Zhifeng Yu

Antibacterial Properties of Magnesium In Vitro and in an In Vivo Model of Implant-Associated Methicillin-Resistant Staphylococcus aureus Infection

ABSTRACT Periprosthetic infection remains a challenging clinical complication. We investigated the antibacterial properties of pure (99.9%) magnesium (Mg) in vitro and in an in vivo rat model of implant-related infection. Mg was highly effective against methicillin-resistant Staphylococcus aureus-induced osteomyelitis and improved new peri-implant bone formation. Bacterial icaA and agr RNAIII transcription levels were also assessed to characterize the mechanism underlying the antibacterial properties of the Mg implant.

Anti-infective efficacy, cytocompatibility and biocompatibility of a 3D-printed osteoconductive composite scaffold functionalized with quaternized chitosan

Contaminated or infected bone defects remain serious challenges in clinical trauma and orthopaedics, and a bone substitute with both osteoconductivity and antibacterial properties represents an improvement for treatment strategy. In this study, quaternized chitosan (hydroxypropyltrimethyl ammonium chloride chitosan, HACC) was grafted to 3D-printed scaffolds composed of polylactide-co-glycolide (PLGA) and hydroxyapatite (HA), in order to design bone engineering scaffolds endowed with antibacterial and osteoconductive properties. We found that both the PLGA/HA/HACC and PLGA/HACC composite scaffolds decreased bacterial adhesion and biofilm formation under in vitro and in vivo conditions. Additionally, ATP leakage assay indicated that immobilizing HACC on the scaffolds could effectively disrupt microbial membranes. Using human bone marrow-derived mesenchymal stem cells (hBMSCs), we demonstrated that HA incorporated scaffolds, including PLGA/HA and PLGA/HA/HACC, favoured cell attachment, proliferation, spreading and osteogenic differentiation compared to HA-free PLGA or PLGA/HACC scaffolds. Finally, an in vivo biocompatibility assay conducted on rats, showed that HA incorporated scaffolds (including PLGA/HA and PLGA/HA/HACC scaffolds) exhibited good neovascularization and tissue integration. Taken together, our findings support the approach for developing porous PLGA/HA/HACC composite scaffold with potential clinical application in the treatment of infected bone. STATEMENT OF SIGNIFICANCE Although plenty of conductive scaffold biomaterials have been exploited to improve bone regeneration under infection, potential tissue toxicity under high concentration and antibiotic-resistance are their main deficiencies. This study indicated that HACC-grafted PLGA/HA composite scaffold prepared using an innovative 3D-printing technique and covalent grafting strategy showed significantly enhanced antibacterial activities, especially against the antibiotic-resistant strains, together with good osteogenic activity and biocompatibility. Therefore, it provides an effective porous composite scaffold to combat the infected bone defect in clinic with decreased risks of bacterial resistance and open a feasible strategy for the modification of scaffold interfaces involved in the bone regeneration and anti-infection.

Structural simulation of adenosine phosphate via plumbagin and zoledronic acid competitively targets JNK/Erk to synergistically attenuate osteoclastogenesis in a breast cancer model

The treatment of breast cancer-induced osteolysis remains a challenge in clinical settings. Here, we explored the effect and mechanism of combined treatment with zoledronic acid (ZA) and plumbagin (PL), a widely investigated component derived from Plumbago zeylanica, against breast cancer-induced osteoclastogenesis. We found that the combined treatment with PL and ZA suppressed cell viability of precursor osteoclasts and synergistically inhibited MDA-MB-231-induced osteoclast formation (combination index=0.28) with the abrogation of recombinant mouse receptor activator of nuclear factor-κB ligand (RANKL)-induced activation of NF-κB/MAPK (nuclear factor-κB/mitogen-activated protein kinase) pathways. Molecular docking suggested a putative binding area within c-Jun N-terminal kinase/extracellular signal-regulated kinase (JNK/Erk) protease active sites through the structural mimicking of adenosine phosphate (ANP) by the spatial combination of PL with ZA. A homogeneous time-resolved fluorescence assay further illustrated the direct competitiveness of the dual drugs against ANP docking to phosphorylated JNK/Erk, contributing to the inhibited downstream expression of c-Jun/c-Fos/NFATc-1 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1). Then, in vivo testing demonstrated that the combined administration of PL and ZA attenuated breast cancer growth in the bone microenvironment. Additionally, these molecules prevented the destruction of proximal tibia, with significant reduction of tartrate-resistant acid phosphatase (TRAcP)-positive osteoclast cells and potentiation of apoptotic cancer cells, to a greater extent when combined than when the drugs were applied independently. Altogether, the combination treatment with PL and ZA could significantly and synergistically suppress osteoclastogenesis and inhibit tumorigenesis both in vitro and in vivo by simulating the spatial structure of ANP to inhibit competitively phosphorylation of c-Jun N-terminal kinase/extracellular signal-regulated kinase (JNK/Erk).

Cytocompatibility with osteogenic cells and enhanced in vivo anti-infection potential of quaternized chitosan-loaded titania nanotubes.

Infection is one of the major causes of failure of orthopedic implants. Our previous study demonstrated that nanotube modification of the implant surface, together with nanotubes loaded with quaternized chitosan (hydroxypropyltrimethyl ammonium chloride chitosan, HACC), could effectively inhibit bacterial adherence and biofilm formation in vitro. Therefore, the aim of this study was to further investigate the in vitro cytocompatibility with osteogenic cells and the in vivo anti-infection activity of titanium implants with HACC-loaded nanotubes (NT-H). The titanium implant (Ti), nanotubes without polymer loading (NT), and nanotubes loaded with chitosan (NT-C) were fabricated and served as controls. Firstly, we evaluated the cytocompatibility of these specimens with human bone marrow-derived mesenchymal stem cells in vitro. The observation of cell attachment, proliferation, spreading, and viability in vitro showed that NT-H has improved osteogenic activity compared with Ti and NT-C. A prophylaxis rat model with implantation in the femoral medullary cavity and inoculation with methicillin-resistant Staphylococcus aureus was established and evaluated by radiographical, microbiological, and histopathological assessments. Our in vivo study demonstrated that NT-H coatings exhibited significant anti-infection capability compared with the Ti and NT-C groups. In conclusion, HACC-loaded nanotubes fabricated on a titanium substrate show good compatibility with osteogenic cells and enhanced anti-infection ability in vivo, providing a good foundation for clinical application to combat orthopedic implant-associated infections.

Augmentation of screw fixation with injectable calcium sulfate bone cement in ovariectomized rats

The objective of this study was to determine the effect of augmenting screw fixation with an injectable calcium sulfate cement (CSC) in the osteoporotic bone of ovariectomized rats. The influence of the calcium sulfate (CS) on bone remodeling and screw anchorage in osteoporotic cancellous bone was systematically investigated using histomorphometric and biomechanical analyses. The femoral condyles of 55 Sprague-Dawley ovariectomized rats were implanted with screw augmented with CS, while the contralateral limb received a nonaugmented screw. At time intervals of 2, 4, 8, 12, and 16 weeks, 11 rats were euthanized. Six pair-matched samples were used for histological analysis, while five pair-matched samples were preserved for biomechanical testing. Histomorphometric data showed that CS augmented screws activated cancellous bone formation, evidenced by a statistically higher (p < 0.05) percentage of osteoid surface at 2, 4, and 8 weeks and a higher rate of bone mineral apposition at 12 weeks compared with nonaugmented screws. The amount of the bone-screw contact at 2, 8, and 12 weeks and of bone ingrowth on the threads at 4 and 8 weeks was greater in the CS group than in the nonaugmented group (p < 0.05), although these parameters increased concomitantly with time for both groups. The CS was resorbed completely at 8 weeks without stimulating fibrous encapsulation on the screw surface. Also, the cement significantly increased the screw pull-out force and the energy to failure at 2, 4, 8, and 12 weeks after implantation, when compared with the control group (p < 0.05). These results imply that augmentation of screw fixation with CS may have the potential to decrease the risk of implant failure in osteoporotic bone.

In vivo evaluation of the anti-infection potential of gentamicin-loaded nanotubes on titania implants

Titanium-based implants have been widely used in orthopedic surgery; however, failures still occur. Our in vitro study has demonstrated that gentamicin-loaded, 80 nm-diameter nanotubes possessed both antibacterial and osteogenic activities. Thus, the aim of this study was to further investigate the in vivo anti-infection effect of the titanium implants with gentamicin-loaded nanotubes. Thirty-six male Sprague Dawley rats were used to establish an implant-associated infection model. A volume of 50 μL Staphylococcus aureus suspension (1×105 CFU/mL) was injected into the medullary cavity of the left femur, and then the titanium rods without modification (Ti), titanium nanotubes without drug loading (NT), and gentamicin-loaded titanium nanotubes (NT-G) were inserted with phosphate-buffered saline-inoculated Ti rods as a blank control. X-ray images were obtained 1 day, 21 days, and 42 days after surgery; micro-computed tomography, microbiological, and histopathological analyses were used to evaluate the infections at the time of sacrifice. Radiographic signs of bone infection, including osteolysis, periosteal reaction, osteosclerosis, and damaged articular surfaces, were demonstrated in the infected Ti group and were slightly alleviated in the NT group but not observed in the NT-G group. Meanwhile, the radiographic and gross bone pathological scores of the NT-G group were significantly lower than those of the infected Ti group (P<0.01). Explant cultures revealed significantly less bacterial growth in the NT-G group than in the Ti and NT groups (P<0.01), and the NT group showed decreased live bacterial growth compared with the Ti group (P<0.01). Confocal laser scanning microscopy, scanning electron microscopy, and histopathological observations further confirmed decreased bacterial burden in the NT-G group compared with the Ti and NT groups. We concluded that the NT-G coatings can significantly prevent the development of implant-associated infections in a rat model; therefore, they may provide an effective drug-loading strategy to combat implant-associated infections in clinic.

Bacterial inhibition potential of quaternised chitosan-coated VICRYL absorbable suture: An in vitro and in vivo study

Summary Background/Objective As a widely used absorbable suture with antibacterial property, triclosan- coated polyglactin suture (Vicryl Plus) has been extensively utilized to reduce the occurrence rate of surgical site infections (SSIs) in orthopaedic surgery. However, the potential toxicity and side-effects of triclosan raised increasing concerns about its biological safety. This study aimed to investigate the antimicrobial activity and biocompatibility of quaternised chitosan-coated Vicryl suture (HV) both in vitro and in vivo. Methods In this study, a modified chitosan derivate, (hydroxypropyltrimethyl ammonium chloride chitosan, HACC), was coated over the surface of the absorbable Vicryl suture. Two standard bacteria strains, Staphylococcus epidermidis (ATCC35984) and methicillin-resistant Staphylococcus aureus (ATCC43300), were selected to evaluate bacterial adhesion and biofilm formation on the sutures at 6, 24 and 48 h in vitro. Additionally, human skin-derived fibroblasts cells were used to test the cytocompatibility of the sutures. Furtherly, sutures contaminated with methicillin-resistant S. aureus were implanted subcutaneously in SD rats in order to confirm the in vivo antibacterial performance and biocompatibility. Results We found that HACC-coated Vicryl suture (HV) exhibited significant anti-bacterial effects on the two tested strains. The bacterial attachment and biofilm formation on the surface of the HV sutures were found to be comparable to that of Vicryl Plus sutures (VP). Moreover, all the four tested sutures presented good cytocompatibility with human skin-derived fibroblasts cells. Histology and immunohistochemistry results indicated that the infections and inflammations were significantly inhibited around the HV and VP sutures. Conclusion In general, the present study demonstrated that the quaternised chitosan coating is a flexible and cost-effective alternative strategy to prevent the suture related surgical site infections in orthopaedic practices.

Changes in the mechanical properties and composition of bone during microdamage repair.

Under normal conditions, loading activities result in microdamage in the living skeleton, which is repaired by bone remodeling. However, microdamage accumulation can affect the mechanical properties of bone and increase the risk of fracture. This study aimed to determine the effect of microdamage on the mechanical properties and composition of bone. Fourteen male goats aged 28 months were used in the present study. Cortical bone screws were placed in the tibiae to induce microdamage around the implant. The goats were euthanized, and 3 bone segments with the screws in each goat were removed at 0 days, 21 days, 4 months, and 8 months after implantation. The bone segments were used for observing microdamage and bone remodeling, as well as nanoindentation and bone composition, separately. Two regions were measured: the first region (R1), located 1.5 mm from the interface between the screw hole and bone; and the second region (R2), located>1.5 mm from the bone-screw interface. Both diffuse and linear microdamage decreased significantly with increasing time after surgery, with the diffuse microdamage disappearing after 8 months. Thus, screw implantation results in increased bone remodeling either in the proximal or distal cortical bone, which repairs the microdamage. Moreover, bone hardness and elastic modulus decreased with microdamage repair, especially in the proximal bone tissue. Bone composition changed greatly during the production and repair of microdamage, especially for the C (Carbon) and Ca (Calcium) in the R1 region. In conclusion, the presence of mechanical microdamage accelerates bone remodeling either in the proximal or distal cortical bone. The bone hardness and elastic modulus decreased with microdamage repair, with the micromechanical properties being restored on complete repair of the microdamage. Changes in bone composition may contribute to changes in bone mechanical properties.

Comparison between Transareola Single-site Endoscopic Thyroidectomy and Minimally Invasive Video-assisted Thyroidectomy

OBJECTIVES: To compare surgical outcomes between transareola single-site endoscopic thyroidectomy (TASSET) and minimally invasive video-assisted thyroidectomy (MIVAT). Methods: Patients with thyroid nodules were randomized to TASSET (n = 24) or MIVAT (n = 24). Surgical outcomes and patient-rated cosmetic results, based on numerical (0 [worst], 10 [best]) and verbal (1 [poor], 4 [excellent]) response scales, were compared. Results: There were no significant differences between groups for age, sex, indication for operation, estimated blood loss, postoperative pain and length of postoperative stay. TASSET was associated with a significantly longer mean ± SD operative time than MIVAT (156.84 ± 41.42 vs. 66.38 ± 17.58 min), and significantly improved cosmetic results according to the numerical (9.63 ± 0.60 vs 7.90 ± 1.38) and verbal response (3.8 ± 0.5 vs 3.1 ± 0.7) scales. Postoperative complaints were comparable between the two approaches, although MIVAT involved a shorter operation time. Conclusions: Patients treated with TASSET had superior cosmetic results compared with those treated with MIVAT.

Bacteria-Targeting Nanoparticles with Microenvironment-Responsive Antibiotic Release To Eliminate Intracellular Staphylococcus aureus and Associated Infection

Staphylococcus aureus ( S. aureus) is a causative agent in life-threatening human diseases that afflict millions of people annually. Traditional antibiotic treatments are becoming less efficient because S. aureus can invade host cells including osteoblasts and macrophages, constituting a reservoir that is relatively protected from antibiotics that can lead to recrudescent infection. We herein report a unique intracellular antibiotic delivery nanoparticle, which is composed of (i) a mesoporous silica nanoparticle (MSN) core loaded with gentamicin, (ii) an infected microenvironment (bacterial toxin)-responsive lipid bilayer surface shell, and (iii) bacteria-targeting peptide ubiquicidin (UBI29-41) that is immobilized on the lipid bilayer surface shell. The lipid material acts as a gate that prevents drug release before the MSNs reach the target cells or tissue, at which point they are degraded by bacterial toxins to rapidly release the drug, thus eliminating efficient bacteria. We confirm rapid drug release in the presence of bacteria in an extracellular model and observe that S. aureus growth is effectively inhibited both in vitro and in vivo of planktonic and intracellular infection. The inflammation-related gene expression in infected preosteoblast or macrophage is also downregulated significantly after treatment by the antibiotic delivery nanoparticles. The antibiotic delivery nanoparticles offer advantages in fighting intracellular pathogens and eliminating the inflammation caused by intracellular bacterial infections.