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Staphylococcus aureus is a primary pathogen responsible for causing postoperative infections as it survives and persists in host cells, including osteoblasts and macrophages. These cells then serve as reservoirs resulting in chronic infections. Most traditional antibiotics have poor effects on intracellular S. aureus because they cannot enter the cell. Herein, a cell-penetrating peptide TAT-KR-12 was derived from the trans-activating transcription (TAT) peptide and KR-12 (residues 18-29 of human cathelicidin LL-37). The TAT acts as a "trojan horse" to deliver KR-12 peptide into the cells to kill S. aureus. Moreover, effective antibacterial properties and biocompatibility were observed in vitro, demonstrating that TAT-KR-12 is effective not only in eliminating planktonic S. aureus, but also in eliminating intracellular S. aureus cells in vitro. TAT-KR-12, as with LL-37, also elicits strong anti-inflammatory activities in LPS-stimulated macrophages, as demonstrated by significant inhibition of NO, TNF-α, and IL-1β expression and secretion from LPS-stimulated RAW264.7 cells. In the subcutaneous infection mouse model of planktonic and intracellular infections, the growth of S. aureus in vivo is evidently inhibited without cytotoxicity. These results suggest that the novel antimicrobial TAT-KR-12 may prove to be an effective treatment option to overcome antibiotic resistance caused by intracellular bacterial infections.

Due to its excellent mechanical and low-friction properties, polyetheretherketone (PEEK) has been widely investigated for use in orthopedic applications over the past decade. However, the bioinertness and poor osteogenic properties of PEEK have hampered its clinical application. In this study, the surface of PEEK was modified by co-treatment with hydrofluoric acid and nitric acid (AFN). The microstructures of the modified PEEK surfaces were investigated using scanning electron microscopy. The water contact angles of the surfaces were also measured. To evaluate their cytocompatibility, PEEK samples were used as substrates to culture rat bone mesenchymal stem cells, and cell adhesion, viability, and expression of specific marker genes were measured. Treatment of PEEK with AFN (PEEK-AFN) was found to enable better osteoblast adhesion, spreading, and proliferation; the activity of alkaline phosphatase (an early osteogenic differentiation marker) was also found to be enhanced post-treatment. Furthermore, PEEK-AFN was able to modulate macrophage polarization and down regulated the expression of proinflammatory factors via inhibiting the NF-κB pathway. Thus, treatment of PEEK with AFN could promote M2 polarization of the macrophages and stimulate the differentiation of osteoblasts. These results provide valuable information that could facilitate the use of PEEK-based composites as bone implant materials.

Polyetheretherketone (PEEK) is considered to be a promising bone implant material owing to its biocompatibility and elastic modulus, which is similar to that of natural bone. However, the clinical potential of PEEK is severely limited by its bioinertness, which leads to poor osseointegration, and the lack of antibacterial properties. In this study, the antimicrobial peptide, KR-12, was immobilized on the surface of PEEK implants with the assistance of polydopamine (PDA) to inhibit bacterial infection as well as to promote osteogenesis and osseointegration. Compared to unmodified PEEK, the PEEK with immobilized KR-12 showed significantly improved antibacterial activity against Staphylococcus aureus (ATCC 25923), both in vitro and in vivo. For the in vitro and in vivo evaluation of the osteogenic properties of modified PEEK, rat bone mesenchymal stem cells (rBMSCs) and a rat femoral defect model were used, respectively. The in vitro studies showed that compared to rBMSCs treated with unmodified PEEK, those treated with KR-12-coated PEEK exhibited improved adhesion, proliferation, and osteogenic differentiation. Moreover, micro-computed tomography and histological analysis suggested that the KR-12 coating promoted osteointegration in vivo in rat femurs. Taken together, these results suggest that the KR-12 coating could improve the antibacterial ability of pure or PDA-coated PEEK against Staphylococcus aureus (ATCC 25923), both in vitro and in vivo. Overall, KR-12 combined with the PDA film coating synergistically induced osteogenic effects both in vitro and in vivo. Thus, the surface-modified material, which exhibits both anti-bacterial and osteointegration properties, shows considerable potential for use as an orthopedic implant.

Background/objective: Orthopaedic implants are important devices aimed at relieving pain and improving mobility. Staphylococcal infection and aseptic loosening are two common events associated with inflammatory osteolysis that lead to implant failures. Bone mass and biomechanical properties are important indicators that could influence patient outcomes after revision surgery. However, the dynamics of bacterial infections and their influence on bone mass and biomechanical properties remain unclear. Hence, in this study, we developed rabbit aseptic inflammation and staphylococcal infection models to determine the effects of coagulase-positive and coagulase-negative bacterial infection, as well as aseptic inflammation, on the mass and biomechanical properties of the bone. Methods: Sixty New Zealand white rabbits were randomly assigned to 6 groups, and each group had 10 rabbits. The medullary cavities in rabbits of each group were injected with phosphate-buffered saline (100 ​μL), titanium (Ti)-wear particles (300 μg/100 ​μL), a low concentration of Staphylococcus epidermidis (105/100 ​μL), a high concentration of S. epidermidis (108/100 ​μL), a low concentration of Staphylococcus aureus (105/100 ​μL), and a high concentration of S. aureus (108/100 ​μL), respectively. At four and eight weeks after surgery, the rabbits were sacrificed, and the tibias on the surgical side were analysed via histopathology, microcomputed tomography, and nanoindentation testing. Results: Histopathological analysis demonstrated that inflammatory responses and bacterial loads caused by high concentrations of staphylococcal infections, particularly coagulase-positive staphylococci, are more detrimental than low concentrations of bacterial infection and Ti-wear particles. Meanwhile, microcomputed tomography and nanoindentation testing showed that high concentrations of S. aureus caused the highest loss in bone mass and most biomechanical function impairment in rabbits experiencing aseptic inflammation and staphylococcal infections. Conclusions: Inflammatory osteolysis caused by a high concentration of coagulase-positive staphylococci is significantly associated with low bone mass and impaired biomechanical properties. The translational potential of this article: It is necessary to obtain an overall assessment of the bone mass and biomechanical properties before revision surgery, especially when S. aureus infection is involved. In addition, a better understanding of these two parameters might help develop a reasonable treatment regimen and reduce the risk of adverse events after revision surgery. Keywords: Biomechanical phenomena, Bone density, Bone resorption, Osteolysis, Staphylococcal infections

Implant infection is one of the most severe complications after orthopedic surgery. The construction of an antibacterial coating on orthopedic implants with release-killing or contact-killing is one of the most efficient strategies to prevent implant-related infections. Here we reported a hydroxypropyltrimethyl ammonium chloride chitosan (HACC) based multilayer modified plasma-sprayed porous titanium coating generated via the layer-by-layer covalent-immobilized method. We demonstrated that the multilayer coating inhibited the colonization and biofilm formation of several bacterial strains, including Staphylococcus aureus (ATCC 25923), methicillin-resistant Staphylococcus aureus (MSRA, ATCC 43300) and clinical isolates of methicillin-resistant Staphylococcus epidermidis (MRSE 287), in vitro. HACC in the multilayer was released slowly with the degradation of the coating under the action of collagenase, further killing the planktonic bacteria, while the remaining HACC could kill the colonized bacteria. In a rat model of femur implants, the HACC-based multilayer-modified TCs effectively controlled the infection caused by MRSA and prevented bone destruction. Therefore, the HACC-based multilayer modified TCs with multiple antimicrobial properties could be a new potential ideal surface modification strategy to prevent implant associated infections.

As technology continues to advance, implants are widely used in orthopedic surgery, such as the fixation of fractures, artificial joints, the reconstruction of the spine and the correction of skeletal deformities [...]

Distal femoral resection in total knee arthroplasty (TKA) is commonly performed using intramedullary jigs with a pre-operatively planned valgus cut angle (VCA). For valgus knees with lateral femoral condyle hypoplasia, the method of determining the accurate VCA has not been clarified. The aim of the present study is to introduce a method that can accurately determine the distal femoral VCA in the valgus knee arthroplasty. Twenty patients with valgus deformity caused by lateral femoral condylar hypoplasia underwent primary TKA with individually measured VCA. The VCA was defined as the acute crossing angle of the anatomical and mechanical axes of the femur on a pre-operative X-ray film, and the two axes almost always crossed at the distal femoral diaphysis, but not the centre of the knee as generally described. The entry point of the femoral intramedullary guide rod was determined on the extension of the femoral anatomical axis and was usually medial to the centre of the knee. According to the pre- and post-operative X-ray films, the mechanical lateral distal femoral angle (mLDFA), and coronal alignment of the femoral components were measured. The post-operative knee pain and function were evaluated using the Visual Analog Scale and Knee Society Score, respectively. The mean VCA measured according to the above method was 6.4° ± 1.0° (4.7–8.2°), and the femoral entry point was located at a mean distance of 7.4 ± 2.1 mm (4.5–10.9 mm) medial to the centre of the knee joint. The mean mLDFA before and after operation was 77.4° ± 5.7° (74–82°) and 88.4° ± 1.7° (86–90°), respectively, showing a statistically significant difference (P

Polyetheretherketone (PEEK) has gained increased attention as an orthopedic implant material in recent years. However, the adverse inflammatory and immunological reactions elicited by the PEEK surface result in inferior osteointegration and affect its clinical utility. To improve osseointegration and enhance the success rate of PEEK implants, the surface can be biofunctionalized to be immunomodulatory. Herein, A-485, a potent and selective catalytic inhibitor of p300/CBP, was loaded into mesoporous bioactive glass that had been immobilized on the PEEK surface, and the immunomodulatory capability and effects of the biofunctionalized PEEK implants on osteointegration were evaluated. In vitro, the biofunctionalized PEEK surface not only suppressed the acute inflammatory response of macrophages, but also inhibited bone resorption and promoted osteogenesis, indicating that it plays critical and complex roles in maintaining normal bone metabolism. Moreover, the biofunctionalized surface induced less fibrous capsule formation and exhibited superior osseointegration properties in vivo. The A-485-loaded MBG-biofunctionalized PEEK surface attenuated inappropriately activated immune responses mediated by macrophages and subsequently inhibited osteoclastogenesis, promoting osteogenesis and yielding superior osseointegration. Therefore, these surface-biofunctionalized PEEK implants, which regulate both immunity and bone metabolism, demonstrate strong potential for clinical application as orthopedic implants.

Covalent immobilisation of antimicrobial peptides on titanium (Ti) surfaces has been widely performed to inhibit bacterial adhesion and biofilm formation, however, it is unclear whether a spacer is necessary in this process for anti-bacteria adhesion, while still exhibiting good biocompatibility. We investigated the antibacterial properties and inflammatory response of Ti and PEGylated Ti with covalently immobilized KR-12 peptide on the surfaces. The KR-12 peptide was derived from LL-37, which is bactericidal and bacteriostatic in solution. Successful covalent immobilisation was determined by X-ray photoelectron spectrometry, contact angle measurements, and atomic force microscopy. KR-12 incorporation significantly decreased the bacterial adhesion to the Ti surface. Compared to the Ti-KR-12, the KR-12 immobilisation via a PEG spacer increased anti-microbial efficiency and reduced in vitro adhesion and biofilm formation of Staphylococcus epidermidis. Activated macrophages deprived from THP-1 cells with long filopodia were observed on the titanium surface, which was in agreement with increased TNF-α and IL-1β secretion. The KR-12-modified Ti surface and PEGylated Ti surface significantly decreased TNF-α and IL-1β secretion and macrophages remained in inactivated round states. The grafting of a PEG spacer prior to KR-12 immobilisation on the Ti surface improved the antibacterial properties and reduced macrophage activation, and may also decrease overall inflammatory responses. Thus, this approach showed the potential to biofunctionalize Ti for anti-bacteria adhesion and reducing macrophage inflammation.

KR-12-a5 is an analogue of the antimicrobial peptide KR-12. Both of these two agents can play key effects in the treatment of infections such as osteomyelitis. Our previous work demonstrated that the osteogenic differentiation of human bone marrow mesenchymal stem cells (HBMSCs) can be enhanced by KR-12. The present study investigated if KR-12-a5 could reverse the adverse effects of lipopolysaccharides (LPS) on HBMSC osteogenesis and the involved molecular mechanisms. We observed the proliferation, cell cycle, and apoptosis of HBMSCs in the presence of KR-12-a5 by a cell counting kit-8 assay and flow cytometry. The osteogenic differentiation of HBMSCs was studied by alkaline phosphatase, Alizarin Red staining, and quantitative assays. Osteogenic differentiation marker levels were detected using real-time quantitative PCR analysis, which demonstrated that KR-12-a5 treatment reversed the inhibition of osteogenesis. Western blot analysis indicated that LPS-activated P38 mitogen-activated protein kinase (MAPK) signaling was inhibited and BMP/Smad pathway was reactivated after KR-12-a5 treatment under induced osteogenic conditions. Furthermore, flow cytometry results demonstrated that KR-12-a5 relieved LPS-induced oxidative stress. Combining the LPS-treated mouse model results, we proved that KR-12-a5 reversed the adverse effects of LPS on HBMSC osteogenic differentiation by influencing the BMP/Smad and P38 MAPK signaling pathways.

Osteoporosis is one of the most common metabolic bone diseases among pre- and post-menopausal women. Despite numerous advances in the treatment of osteoporosis in recent years, the outcomes remain poor due to severe side effects. In this study, we investigated whether A-485, a highly selective catalytic p300/CBP inhibitor, could attenuate RANKL-induced osteoclast differentiation and explored the underlying molecular mechanisms. The protective role of A-485 in osteoporosis was verified using a mouse model of ovariectomy (OVX)-induced bone loss and micro-CT scanning. A-485 inhibited RANKL-induced osteoclast differentiation in vitro by reducing the number of tartrate-resistant acid phosphatase-positive osteoclasts without inducing significant cytotoxicity. In particular, A-485 dose-dependently disrupted F-actin ring formation and downregulated the expression of genes associated with osteoclast differentiation, such as CTSK, c-Fos, TRAF6, VATPs-d2, DC-STAMP, and NFATc1, in a time- and dose-dependent manner. Moreover, A-485 inhibited the RANKL-induced phosphorylation of MAPK pathways and attenuated OVX-induced bone loss in the mouse model while rescuing the loss of bone mineral density. Our in vitro and in vivo findings suggest for the first time that A-485 has the potential to prevent postmenopausal osteoporosis and could therefore be considered as a therapeutic molecule against osteoporosis.

Infection and poor bone-implant integration are the two main reasons for titanium (Ti) implant failure. Here, we investigated the feasibility of functionalizing Ti with the antimicrobial peptide, KR-12, derived from the human cationic antimicrobial peptide. The minimal inhibitory concentration and cell viability effects of KR-12 were investigated prior to immobilization on the Ti surface. The results showed that KR-12 possessed a wide anti-bacterial spectrum with no cytotoxicity to human bone marrow mesenchymal stem cells (hBMSCs). Successful covalent immobilization of KR-12 onto an amine-functionalized Ti (Ti-KR-12) surface was characterized by X-ray photoelectron spectroscopy. Gram-positive bacteria, Staphylococcus epidermidis and methicillin resistant Staphylococcus epidermidis were employed for antibacterial characterization. Ti-KR-12 substrates could significantly inhibit adhesion and colonization of common pathogenic bacteria and the drug resistance of pathogenic bacteria. The results of the CCK-8 assay, confocal laser scanning microscopy, and scanning electron microscopy showed that KR-12 covalently immobilized on Ti improved adhesion and proliferation of hBMSCs. The osteogenic differentiation of hBMSCs on samples was investigated by alkaline phosphatase staining, sirius red staining, alizarin red staining, and real-time PCR. The staining and real-time PCR results demonstrated that hBMSCs grown on Ti-KR-12 surfaces for 10 and 14 days under conditions inducing osteogenic differentiation displayed significantly higher alkaline phosphatase activity, larger extracellular matrix calcium deposition area, and higher expression of alkaline phosphatase, osteocalcin, osteopontin, and collagen type-1 mRNA than bare Ti. Our results demonstrated the KR-12 peptide was suitable for improving the biological properties of bioinert titanium. KR-12 showed antibacterial activity and the capability to promote cell proliferation and Ti-KR-12 surfaces significantly decreased bacteria adhesion, whilst promoting the osteogenic differentiation of hBMSCs.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are one of the most serious surgery complications, and their prevention is of utmost importance. Flufenamic acid is a non-steroid anti-inflammatory drug approved for clinical use to relieve inflammation and pain in rheumatoid arthritis patients. In this study, we explored the antibacterial efficacy of flufenamic acid and the mechanisms underlying this effect. By using minimal inhibitory concentration (MIC), time-kill, resistance induction assays, and the antibiotic synergy test, we demonstrated that flufenamic acid inhibited the growth of methicillin-resistant staphylococci and did not induce resistance when it was used at the MIC. Furthermore, flufenamic acid acted synergistically with the beta-lactam antibiotic oxacillin and did not show significant toxicity toward mammalian cells. The biofilm inhibition assay revealed that flufenamic acid could prevent biofilm formation on medical implants and destroy the ultrastructure of the bacterial cell wall. RNA sequencing and quantitative RT-PCR indicated that flufenamic acid inhibited the expression of genes associated with peptidoglycan biosynthesis, beta-lactam resistance, quorum sensing, and biofilm formation. Furthermore, flufenamic acid efficiently ameliorated a local infection caused by MRSA in mice. In conclusion, flufenamic acid may be a potent therapeutic compound against MRSA infections and a promising candidate for antimicrobial coating of implants and surgical devices.

Postmenopausal osteoporosis is one of the most common types of osteoporosis resulting from estrogen deficiency in elderly women. In addition, hypertension is another common disease in the elderly, and it has become an independent risk factor for osteoporosis and osteoporotic fractures. Here, we report for the first time that felodipine, a first-line antihypertensive agent, significantly prevents postmenopausal osteoporosis in addition to its vasodilation properties. Quantitative RT-PCR analysis revealed that treatment with felodipine significantly downregulated the genes associated with osteoclast differentiation. RNA-sequencing and western blotting suggested that felodipine could inhibit bone resorption by suppressing MAPK pathway phosphorylation. Moreover, micro-CT scanning and histological analysis in an ovariectomy (OVX)-induced bone-loss mouse model indicated that felodipine might be a potent drug for preventing osteoporotic fractures. Therefore, this study proposes an attractive and promising agent with vasodilation properties to treat postmenopausal osteoporosis.

Bacitracin, a widely used metallopeptide antibiotic, has been reported to be locally used in treating wounds without systemic adverse reactions. Our preliminary study showed that bacitracin might enhance the osteogenic differentiation of human bone marrow mesenchymal stem cells (HBMSCs). The present study investigated whether bacitracin affects the osteogenic differentiation of HBMSCs and the molecular mechanisms involved. The proliferation of HBMSCs in the presence of bacitracin was examined using a cell counting kit-8 (CCK-8) assay. The effects of bacitracin on the cell cycle and apoptosis of HBMSCs were observed using flow cytometry assay. Staining and quantitative assays for alkaline phosphatase (ALP) staining, collagen deposition (Sirius Red), and mineralization (Alizarin Red) were used to study osteogenic differentiation of HBMSCs. The expression of osteogenic differentiation markers was detected using quantitative reverse transcription polymerase chain reaction (RT-qPCR) analyses. The activation of related signaling pathways was examined using a luciferase reporter assay and western blotting. Bacitracin treatment increased osteogenic differentiation of HBMSCs without cytotoxicity and did not adversely affect cell cycle progression or apoptosis. The luciferase reporter assay showed that bacitracin activated the transcription of bone morphogenetic protein-2 (BMP2) gene, a key gene in the BMP2/Smad signaling axis. Western blotting indicated that this axis was markedly activated by bacitracin stimulation of osteogenesis. Moreover, the activation of Smad phosphorylation and osteogenic differentiation by bacitracin was inhibited by a transforming growth factor (TGF)-β/Smad inhibitor (LDN-193189 HCl) and small interfering RNA (siRNA) gene silencing (si-BMP2). In conclusion, our results suggest that bacitracin can promote osteogenesis of HBMSCs by activating the BMP2/Smad signaling axis.

Infection is one of the most important causes of titanium implant failure in vivo . A developing prophylactic method involves the immobilization of antibiotics, especially vancomycin, onto the surface of the titanium implant. However, these methods have a limited effect in curbing multiple bacterial infections due to antibiotic specificity. In the current study, enoxacin was covalently bound to an amine-functionalized Ti surface by use of a polyethylene glycol (PEG) spacer, and the bactericidal effectiveness was investigated in vitro and in vivo . The titanium surface was amine functionalized with 3-aminopropyltriethoxysilane (APTES), through which PEG spacer molecules were covalently immobilized onto the titanium, and then the enoxacin was covalently bound to the PEG, which was confirmed by X-ray photoelectron spectrometry (XPS). A spread plate assay, confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM) were used to characterize the antimicrobial activity. For the in vivo study, Ti implants were inoculated with methicillin-resistant Staphylococcus aureus (MRSA) and implanted into the femoral medullary cavity of rats. The degree of infection was assessed by radiography, micro-computed tomography, and determination of the counts of adherent bacteria 3 weeks after surgery. Our data demonstrate that the enoxacin-modified PEGylated Ti surface effectively prevented bacterial colonization without compromising cell viability, adhesion, or proliferation in vitro . Furthermore, it prevented MRSA infection of the Ti implants in vivo . Taken together, our results demonstrate that the use of enoxacin-modified Ti is a potential approach to the alleviation of infections of Ti implants by multiple bacterial species.

Bacitracin immobilized on the titanium (Ti) surface significantly improves anti-bacterial activity and biocompatibility in vitro. In the current study, we investigated the biologic performance (bactericidal effect and bone-implant integration) of bacitracin-modified Ti in vivo. A rat osteomyelitis model with femoral medullary cavity placement of Ti rods was employed to analyze the prophylactic effect of bacitracin-modified Ti (Ti-BC). Thirty-six female Sprague Dawley (SD) rats were used to establish the Ti implant-associated infection. The Ti and Ti-BC rods were incubated with and without Staphylococcus aureus to mimic the contaminated Ti rod and were implanted into the medullary cavity of the left femur, and sterile Ti rods were used as the blank control. After 3 weeks, the bone pathology was evaluated using X-ray and micro-computed tomography (micro-CT) analysis. For the investigation of the Ti-BC implant osseointegration in vivo, fifteen SD rats were divided into three groups (N=5), namely Ti, Ti-dopamine immobilized (Ti-DOPA), and Ti-BC. Ti rods were implanted into the left femoral cavity and micro-CT and histological evaluation was conducted after 12 weeks. The in vivo study indicated that Ti-immobilized bacitracin owned the prophylaxis potential for the infection associated with the Ti implants and allowed for the osseointegration. Thus, the multiple biofunctionalized Ti implants could be realized via immobilization of bacitracin, making them promising candidates for preventing the Ti implant-associated infections while retaining the osseointegration effects.