Identification of key candidate genes and pathways in rheumatoid arthritis and osteoarthritis by integrated bioinformatical analysis.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are the most common joint disorders. Although they have shown analogous clinical manifestations, the pathogenesis of RA and OA are different. In this study, we used the online Gene Expression Omnibus (GEO) microarray expression profiling dataset GSE153015 to identify gene signatures between RA and OA joints. The relevant data on 8 subjects obtained from large joints of RA patients (RA-LJ), 8 subjects obtained from small joints of RA patients (RA-SJ), and 4 subjects with OA were investigated. Differentially expressed genes (DEGs) were screened. Functional enrichment analysis of DEGs including the Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified, which were mainly associated with T cell activation or chemokine activity. Besides, protein-protein interaction (PPI) network analysis was performed, and key modules were identified. Hub genes of RA-LJ and OA groups were screened, they were CD8A, GZMB, CCL5, CD2, and CXCL9, whereas CD8A, CD2, IL7R, CD27, and GZMB were hub genes of RA-SJ and OA group. The novel DEGs and functional pathways between RA and OA identified in this study may provide new insight into the underlying molecular mechanisms and therapeutic strategies of RA and OA.

Approaches to Biofunctionalize Polyetheretherketone for Antibacterial: A Review.

Due to excellent mechanical properties and similar elastic modulus compared with human cortical bone, polyetheretherketone (PEEK) has become one of the most promising orthopedic implant materials. However, implant-associated infections (IAIs) remain a challenging issue since PEEK is bio-inert. In order to fabricate an antibacterial bio-functional surface, modifications of PEEK had been widely investigated. This review summarizes the modification strategies to biofunctionalize PEEK for antibacterial. We will begin with reviewing different approaches, such as surface-coating modifications and controlled release of antimicrobials. Furthermore, blending modifications and 3D printing technology were discussed. Finally, we compare the effects among different approaches. We aimed to provide an in-depth understanding of the antibacterial modification and optimize the design of the PEEK orthopedic implant.

Bone infection site targeting nanoparticle-antibiotics delivery vehicle to enhance treatment efficacy of orthopedic implant related infection.

Orthopedic implants account for 99% of orthopedic surgeries, however, orthopedic implant-related infection is one of the most serious complications owing to the potential for limb-threatening sequelae and mortality. Current antibiotic treatments still lack the capacity to target bone infection sites, thereby resulting in unsatisfactory therapeutic effects. Here, the bone infection site targeting efficacy of D6 and UBI29-41 peptides was investigated, and bone-and-bacteria dual-targeted nanoparticles (NPs) with D6 and UBI29-41 peptides were first fabricated to target bone infection site and control the release of vancomycin in bone infection site. The results of this study demonstrated that the bone-and-bacteria dual-targeted mesoporous silica NPs exhibit excellent bone and bacteria targeting efficacy, excellent biocompatibility and effective antibacterial properties in vitro. Furthermore, in a rat model of orthopedic implant-related infection with methicillin-resistant Staphylococcus aureus, the growth of bacteria was evidently inhibited without cytotoxicity, thus realizing the early treatment of implant-related infection. Hence, the bone-and-bacteria dual-targeted molecule-modified NPs may target bacteria-infected bone sites and act as ideal candidates for the therapy of orthopedic implant-related infections.

p300/CBP inhibitor A-485 inhibits the differentiation of osteoclasts and protects against osteoporotic bone loss.

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.

Hydrofluoric acid and nitric acid cotreatment for biofunctionalization of polyetheretherketone in M2 macrophage polarization and osteogenesis.

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.

Overcoming Planktonic and Intracellular Staphylococcus aureus-Associated Infection with a Cell-Penetrating Peptide-Conjugated Antimicrobial Peptide.

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-1ß expression and secretion from LPS-stimulated RAW264.7 cells. In the subcutaneous infection mouse model of planktonic and intracellular infections, the growth of S. aureusin 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.

Antibacterial and antibiofilm effects of flufenamic acid against methicillin-resistant Staphylococcus aureus.

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.

Trends in Bone Mineral Density, Osteoporosis, and Osteopenia Among U.S. Adults With Prediabetes, 2005-2014.

OBJECTIVE: We aimed to evaluate trends in bone mineral density (BMD) and the prevalence of osteoporosis/osteopenia in U.S. adults with prediabetes and normal glucose regulation (NGR) and further investigate the association among prediabetes, osteopenia/osteoporosis, and fracture. RESEARCH DESIGN AND METHODS: We collected and analyzed data from the U.S. National Health and Nutrition Examination Surveys during the period from 2005 to 2014. Femoral neck and lumbar spine BMD data were available for 5,310 adults with prediabetes and 5,162 adults with NGR >40 years old. RESULTS: A shift was observed toward a lower BMD and a higher prevalence of osteopenia/osteoporosis at the femoral neck and lumbar spine in U.S. adults >40 years old with prediabetes since 2005, especially in men <60 and women ≥60 years old. A shift toward a higher prevalence of osteopenia/osteoporosis at the femoral neck was also observed in adults >40 years old with NGR. Moreover, prediabetes was associated with a higher prevalence of hip fracture, although participants with prediabetes had higher BMD and a lower prevalence of osteopenia/osteoporosis at the femoral neck. CONCLUSIONS: There was a declining trend in BMD from 2005 to 2014 in U.S. adults >40 years old with prediabetes and NGR, and this trend was more significant in men <60 years old. Populations with prediabetes may be exposed to relatively higher BMD but a higher prevalence of fracture.

Effects of staphylococcal infection and aseptic inflammation on bone mass and biomechanical properties in a rabbit model.

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 â€‹µL), titanium (Ti)-wear particles (300 µg/100 â€‹µL), a low concentration of Staphylococcus epidermidis (105/100 â€‹µL), a high concentration of S. epidermidis (108/100 â€‹µL), a low concentration of Staphylococcus aureus (105/100 â€‹µL), and a high concentration of S. aureus (108/100 â€‹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.

Flufenamic acid inhibits osteoclast formation and bone resorption and act against estrogen-dependent bone loss in mice.

Postmenopausal osteoporosis is one of the most common types of osteoporosis resulting from estrogen deficiency in elderly women. Nonsteroidal anti-inflammatory drugs (NSAIDs) are important drugs for pain relief in patients with osteoporosis. In this study, we report for the first time that flufenamic acid, a clinically approved and widely used NSAID, not only has analgesic properties but also shows a significant effect in terms of preventing postmenopausal osteoporosis. Quantitative RT-PCR analysis showed that treatment with flufenamic acid significantly downregulated the genes associated with osteoclast differentiation. Meanwhile, RNA-sequencing and western blot analyses suggested that flufenamic acid could inhibit the bone resorption by suppressing the phosphorylation of MAPK pathways. Moreover, an ovariectomy (OVX)-induced bone-loss mouse model indicated that flufenamic acid might be a potent drug for preventing osteoporotic fractures, as verified by micro-CT scanning and histological analysis. Therefore, this study proposes an attractive and potent drug with analgesic properties for the prevention of postmenopausal osteoporosis.

Felodipine blocks osteoclast differentiation and ameliorates estrogen-dependent bone loss in mice by modulating p38 signaling pathway.

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.

KR-12-a5 Reverses Adverse Effects of Lipopolysaccharides on HBMSC Osteogenic Differentiation by Influencing BMP/Smad and P38 MAPK Signaling Pathways.

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.

Improved antibacterial properties of collagen I/hyaluronic acid/quaternized chitosan multilayer modified titanium coatings with both contact-killing and release-killing functions.

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.

Immobilization of type I collagen/hyaluronic acid multilayer coating on enoxacin loaded titania nanotubes for improved osteogenesis and osseointegration in ovariectomized rats.

Titania nanotubes (Ti-NTs) have been proven to be good drug carriers and can release drugs efficiently around implants. Enoxacin (EN) is a broad-spectrum antibiotic that has the ability of anti-osteoclastogenesis. Immobilization of extracellular matrix components on the surface of the material can greatly enhance the biological activity of the implant and slow down the release rate of the drug in Ti-NTs. In the present study, a material system that provided uniform drug release, promoted osteogenesis, and inhibited osteoclast was designed and developed. Scanning electron microscopy, X-ray photoelectron spectroscopy, and water contact angle measurements were used for material surface characterization. Enoxacin release was detected by high performance liquid chromatography. Alkaline phosphatase and Alizarin Red staining were used to evaluate the osteogenic differentiation of rat bone marrow mesenchymal stem cells. Tartrate-resistant acid phosphatase staining and bone absorption assay were applied to osteoclastogenesis experiments. A drug delivery system based on Ti-NTs and type I collagen /hyaluronic acid multilayer coating (Ti-NT+EN+Col/HyA) with predominant biocompatibility, osteogenic property, and anti-osteoclastogenesis ability was successfully constructed. These excellent biological properties were further validated in an ovariectomized rat model. The results of the study indicate that Ti-NT+EN+Col/HyA is a potential material for future orthopedic implants.

Bacitracin promotes osteogenic differentiation of human bone marrow mesenchymal stem cells by stimulating the bone morphogenetic protein-2/Smad axis.

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)-ß/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.

The antimicrobial peptide KR-12 promotes the osteogenic differentiation of human bone marrow stem cells by stimulating BMP/SMAD signaling.

KR-12 is the smallest fragment of human antimicrobial peptide cathelicidin (LL-37), and could play key roles in the treatment of multiple infections, including osteomyelitis. Our preliminary work found that KR-12 enhances the osteogenic differentiation of human bone marrow mesenchymal stem cells (HBMSCs). The present study investigated whether KR-12 affects HBMSC osteogenic differentiation, as well as the molecular mechanisms involved. HBMSC proliferation in the presence of KR-12 was observed with a cell counting 8 assay, and its effects on HBMSC cell cycle progression and apoptosis were examined by flow cytometry. Alkaline phosphatase, Sirius Red, and Alizarin Red staining and quantitative assays were used to study the osteogenic differentiation of HBMSCs. The expression of osteogenic differentiation markers was detected by real-time quantitative PCR analysis. The activation of potentially related pathways was examined by luciferase reporter assay and western blot analysis. KR-12 treatment increased the osteogenic differentiation of HBMSCs without cytotoxicity and did not influence the cell cycle or induce apoptosis. Luciferase reporter assays showed that KR-12 activated the transcription of bone morphogenetic protein 2 (BMP2), a key gene in the BMP/SMAD pathway. Western blot analysis indicated that BMP/SMAD signaling was markedly activated by KR-12 stimulation in osteogenic induction conditions. SMAD phosphorylation was activated by KR-12 treatment, and was inhibited by both a transforming growth factor-ß/SMAD inhibitor (LDN-193189 HCL) and BMP2 small interfering RNA (si-BMP2). LDN-193189 HCL and si-BMP2 treatment also abolished the KR-12-induced osteogenic differentiation of HBMSCs. In conclusion, our results suggest that KR-12 promotes HBMSC osteogenesis through the activation of BMP/SMAD signaling.

Immobilizing bacitracin on titanium for prophylaxis of infections and for improving osteoinductivity: An in vivo study.

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.