Quaternised chitosan coating on titanium provides a self-protective surface that prevents bacterial colonisation and implant-associated infections

Abstract

Implant-related infection is a serious and devastating complication in orthopaedic surgery. The original surfaces of an orthopaedic implant provide ideal substrata for bacterial colonisation and biofilm formation. To reduce this risk, we covalently bonded a titanium implant surface to quaternised chitosan (hydroxypropyltrimethyl ammonium chloride chitosan (HACC)). In vitro, Staphylococcus epidermidis ATCC 35984 was prevented from adhering to and colonising the coated surface. Then, the medullary cavities of rat femora were contaminated with 10⁵ colony-forming units/10 μl of S. epidermidis, and titanium rods were simultaneously implanted into the medullary canals. Implant-associated infection occurred in the control group with uncoated titanium implants, while infection was prevented in the group with titanium-coated implants, as revealed by X-ray, blood and bone culture, and histological analyses. Good bone osseointegration was demonstrated around the coated implants. We conclude that quaternised chitosan coating on titanium could provide a self-protective surface that prevents bacterial colonisation and implant-associated infections.

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Introduction

Orthopaedic implant-related infections are potentially severe and costly complications^1,2 and the second most common cause of orthopaedic implant failure.³ The use of orthopaedic implants continues to grow due to the ageing and increasingly more active and obese population. The treatment of implant-associated complications is exceedingly difficult.⁴ Most of these complications are caused by staphylococcal species⁵ that can form extracellular anionic polysaccharide biofilms on implants and block the penetration of immune cells and antibiotics, necessitating secondary revision surgery to remove all of the implanted materials.^6,7

Current strategies aimed at preventing implant-related infections focus primarily on the use of antimicrobial therapies, including systemic antibiotic prophylaxis and the local administration of antimicrobial agents through irrigation of the surgical site or placement of antibiotic carriers.⁸ Antibiotic resistance has become a serious issue; thus, these antibiotic-based strategies may not be a good choice for preventing nosocomial infections.⁹ As an alternative to antibiotics, different anti-infective agents have been used to coat implant surfaces, including disinfectants such as nitric oxide,^10,11 antimicrobial peptides,¹² materials that release metals,^13,14 and negatively charged hydrophilic polysaccharides such as hyaluronan and heparin.^15,16 As the osseointegration of orthopaedic implants is also important in the functional reconstruction of damaged tissue, it is suggested that coated materials have good biocompatibility with osteogenic cells.

Chitosan has good biocompatibility and limited antibacterial activity,^17–20 and several studies have demonstrated improvements in the antibacterial effects of chitosan through its modification.^21–27 In a previous study, we synthesised quaternised chitosan (hydroxypropyltrimethyl ammonium chloride chitosan [HACC]) with different degrees of substitution (6%, 18%, and 44%) by reacting chitosan with glycidyl trimethylammonium chloride. Our results indicated that HACC with 18% or 44% substitution exhibited significantly improved antibacterial activity compared with unmodified chitosan. HACC 18% was nontoxic to L-929 cells and had good biocompatibility with osteogenic cells, while HACC 44% was slightly cytotoxic and inhibited the proliferation and differentiation of human bone marrow-derived mesenchymal stem cells.²⁸ Further study has demonstrated that HACC can inhibit icaA transcription and biofilm formation by Staphylococcus on titanium.²⁹ Thus, HACC 18% has potential as an antibacterial coating material.

In this study, HACC 18% was covalently bonded to a titanium implant surface using the silanisation of aminopropyltriethoxysilane (APTES) via the bifunctional cross-linker glutaraldehyde. We chose to evaluate the adhesion and colonisation of Staphylococcus epidermidis on this coated surface in vitro. We then evaluated both the antimicrobial efficiency and interface osseointegration of the coated surface in vivo.

Materials and methods

Materials

Titanium (Ti) plates with nominal dimensions of Φ 5 mm × 1 mm and Ti rods (2 mm × 15 mm) were purchased (Sh-puwei, China). HACC with an 18% degree of substitution (DS) with quaternary ammonium was synthesised and successfully characterised by Fourier-transformed infrared (FT-IR) and ¹H nuclear magnetic resonance (NMR) spectroscopy.²⁸ Aminopropyltriethoxysilane (APTES) with 96% purity was purchased from Shanghai Enchang Industry & Trade Co., Ltd. Other chemicals were of analytical grade. Ultra-pure water (>18.2 MΩ cm, Millipore Milli-Q system) was used in the experiments.

Preparation of HACC coating

The surface modification procedure was adapted from the work of Martin et al.³⁰ Briefly, after surface treatment, the Ti plates and rods were placed in a mixture of concentrated sulphuric acid and hydrogen peroxide for surface passivation. The mixture-treated substrates were referred to as M − Ti foils. The M − Ti foils were submerged in a 2% (v/v) solution of APTES in toluene in sealed containers and allowed to react for 24 h. The APTES-treated substrates were referred to as A-M-Ti foils. The A-M-Ti plates and rods were slowly placed into a water–glutaraldehyde solution (2%, v/v), and the containers were then sealed and left for 24 h. The samples were then rinsed with ultra-pure water three times and dried under nitrogen flow at room temperature; these samples were then named Glu-A-M-Ti plates and rods. The final process involved HACC deposition. The HACC solution was prepared using 1% w/v HACC, 2% v/v acetic acid, and 97% ultra-pure water and stirred overnight. The Glu-A-M-Ti plates and rods were placed in a sealed vial containing 40 ml of HACC solution. After a 24 h incubation at room temperature, the samples were extensively washed with 2% v/v acetic acid and ultra-pure water several times. The process of glutaraldehyde and HACC deposition was repeated 5 times, and HACC coatings with several molecules thickness were prepared. These samples were then dried under nitrogen flow at room temperature and stored in N₂. The resulting substrates were referred to as HA-Glu-A-M-Ti (HTi) plates and rods.

Characterisation of the coating

An ESCALAB 250 X-ray photoelectron spectra (XPS) system (Thermo Fisher Scientific Inc.) was used to analyse the chemical composition of each substrate. XPS data were obtained using a monochromatic Al Kα X-ray source (1486.6 eV photons) operating at 150 W and 15 kV. Survey spectra were gathered using an average of 5 scans with pass energy of 70 eV and running from 1100 to 0 eV. To determine charge effects, the adventitious C 1s peak at 284.6 eV was used as a reference. The XPS data were collected and averaged using ESCALAB Surface Analysis Software, Avantage Data System Package (Thermo Fisher Scientific Inc.). Other details of the XPS measurements are similar to those described previously.²⁸

FT-IR spectra of each titanium surface following each reaction step, recorded in attenuated total reflection (ATR) mode, were obtained using a 45° Ge crystal and Varian 640-IR Spectrophotometer with a 16 cm⁻¹ resolution. Substrates were pressed against the crystal using a micrometre pressure clamp at approximately 30 kPa. The M − Ti plates and rods were used as a reference.

Bacterial adhesion test in vitro

HTi and Ti plates with dimensions of Φ 5 mm × 1 mm were sterilised with ethylene oxide, placed into the wells of 24-well microtitre plates, and washed twice with phosphate-buffered saline (PBS). S. epidermidis ATCC 35984 cells (kindly provided by Prof. Di Qu, Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, Shanghai, China) were resuspended at a density of 1.0 × 10⁶ colony-forming units (CFU) per ml in fresh TSBG. Aliquots (1 ml) of these cell suspensions were seeded onto the wells of HTi and Ti plates. These 24-well microtitre plates were incubated at 37 °C for 1, 2, 3, 4, or 5 h, and non-adherent bacteria were removed by gently washing the plates three times with PBS. Adherent bacteria were stained using the Live/Dead® BacLight™ Viability Kit (Molecular Probes, Eugene, OR) for 15 min at room temperature in the dark, followed by three PBS washes to remove nonspecific staining. Fluorescent adherent bacteria were visualised by confocal laser scanning microscopy (Leica TCS SP2; Leica Microsystems, Heidelberg, Germany). Leica confocal software was used to analyse the biofilm images. Images were acquired from random locations within the biofilm formed on the upper side of the titanium plates.

Animals and operative procedure

All experiments were approved by the Animal Ethics Committee of Shanghai Jiaotong University School of Medicine. Twenty 5 month-old female Sprague-Dawley rats were used in this study. Under general anaesthesia, the left hind knees of the rats were prepared using a sterile technique. Straight incisions of 5 mm were made on the lateral sides of the knees. The fibres of the tensor fascia lata were cut longitudinally, and the intercondylar fossae of the distal femora were exposed. With a hand-driven titanium burr, a 2 mm (diameter) × 15 mm (depth) hole was drilled through cortical and cancellous bone to access the medullary cavity. The hole was subsequently washed with saline solution and dried with gauze. Ten microlitres of PBS containing 10⁵ CFU of S. epidermidis (ATCC 35984) was injected with a 50 μl microsyringe (Hamilton, IL, USA) for contamination of the medullary cavity. HTi or Ti rods were immediately inserted into 10 animals in each group. Finally, the wound was closed by suturing.

General assessment

On days 0, 7, 14, 21, 28, 35, and 42 after surgery, body weight and body temperature were measured. Each animal was euthanised 42 days after surgery, according to the approved protocol. Briefly, the femora with implants were excised, and the thigh and adjacent hip and knee joints were evaluated for any signs of inflammation or swelling before being dissected free of soft tissue. Anteroposteriorly, soft X-ray radiographs of the femora were taken (26 kV_p, 12 s; Faxitron, USA). After longitudinal sectioning of the femora, the intramedullary cavity was assessed for pus, abscess formation, cortical lysis, and joint effusion of the adjacent knee joint.

Microbiological evaluation

Fluorescent staining of the implants. Five random femora from every group were longitudinally split into two halves. One half was fixed in a 2.5% glutaraldehyde solution at 4 °C for scanning electron microscopy. The other half was dipped in liquid nitrogen and submitted for pathogen culture and identification, as described below. The HTi and Ti rods were carefully removed, washed three times in PBS, and stained using the Live/Dead® BacLight™ Viability Kit (Molecular Probes, Eugene, OR, USA) for 15 min at room temperature in the dark, followed by three PBS washes to remove nonspecific staining. Fluorescent adherent bacteria were visualised by confocal laser scanning microscopy (Leica TCS SP2; Leica Microsystems).

Scanning electron microscopy (SEM). Five femora halves from each group were submitted for SEM examination in 2.5% glutaraldehyde solution for 2 h at 4 °C. Then, samples were washed twice with 0.01 M PBS for 1 h and subsequently fixed with 0.1% osmium tetraoxide for 1 h, followed by dehydration by replacing the buffer with increasing concentrations of ethanol (30%, 50%, 70%, 80%, 90%, 95%, and 100%) for 10 min each. After critical-point drying and coating by gold sputtering, samples were examined using a scanning electron microscope (JEOL JSM-6360LV; JEOL Ltd., Tokyo, Japan).

Pathogen culture and identification. The other five femora halves from each group were frozen in liquid nitrogen, crushed into fragments, and pulverised in a sterile bone mill (SPEX CertiPrep, Metuchen, USA). Two hundred milligrams of bone powder was agitated in 2 ml of sterile PBS for 2 min by vortexing. The suspension was centrifuged for 10 s (10 000 rpm), and a 0.1 ml sample of each suspension was taken to determine the bacterial number by serial dilutions with triplicate plating on Todd-Hewitt agar plates (countable range: 30–300 CFU per plate). Then, the test tubes were incubated at 37 °C in a shaking incubator operating at 100 cycles per min. Two plates were used per testing solution. After incubation for 24 h at 37 °C, the surviving colonies were counted, and the mean number of cells on duplicate plates was calculated.

Bacteria isolated from swabs, femoral cultures, and rods were identified by a fully automated laboratory system (Vitek-32, BioMerieux, Marcy, France). Additionally, for all 20 rats, 1 ml of blood (collected by puncture of the right atrium), the right femora, and samples of the liver and spleen were taken for pathogen culture and identification to determine whether the infection had been systemically transmitted.

Bone histology

The other five femora from each group, with the implanted rods, were fixed in 4% paraformaldehyde for 48 h. Each specimen was rinsed with running water for 24 h and dehydrated in a graded series of ethanol. They were then dehydrated in xylene and infiltrated and embedded in methylmethacrylate.³¹ These femora were longitudinally sectioned in a sagittal plane into 220 μm-thick slices using a diamond saw (Leica SP1600; Leica Instruments, Nussloch, Germany). The sections were then hand-ground to 50 μm and stained with Gram stain, Stevenel's blue, and van Gieson's picro-fuchsin.

Statistical analysis

The results are expressed as means ± SD. All of the data analyses were performed using SAS (version 8.2; SAS Institute, Cary, NC, USA). Statistical differences were determined by an analysis of variance (ANOVA). The level of significance was set at p < 0.05 (two-tailed).

Results

Characterisation of HACC coating

The chemical composition of the surfaces after each stage of surface functionalisation was determined by XPS. The XPS wide-scan spectra of M − Ti, A-M-Ti, Glu-A-M-Ti, and HA-Glu-A-M-Ti are shown in Fig. 1. As shown in Fig. 1(a), the predominant components of the M − Ti plates and rods were C 1s, O 1s, and Ti 2p. Carbon is typically present due to unavoidable hydrocarbon contamination, and it is conveniently and routinely used as an internal reference at 284.8 eV for calibrating peak positions.³² A small amount of N was also observed, which was most likely due to unavoidable adventitious contamination. In the wide spectrum of APTES-treated Ti (Fig. 1(b)), an increase in the intensity of N 1s indicated the successful deposition of APTES on the Ti substrates. In Fig. 1(c), a small decrease in N 1s and significant decrease in Ti 2p signals demonstrated that the amino groups on the APTES-treated surface reacted with the aldehyde groups in the glutaraldehyde solution. The significant increase in C 1s and conspicuous decrease in Ti 2p intensity shown in Fig. 1(d) confirmed that HACC covalently attached to the Glu-A-M-Ti surfaces, which was consistent with the discernable increase in N 1s intensity.

Fig. 1 XPS wide scan spectra of (a) M-Ti, (b) A-M-Ti, (c) Glu-A-M-Ti and (d).

The FTIR spectra of functionalised Ti are shown in Fig. 2. In Fig. 2(a), the absorption bands at 1566 cm⁻¹ were generally due to cyclic structures of the amino nitrogen atoms coordinated to silicon atoms, while the absorption bands near 1508 cm⁻¹ could be attributed to the symmetric NH₃⁺ deformation mode of cyclic, internal zwitterions.^33,34 The absorption bands near 1570 cm⁻¹ for the amine groups (–NH₂) as well as the disappearance of the absorption bands at 1508 cm⁻¹ indicated that the majority of the NH₃⁺ groups reacted with the aldehyde group of glutaraldehyde. In Fig. 2(c), the broad peak around 3370 cm⁻¹ could be attributed to the stretching vibration of the hydroxyls of HACC.³⁵ The much broader and sharper absorption bands between 958 and 1155 cm⁻¹ and the absorption bands at 1160 cm⁻¹ (asymmetric stretching of the C–O–C bridge) are characteristic of the saccharide structure of HACC.³⁵ The amide I absorption (ν C=O, around 1665 cm⁻¹) of the acetyl-glucosamine units of HACC was also observed.³³ However, the amide II absorption (δ N–H, around 1536 cm⁻¹ (ref. 33, 34 and 36)) was not obviously observed, and it may have overlapped with the deformation vibration of the amine groups (–NH₂) of HACC around 1570 cm⁻¹.

Fig. 2 FT-IR/ATR spectra of (a) A-M-Ti, (b) Glu-A-M-Ti and (c) HA-Glu-A-M-Ti.

HTi inhibits S. epidermidis adhesion and proliferation

To evaluate the bacterial adhesion and proliferation on HTi plates, surfaces were challenged with S. epidermidis for 1, 2, 3, 4, and 5 h. The adherent bacteria were fluorescently stained by the Live/Dead® BacLight™ Viability Kit, resulting in live cells appearing green and dead cells appearing red. Confocal laser scanning microscopy was used to assess the viability of bacterial growth on HTi compared with Ti plates. The images showed that green bacterial clusters on the Ti plates increased with time, but few fluorescent bacterial colonies were present on the surface of HTi plates at each time point, which suggested that HTi plates prevented bacterial adhesion and proliferation (Fig. 3(a)). Three viewing areas were randomly selected from each HTi and Ti plate for quantitative analysis. The green fluorescence data from the bacterial CFU on the Ti plates were normalised to 100% at various time points, and the data from the bacterial CFU on the HTi plates were expressed as a percentage of this control. The numbers of adherent bacteria were significantly lower (p < 0.01) on the HTi plates than on the control Ti plates at each time point, as shown in Fig. 3(b).

Fig. 3 HTi plates inhibit bacterial colonization in vitro. Ti (a) and HTi (b) plates were incubated with S. epidermidis and bacterial colonization was fluorescent staining for total cells (a) after incubation time of 1 h (1), 2 h (2), 3 h (3), 4 h (4), and 5 h (5). The HTi plates showed significantly fewer adherent colonies at each time point than Ti plates (control), *p < 0.05 (b).

General signs

Of the twenty rats, ten received an uncoated Ti rod, and other received an antiseptic-coated HTi rod. Soft X-ray images of the Ti rod group showed radiographic signs of osseous destruction in all animals, with an increase in osteolytic lesions, periosteal elevation, new bone formation, and consecutive deformity after 14 days (Fig. 4(a)), which were more obvious after 42 days (Fig. 5(a1)), whereas soft X-ray images of all animals in the HTi rod group were free of radiographic signs of infection after 14 days (Fig. 4(b)) and showed no change after 42 days (Fig. 5(b1)). Intramedullary pus formation was revealed in all five animals in the Ti rod group (Fig. 5(a2)) but not in any animals in the HTi rod group (Fig. 5(b2)). All animals showed few signs of systemic inflammation and no significantly increased body temperature or decreased body weight during the entire observation period of 42 days (Table 1).

Fig. 4 Representative X-ray radiographs of implanted femora after 14 days. Images of all animals of group Ti rods (a) showed radiographic signs of osseous destruction with increased periosteal elevation, reactive new bone formation and consecutive deformity. Whereas images of all animals of group HTi rods (b) were free of infected radiographic signs.

Fig. 5 Radiological images of femora in anterior–posterior view showed clear signs of osseous destruction and periosteal new bone formation (a1), while no signs of infection in (b1); clinical signs of infection in the intramedullary cavity with an Ti implant was shown with yellow pus formation (a2) compared to physiological appearance with a HTi implant (b2) after longitudinal section of the femora. Fluorescent staining of implants showing thick green clusters formed on the surfaces of a Ti implant (a3), while only discontinuous red dead bacteria adhered on an HTi implant (b3).

Table 1 The general signs of systemic inflammation with body temperature or body weight during the entire observation period of 42 days

	Day 0	Day 3	Day 7	Day 14	Day 21	Day 28	Day 35	Day 42
Body weight/g
Ti (control)	311.5 ± 13.6	315.9 ± 15.6	331.5 ± 20.1	352.2 ± 22.7	379.4 ± 34.2	412.7 ± 29.3	426.7 ± 23.1	449.6 ± 17.7
HTi	311.7 ± 15.3	314.5 ± 16.9	325.9 ± 22.3	342.1 ± 24.1	373.5 ± 38.2	408.4 ± 31.4	423.3 ± 29.4	445.8 ± 28.3
Temperature/°C
Ti (control)	35.4 ± 1.1	36.4 ± 1.1	36.3 ± 0.8	37.4 ± 0.2	37.1 ± 0.3	36.4 ± 0.4	36.5 ± 0.4	36.5 ± 0.7
HTi	36.3 ± 0.2	37.1 ± 0.1	37.2 ± 0.2	37.5 ± 0.3	36.9 ± 0.2	36.3 ± 0.3	36.4 ± 0.1	36.0 ± 0.8

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Microbiological evaluation

Fluorescence staining of implants. Fluorescence staining was used to assess the viability of bacteria on Ti and HTi rods 42 days after the operation. The results showed that thick green clusters formed on the surfaces of all five Ti rods (Fig. 5(a3)), while only discontinuous red (dead) bacteria adhered to all five HTi rods (Fig. 5(b3)).

Scanning electron microscopy. SEM was performed to identify bacterial growth in the rat femoral epiphysis around the HTi and Ti rods 42 days after the operation. Micrographs of all five groups of Ti rods showed migration and growth of bacteria in trabecular bone (Fig. 6(a1 and a2)). No signs of bacterial growth were found in the trabecular bone of any of the five animals with HTi rods (Fig. 6(b1 and b2)).

Fig. 6 Scanning electron microscopy (SEM) images showed that there were spherical bacterial growth in trabecular bone around the uncoated titanium rods (a1 and a2), while no spherical bacteria growth in the bone around coated titanium rods (b1 and b2). 1500× (a1 and b1), 5000× (a2 and b2).

Identification of bacteria. We found no surviving colonies in samples from the HTi rod group after 24 h of incubation. All samples (cultured on agar plates) in the Ti rod group showed massive bacterial growth, with a mean of 3.8 × 10⁶ CFU per gram of bone. All the recovered bacteria were confirmed by the fully automated Vitek-32 laboratory system (BioMerieux, Marcy, France) to be the bacterial inoculum used for infection, demonstrating the absence of other microorganisms at the site of infection.

No bacteria were detected from the liver, spleen, blood, or right femora control (which had not undergone surgery) in any of the experimental groups. This result indicates that the infection did not spread but was confined to the site of surgery.

Histological appearance. Representative images of undecalcified sections of metaphyseal bone tissue around the implants are shown in Fig. 7. All five histological slices from the Ti rod group showed that there was no good osseointegration but rather fibrous tissue membranes between uncoated titanium rods (Fig. 7(a1)), and typical signs of bone infection with Gram-stained blue S. epidermidis were seen in the bone (Fig. 7(a2)). All five histological slices from the HTi rod group were well integrated with the surrounding bone (Fig. 7(b1)) and showed no detectable signs of bone infection, such as the development of abscess lesions or the destruction of cortical and cancellous bone (Fig. 7(b2)).

Fig. 7 Representative image of undecalcified sections of metaphyseal bone tissue around implants. There were fibrous tissue membranes between uncoated titanium rods (a1) and bone and Gram stained blue S. epidermidis in the bone (a2). The coated titanium rods were well-integrated with the surrounding bone (b1) and no bacterial growth in the bone (b2). Gram staining and van Gieson-picric acid fuchsin staining. 5× (a1 and b1), 200× (a2 and b2).

Discussion

The strategy of HACC immobilisation involves (i) pretreatment of titanium surfaces with a mixture of sulphuric acid and hydrogen peroxide, (ii) silanisation of an aminofunctional organosilane (APTES) onto the surface of titanium, (iii) substitution of the terminal amine groups for a bifunctional cross-linker (glutaraldehyde) with two aldehyde end groups, and (iv) reaction of these end groups with the amino groups of HACC. Callen³⁷ found that nitric acid passivation of Ti₆A₁₄V reduced the thickness of the surface oxide layer and increased trace element release. Nanci³⁸ also revealed that an oxidation procedure using a mixture of sulphuric acid and hydrogen peroxide resulted in an increase in the roughness of the titanium surface and the generation of TiO₂ with high compositional homogeneity. Based on these findings, we selected the mixture of sulphuric acid and hydrogen peroxide for the pretreatment of titanium surfaces. Silanization is a critical step for the subsequent reproducibility of the chemical depositions, and APTES was chosen to silanize the titanium surface. The silanol group of APTES was used for covalent attachment to the TiO₂ surface, and the primary amine group at the opposite end was transformed into a Schiff base by reaction with one aldehyde group of glutaraldehyde. Many research papers have reported the use of APTES for the covalent attachment of proteins, cell-adhesive peptides containing the Arg-Gly-Asp (RGD) of Arg-Gly-Asp-Cys (RGDC), and other molecules onto titanium surfaces.^39,40 Glutaraldehyde was chosen as the cross-linker to link HACC to APTES. Glutaraldehyde is a conventional cross-linker widely used for protein and chitosan cross-linking. We assume that silanisation of APTES and the use of glutaraldehyde did not significantly affect the in vivo evaluation of biological activity of HACC-functionalised titanium.

Surfaces of titanium modified with HACC prevented the initial attachment by bacteria in vitro and infection in vivo in this study. Additionally, our results indicate that the mechanism of antibacterial activity of the coating may not be completely identical in vitro and in vivo. In vitro, as shown in the test of bacterial adhesion (Fig. 3), the adhesion-resistant coating of covalently bonded HACC to metal surfaces was unfavourable for bacterial adhesion. After implantation, some HACC may have been released into the surrounding mini-environments and was antiseptic against the S. epidermidis in the bone marrow cavity (Fig. 5(b2)). Our previous study has been proved that covalent-immobilized macromolecule possess outstanding stability of immobilization, and will be slow degraded under the action of enzymes.⁴¹ During the long study period, some bacteria attached to the surface of the coated titanium, but they were all dead within the HACC that was released or grafted onto the metal surfaces (Fig. 5(b3)). These two mechanisms of antibacterial activity make this coating particularly suitable for the case of remodelling due to infection, which may incur a high risk of bacterial residues in the bone marrow cavity.

Bacteria can attach to metal implants, leading to implant-associated infection. Many antibacterial coatings, such as antibiotic-loaded coatings,⁴² silver-loaded coatings,⁴³ and some polymer coatings,⁴⁴ have shown good potential in recent years, but these coatings are still not widely used in the clinic because of various inherent shortcomings. Antibiotic-loaded coatings are not optimal because the bacteria in the vicinity of the implant could develop resistance to the antibiotics,⁴⁵ as well as because the antibiotic delivery time at effective concentrations is relatively short and some types of antibiotics may harm cell functions.⁴⁶ The inherent shortcomings of silver-loaded coatings are their cytotoxicity⁴⁷ and the fact that silver compromises the physical properties of titanium, such as its corrosion resistance.⁴⁸

A new strategy of prophylaxis is to make the surfaces unfavourable for bacterial adhesion.⁴⁹ However, many strategies for developing antibacterial adhesion-resistant surfaces do not take into account the problem of bone tissue integration. When any orthopaedic device is used, the surface should not impair peri-implant bone growth and should preferably benefit tissue integration.⁵⁰ Some polymer coatings can significantly reduce the adhesion of S. aureus and S. epidermidis and not impair osteoblast functions on these surfaces.⁴ According to our previous studies,^28,29 HACC 18% significantly prevents biofilm formation and bacterial survival on titanium surfaces, reduces bacterial viability in pre-existing biofilms, and is biocompatible with osteogenic cells. It is reported that osteoblasts and bacteria will compete with each other to adhere to the surface of the implant, thus the inhibition of bacteria growth will also benefit the attachment of osteogenic cells and osteointegration of the implant.⁵¹ The results of the current animal experiments also show that this coating is conducive to bone integration in the metaphysical regions (Fig. 7).

Conclusions

In summary, we have documented a new approach to limiting or even preventing bacterial colonisation of an implant material that is widely used in orthopaedic devices. A quaternised chitosan coating on titanium implants could provide a self-protective surface that decreases infection rates associated with orthopaedic implantation. It is important to note that the quaternised chitosan coating can also promote the osseointegration of the implant, which is important for the functional reconstruction of damaged tissue.

Acknowledgements

This research was supported by a grants from the National Natural Science Foundation of China (31271015, 81401819), grants from the Science and Technology Commission of Shanghai Municipality (13JC1403900, 13DZ2294000). Programs Supported by the Ningbo Natural Science Foundation (2012A610223, 2012C50001), and the foundation project for medical science and technology of Zhejiang Province (2013KYA179, 2013KYB232).

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Footnote

† Both authors equally contributed to this work.

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