
11 Wei X. Pan Y. Wang M. Lin H. Jiang L. Lin D. Cheng H. Comparative analysis of leaching residual monomer and biological effects of four types of conventional and CAD/CAM dental polymers: an in vitro study Clin. Oral Invest. 26 2022 2887 2898
12 Jin Z. Ding G. Yang G. Rapid detection of antibiotic resistance genes in lactic acid bacteria using PMMA-based microreactor arrays Appl. Microbiol. Biotechnol. 104 14 2020 6375 6383 32488313
13 Li Y. Wang W. Han J. Li G. Zhang W. Yang L. Li W. Synthesis of silver- and strontium-substituted hydroxyapatite with combined osteogenic and antibacterial activities Biol. Trace Elem. Res. 200 2022 2931 2942
14 Wang X. Peng Y. Wu Y. Cao S. Deng H. Cao Z. Chitosan/silk fibroin composite bilayer PCL nanofibrous mats for bone regeneration with enhanced antibacterial properties and improved osteogenic potential Int. J. Biol. Macromol. 230 2023 123265
15 Wu Y. Huo S. Liu S. Hong Q. Wang Y. Lyu Z. Cu-Sr bilayer bioactive glass nanoparticles/polydopamine functionalized polyetheretherketone enhances osteogenic activity and prevents implant-associated infections through spatiotemporal immunomodulation Adv. Healthcare Mater. 12 2023 e2301772
16 Orihuela-Fuchs V.A. Fuentes-Figueroa S. Infection rate in open fractures adjusted for the degree of exposure Acta. Ortop. Mex. 27 2013 293 298 24701769
17 Higgin R. Dean M. Qureshi A. Hancock N. Outcomes following the delayed management of open tibial fractures Injury 52 2021 2434 2438 34158158
18 Hudson M.C. Ramp W.K. Frankenburg K.P. Staphylococcus aureus adhesion to bone matrix and bone-associated biomaterials FEMS Microbiol. Lett. 173 1999 279 284 10227156
19 Ketonis C. Barr S. Adams C.S. Hickok N.J. Parvizi J. Bacterial colonization of bone allografts: establishment and effects of antibiotics Clin. Orthop. Relat. Res. 468 2010 2113 2121 20361282
20 Al-Ahmad A. Wiedmann-Al-Ahmad M. Carvalho C. Lang M. Follo M. Braun G. Wittmer A. Mülhaupt R. Hellwig E. Bacterial and Candida albicans adhesion on rapid prototyping-produced 3D-scaffolds manufactured as bone replacement materials J. Biomed. Mater. Res. A. 87 2008 933 943 18228269
21 Zhang T. Wei Q. Zhou H. Jing Z. Liu X. Zheng Y. Cai H. Wei F. Jiang L. Yu M. Cheng Y. Fan D. Zhou W. Lin X. Leng H. Li J. Li X. Wang C. Tian Y. Liu Z. Three-dimensional-printed individualized porous implants: a new “implant-bone” interface fusion concept for large bone defect treatment Bioact. Mater. 6 2021 3659 3670 33898870
22 Thelen S. Barthelat F. Brinson L.C. Mechanics considerations for microporous titanium as an orthopedic implant material J. Biomed. Mater. Res. A. 69 2004 601 610 15162401
23 Xie K. Guo Y. Zhao S. Wang L. Wu J. Tan J. Yang Y. Wu W. Jiang W. Hao Y. Partially melted Ti6Al4V particles increase bacterial adhesion and inhibit osteogenic activity on 3D-printed implants: an in vitro study Clin. Orthop. Relat. Res. 477 2019 2772 2782 31764350
24 Zhou J. Georgas E. Su Y. Zhou J. Kröger N. Benn F. Kopp A. Qin Y. Zhu D. Evolution from bioinert to bioresorbable: in vivo comparative study of additively manufactured metal bone scaffolds Adv. Sci. 10 2023 e2302702
25 Zhang T. Zhou W. Jia Z. Wei Q. Fan D. Yan J. Yin C. Cheng Y. Cai H. Liu X. Zhou H. Yang X. Zheng Y. Liu Z. Polydopamine-assisted functionalization of heparin and vancomycin onto microarc-oxidized 3D printed porous Ti6Al4V for improved hemocompatibility, osteogenic and anti-infection potencies Sci. China Mater. 61 2018 579 592
26 Zhang T. Wei Q. Zhou H. Zhou W. Fan D. Lin X. Jing Z. Cai H. Cheng Y. Liu X. Li W. Song C. Tian Y. Xu N. Zheng Y. Liu Z. Sustainable release of vancomycin from micro-arc oxidised 3D-printed porous Ti6Al4V for treating methicillin-resistant Staphylococcus aureus bone infection and enhancing osteogenesis in a rabbit tibia osteomyelitis model Biomater. Sci. 8 2020 3106 3115 32350485
27 Watanabe Y. Harada N. Sato K. Abe S. Yamanaka K. Matushita T. Stem cell therapy: is there a future for reconstruction of large bone defects? Injury 47 2016 S47 S51
28 Durham E.L. Howie R.N. Hall S. Larson N. Oakes B. Houck R. Grey Z. Steed M. LaRue A.C. Muise-Helmericks R. Cray J. Optimizing bone wound healing using BMP2 with absorbable collagen sponge and Talymed nanofiber scaffold J. Transl. Med. 16 2018 321 30463618
29 El-Rashidy A.A. Roether J.A. Harhaus L. Kneser U. Boccaccini A.R. Regenerating bone with bioactive glass scaffolds: a review of in vivo studies in bone defect models, Acta Biomaterials 62 2017 1 28
30 Yang Y. Chu L. Yang S. Zhang H. Qin L. Guillaume O. Eglin D. Richards R.G. Tang T. Dual-functional 3D-printed composite scaffold for inhibiting bacterial infection and promoting bone regeneration in infected bone defect models Acta Biomater. 79 2018 265 275 30125670
31 Zhang S. Guo Y. Dong Y. Wu Y. Cheng L. Wang Y. Xing M. Yuan Q. A novel nanosilver/nanosilica hydrogel for bone regeneration in infected bone defects ACS Appl. Mater. Interfaces 8 2016 13242 13250 27167643
32 Pearson J.J. Gerken N. Bae C. Lee K.B. Satsangi A. McBride S. Appleford M.R. Dean D.D. Hollinger J.O. Ong J.L. Guda T. In vivo hydroxyapatite scaffold performance in infected bone defects J. Biomed. Mater. Res. B Appl. Biomater. 108 2019 1157 1166 31410993
33 Chu W. Liu Z. Gan Y. Chang Y. Jiao X. Jiang W. Dai K. Use of a novel Screen-Enrich-Combine(-biomaterials) Circulating System to fill a 3D-printed open Ti6Al4V frame with mesenchymal stem cells/β-tricalcium phosphate to repair complex anatomical bone defects in load-bearing areas Ann. Transl. Med. 9 2021 454 33850851
34 Xu N. Wei F. Liu X. Jiang L. Cai H. Li Z. Yu M. Wu F. Liu Z. Reconstruction of the upper cervical spine using a personalized 3D-printed vertebral body in an adolescent with ewing sarcoma Spine 41 2016 E50 E54 26335676
35 Bu Y. Zhang L. Sun G. Su F. Liu J. Yang F. Tang P. Wu D. Tetra-PEG based hydrogel sealants for in vivo visceral hemostasis Adv. Mater. 31 2019 e1901580
36 Wang C. Xu D. Li S. Yi C. Zhang X. He Y. Yu D. Effect of pore size on the physicochemical properties and osteogenesis of Ti6Al4V porous scaffolds with bionic structure ACS Omega 5 2020 28684 28692 33195921
37 Matena J. Petersen S. Gieseke M. SLM produced porous titanium implant improvements for enhanced vascularization and osteoblast seeding Int. J. Mol. Sci. 16 2015 7478 7492 25849656
38 Castro N.J. Tan W.N. Shen C. Zhang L.G. Simulated body fluid nucleation of three-dimensional printed elastomeric scaffolds for enhanced osteogenesis, tissue Eng. Part. A. 22 2016 940 948
39 Gao Y. Huang J. Zhang L. Zhu Y. Yang P. Xue L. Wang N. He W. A three-dimensional phenolic-based carbon anode for microbial electrochemical system with customized macroscopic pore structure to promote interior bacteria colonization Sci. Total Environ. 859 2023 160131
40 Menken L.G. Fleuriscar J. Weiner T. Berliner Z.P. Rodriguez J.A. Aseptic tibial implant loosening after total knee arthroplasty: preventable? J. Am. Acad. Orthop. Surg. 29 2021 326 330 33828053
41 Kellish A.S. Shahi A. Jr J.A.R. Usmani K. Boniello M. Oliashirazi A. Graf K. Dolch H. Fuller D. Mashru R.P. Implant removal due to infection after open reduction and internal fixation: trends and predictors Arch. Bone. Jt. Surg. 10 2022 514 524 35928909
42 Zhang M. Gregory T. Hansen U. Cheng C. Effect of stress-shielding-induced bone resorption on glenoid loosening in reverse total shoulder arthroplasty J. Orthop. Res. 38 2020 1566 1574 32374418
43 Chen D. Li D. Pan K. Gao S. Wang B. Sun M. Zhao C. Liu X. Li N. Strength enhancement and modulus modulation in auxetic meta-biomaterials produced by selective laser melting Acta Biomater. 153 2022 596 613 36162764
44 Szabo E. Rimnac C. Biomechanics of immature human cortical bone: a systematic review J. Mech. Behav. Biomed. Mater. 125 2021 104889
45 Yoon S. Schiffer A. Jang I.G. Lee S. Kim T. Predictions of the elastic modulus of trabecular bone in the femoral head and the intertrochanter: a solitary wave-based approach Biomech. Model. Mechanobiol. 20 2021 1733 1749 34110537
46 Ashar H. Singh A. Ektate K. More S. Ranjan A. Treating methicillin-resistant Staphylococcus aureus (MRSA) bone infection with focused ultrasound combined thermally sensitive liposomes Int. J. Hyperther. 40 2023 2211278
47 Fu P. Nijiati Y. L T. Wu X. Wang Z. Zhou J. Wang C. Ning B. Clinical and molecular characteristics of methicillin-resistant Staphylococcus aureus in bone and joint infection among children Ann. Clin. Microbiol. Antimicrob. 22 2023 104 37993871
48 Rosova L.V. Godovykh N.V. The microbiological study of purulent focus of inflammation in patients with chronic osteomyelitis of long bones, Klin Lab. Diagn 61 10 2016 727 730
49 Okumura N. Hayakawa K. Yamamoto K. Yamada G. Mezaki K. Ohmagari N. Effectiveness of oral cephalexin in antibiotic-course completion for methicillin-susceptible Staphylococcus aureus-induced bacteremic vertebral osteomyelitis BMC Infect. Dis. 23 2023 307 37158826
50 Kim J. Park S.Y. Sohn K.M. Kim B. Joo E. Methicillin resistance increased the risk of treatment failure in native joint septic arthritis caused by Staphylococcus aureus Antibiotics 12 2023 1628 37998830
51 Wang J. Shan S. Li D. Zhang Z. Ma Q. Long-term influence of chloroxylenol on anaerobic microbial community: performance, microbial interaction, and antibiotic resistance gene behaviors Sci. Total Environ. 897 2023 165330
52 Fan N.S. Bai Y.H. Chen Q.Q. Shen Y. Huang B. Jin R. Deciphering the toxic effects of antibiotics on denitrification: process performance, microbial community and antibiotic resistance genes J. Environ. Manag. 262 2020 110375
53 Han H. Yan H. King K.Y. Broad-spectrum antibiotics deplete bone marrow regulatory T cells Cells 10 2021 277 33573218
54 Jing Y. Chen X. Li K. Liu Y. Zhang Z. Chen Y. Liu Y. Wang Y. Lin S.H. Diao L. Wang J. Lou Y. Johnson D.B. Chen X. Liu H. Han L. Association of antibiotic treatment with immune-related adverse events in patients with cancer receiving immunotherapy J. Immunother. Cancer 10 2022 e003779
55 Poelstra K.A. Barekzi N.A. Rediske A.M. Felts A.G. Slunt J.B. Grainger D.W. Prophylactic treatment of gram-positive and gram-negative abdominal implant infections using locally delivered polyclonal antibodies J. Biomed. Mater. Res. 60 2002 206 215 11835177
56 Erben J. Jirkovec R. Kalous T. Klicova M. Chvojka J. Affiliations expand, the combination of hydrogels with 3D fibrous scaffolds based on electrospinning and meltblown technology Bioengineering 9 2022 660 36354571
57 Chang J. Tasellari A. Wagner J.L. Scheetz M.H. Contemporary pharmacologic treatments of MRSA for hospitalized adults: rationale for vancomycin versus non-vancomycin therapies as first line agents Expert Rev. Anti Infect. Ther. 21 2023 1309 1325 37876291
58 Kankilic B. Bayramli E. Korkusuz P. Eroglu H. Sener B. Mutlu P. Korkusuz F. Vancomycin containing PDLLA and PLGA/β-TCP inhibit biofilm formation but do not stimulate osteogenic transformation of human mesenchymal stem cells Front. Surg. 9 2022 885241
59 Xiu P. Jia Z. Lv J. Yin C. Cheng Y. Zhang K. Song C. Leng H. Zheng Y. Cai H. Liu Z. Tailored surface treatment of 3D printed porous Ti6Al4V by microarc oxidation for enhanced osseointegration via optimized bone in-growth patterns and interlocked bone/implant interface ACS Appl. Mater. Interfaces 8 2022 17964 17975
60 Jing X. Xu C. Su W. Ding Q. Ye B. Su Y. Yu K. Zeng L. Yang X. Qu Y. Chen K. Sun T. Luo Z. Guo X. Photosensitive and conductive hydrogel induced innerved bone regeneration for infected bone defect repair Adv. Healthcare Mater. 12 2023 e2201349
61 Ji Z. Su J. Hou Y. Yao Z. Yu B. Zhang X. EGFR/FAK and c-Sre signalling pathways mediate the intemalisation of Staphylococcus aureus by osteoblasts Cell Microbiol. 22 2020 el3240
62 Mouton W. Josse J. Jacqueline C. Abad L. Trouillet-Assant S. Caillon J. Bouvard D. Bouchet M. Laurent F. Diot A. Staphylococcus aureus intemalization impairs osteoblastic activity and early differentiation process Sci. Rep. 11 2021 17685
63 Ren L. Wang H. He X. Song M. C X. Xu Y. Staphylococcus aureus Protein A induces osteoclastogenesis via the NFkappaB signaling pathway Mol. Med. Rep. 16 2017 6020 6028 28849198
64 Wang Y. Liu X. Dou C. Cao Z. Liu C. Dong S. Fei J. Staphylococcal protein A promotes osteoclastogenesis through MAPK signaling during bone infection J. Cell. Physiol. 232 2017 2396 2406 28185243
65 Nune K.C. Li S. Misra R. Advancements in three-dimensional titanium alloy mesh scaffolds fabricated by electron beam melting for biomedical devices: mechanical and biological aspects Sci. China Mater. 61 2018 455 474
66 Niinomi M. Mechanical biocompatibilities of titanium alloys for biomedical applications J. Mech. Behav. Biomed. Mater. 1 2008 30 42 19627769
67 Jin P. Liu L. Cheng L. Chen X. Xi S. Jiang T. Calcium-to-phosphorus releasing ratio affects osteoinductivity and osteoconductivity of calcium phosphate bioceramics in bone tissue engineering Biomed. Eng. Online 22 2023 12 36759894
