Single cell transcriptome profiling of infrapatellar fat pad highlights the role of interstitial inflammatory fibroblasts in osteoarthritis.

OBJECTIVES: Osteoarthritis (OA) is a whole-joint disease in which the role of the infrapatellar fat pad (IFP) in its pathogenesis is unclear. Our study explored the cellular heterogeneity of IFP to understand OA and identify therapeutic targets. METHODS: Single-cell and single-nuclei RNA sequencing were used to analyze 10 IFP samples, comprising 5 from OA patients and 5 from healthy controls. Analyses included differential gene expression, enrichment, pseudotime trajectory, and cellular communication, along with comparative studies with visceral and subcutaneous fats. Key subcluster and pathways were validated using multiplex immunohistochemistry. RESULTS: The scRNA-seq performed on the IFPs of the OA and control group profiled the gene expressions of over 49,674 cells belonging to 11 major cell types. We discovered that adipose stem and progenitor cells (ASPCs), contributing to the formation of both adipocytes and synovial-lining fibroblasts (SLF). Interstitial inflammatory fibroblasts (iiFBs) were a subcluster of ASPCs that exhibit notable pro-inflammatory and proliferative characteristics. We identified four adipocyte subtypes, with one subtype showing a reduced lipid synthesis ability. Furthermore, iiFBs modulated the activities of macrophages and T cells in the IFP. Compared to subcutaneous and visceral adipose tissues, iiFBs represented a distinctive subpopulation of ASPCs in IFP that regulated cartilage proliferation through the MK pathway. CONCLUSION: This study presents a comprehensive single-cell transcriptomic atlas of IFP, uncovering its complex cellular landscape and potential impact on OA progression. Our findings highlight the role of iiFBs in OA, especially through MK pathway, opening new avenues for understanding OA pathogenesis and developing novel targeted therapies.

CXCL chemokines-mediated communication between macrophages and BMSCs on titanium surface promotes osteogenesis via the actin cytoskeleton pathway.

The refined functional cell subtypes in the immune microenvironment of specific titanium (Ti) surface and their collaborative role in promoting bone marrow mesenchymal stem cells (BMSCs) driven bone integration need to be comprehensively characterized. This study employed a simplified co-culture system to investigate the dynamic, temporal crosstalk between macrophages and BMSCs on the Ti surface. The M2-like sub-phenotype of macrophages, characterized by secretion of CXCL chemokines, emerges as a crucial mediator for promoting BMSC osteogenic differentiation and bone integration in the Ti surface microenvironment. Importantly, these two cells maintain their distinct functional phenotypes through a mutually regulatory interplay. The secretion of CXCL3, CXCL6, and CXCL14 by M2-like macrophages plays a pivotal role. The process activates CXCR2 and CCR1 receptors, triggering downstream regulatory effects on the actin cytoskeleton pathway within BMSCs, ultimately fostering osteogenic differentiation. Reciprocally, BMSCs secrete pleiotrophin (PTN), a key player in regulating macrophage differentiation. This secretion maintains the M2-like phenotype via the Sdc3 receptor-mediated cell adhesion molecules pathway. Our findings provide a novel insight into the intricate communication and mutual regulatory mechanisms operating between BMSCs and macrophages on the Ti surface, highlight specific molecular events governing cell-cell interactions in the osteointegration, inform the surface design of orthopedic implants, and advance our understanding of osteointegration.

Using a degradable three-layer sandwich-type coating to prevent titanium implant infection with the combined efficient bactericidal ability and fast immune remodeling property.

Titanium (Ti) implant-associated infections are a challenge in orthopedic surgery, for which a series of antibacterial coatings have been designed and fabricated to reduce the risk of bacterial contamination. Herein, we created a degradable three-layer sandwich-type coating to achieve long-term antibacterial effects while simultaneously reconstructing the local immune microenvironment. The vancomycin (Van)-loaded vaterite coating constitutes the outer and inner layers, whereas Interleukin-12 (IL-12)-containing liposomes embedded in sodium alginate constitutes the middle layer. Van, released from the vaterite, demonstrated a favorable and rapid bactericidal ability against the representative methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains. The released IL-12 exhibited the desired immune reconstitution abilities, actively facilitating defenses against subsequent bacterial invasions. Furthermore, the biocompatibility and cell-binding feature of the multifunctional coating was beneficial for achieving solid interface intergradation. Overall, the benefits of the three-layer sandwich-type coating, including the convenient fabrication process, efficient antimicrobial activity, fast immune remodeling property, fine cell-binding feature, and biodegradability, highlight its promising translational potential in preventing implant infection. STATEMENT OF SIGNIFICANCE: To prevent titanium implant infections, researchers have designed various antibacterial coatings. However, most of these coatings focused only on killing the invading bacteria over a limited postoperative period. However, the local immune microenvironment is compromised during surgery. Local immune deflection impedes the ability of the local immune defenses to clear bacteria and limits immune memory building from active defense against long-term subsequent bacterial invasions. Furthermore, these coatings are usually nondegradable and differ substantially from bone components, thereby impairing the integration of the coating and bone interface and generating concerns about implant stability and bacterial contamination. In this work, we synthesized a degradable coating that provides sustained antibacterial activity, promotes immune reconstitution, and simultaneously achieves solid bone integration.

A self-assembled bilayer polypeptide-engineered hydrogel for spatiotemporal modulation of bactericidal and anti-inflammation process in osteomyelitis treatment.

BACKGROUND: Drug resistance of pathogens and immunosuppression are the main causes of clinical stagnation of osteomyelitis. The ideal treatment strategy for osteomyelitis is to achieve both efficient antibacterial and bone healing through spatiotemporal modulation of immune microenvironment. METHODS: In this study, a bilayer hydrogel based on genetically engineered polypeptide AC10A and AC10ARGD was prepared by self-assembly. Ag2S QDs@DSPE-mPEG2000-Ce6/Aptamer (AD-Ce6/Apt) was loaded in the top layer AC10A hydrogel (AA) for antibacterial, and bone marrow-derived mesenchymal stem cells (BMSCs) were loaded in the lower layer AC10ARGD hydrogel (MAR) for bone healing. The AD-Ce6/Apt can be released from the AA hydrogel to target S. aureus before bacterial biofilm formation and achieved significant bactericidal effect under irradiation with a 660 nm laser. Moreover, AD-Ce6/Apt can induce M1 type polarization of macrophages to activate the immune system and eliminate residual bacteria. Subsequently, BMSCs released from the MAR hydrogel can differentiate into osteoblasts and promote the formation of an anti-inflammatory microenvironment by regulating the M2 type polarization of macrophages. The bilayer AA-MAR hydrogel possessed good biocompatibility. RESULTS: The in vitro and in vivo results showed that the AA-MAR hydrogel not only realized efficient photodynamic therapy of S. aureus infection, but also promoted the transformation of immune microenvironment to fulfill the different needs of each stage, which ultimately improved bone regeneration and mechanical properties post-surgery. CONCLUSION: This work presents an approach for spatiotemporal modulation of immune microenvironment in the treatment of osteomyelitis.

Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Promotes Inflammation and Accelerates Osteoarthritis by Activating ß-Catenin.

Osteoarthritis (OA) is a chronic articular disease characterized by cartilage degradation, subchondral bone remodeling and osteophyte formation. Src homology 2 domain-containing protein tyrosine phosphatase (SHP2) has not been fully investigated in the pathogenesis of OA. In this study, we found that SHP2 expression was significantly increased after interleukin-1ß (IL-1ß) treatment in primary mouse chondrocytes. Inhibition of SHP2 using siRNA reduced MMP3, MMP13 levels, but increased AGGRECAN, COL2A1, SOX9 expression in vitro. On the contrary, overexpression of SHP2 exerted the opposite results and promoted cartilage degradation. Mechanistically, SHP2 activated Wnt/ß-catenin signaling possibly through directly binding to ß-catenin. SHP2 also induced inflammation through activating Mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways. Our in vivo studies showed that SHP2 knockdown effectively delayed cartilage destruction and reduced osteophyte formation in the mouse model of OA induced by destabilization of the medial meniscus (DMM). Altogether, our study identifies that SHP2 is a novel and potential therapeutic target of OA.

Two reactive behaviors of chondrocytes in an IL-1ß-induced inflammatory environment revealed by the single-cell RNA sequencing.

OBJECTIVE: To investigate the heterogeneous responses of in vitro expanded chondrocytes, which were cultured in an interleukin (IL)-1ß -induced inflammatory environment. METHOD: Human articular chondrocytes were expanded, in vitro, for 13 days and treated with IL-1ß for 0, 24, and 48 h. Cells were collected and subjected to single-cell RNA sequencing. Multiple bioinformatics tools were used to determine the signatures that define chondrocyte physiology. RESULTS: Two major cell clusters with distinct expression patterns were identified at the initial phase and were with heterogeneous variation that coincides with inflammation progress. They transformed into two terminal cell clusters one of which exhibited OA-phenotype and proinflammatory characteristics through two paths, "response-to-inflammation" and "atypical response-to-inflammation", respectively. The involved cell clusters exhibited intrinsic relationship with cell types within native cartilage from OA patients. Genes controlling cell transformation to OA-phenotype were relating to the tumor necrosis factor (TNF) signaling pathway via NFKB, up-regulated KRAS signaling and the IL2/STAT5 signaling pathway and pathways relating to apoptosis and reactive oxygen species. CONCLUSION: The in vitro expanded chondrocytes under IL-1ß-induced inflammatory progression behave heterogeneously. One of the initial cell clusters could transform into a proinflammatory subpopulation through a termed response-to-inflammation path, which may serve as the core target to alleviate OA progression.

Sprayable hydrogel dressing accelerates wound healing with combined reactive oxygen species-scavenging and antibacterial abilities.

Wound management poses a considerable economic burden on the global healthcare system, considering the impacts of wound infection, delayed healing and scar formation. To this end, multifunctional dressings based on hydrogels have been developed to stimulate skin healing. Herein, we describe the design, fabrication, and characterization of a sprayable hydrogel-based wound dressing loaded with cerium oxide nanoparticles (CeONs) and an antimicrobial peptide (AMP), for combined reactive oxygen species (ROS)-scavenging and antibacterial properties. We adopted a mussel-inspired strategy to chemically conjugate gelatin with dopamine motifs and prepared a hydrogel dressing with improved binding affinity to wet skin surfaces. Additionally, the release of AMP from the hydrogel demonstrated rapid release ablation and contact ablation against four representative bacterial strains, confirming the desired antimicrobial activities. Moreover, the CeONs-loaded hydrogel dressing exhibited favorable ROS-scavenging abilities. The biocompatibility of the multifunctional hydrogel dressing was further proven in vitro by culturing with HaCaT cells. Overall, the benefits of the developed hydrogel wound dressing, including sprayability, adhesiveness, antimicrobial activity, as well as ROS-scavenging and skin-remodeling ability, highlight its promissing translational potentials in wound management. STATEMENT OF SIGNIFICANCE: Various hydrogel-based wound-dressing materials have been developed to stimulate wound healing. However, from the clinical perspective, few of the current wound dressings meet all the intended multifunctional requirements of preventing infection, promoting rapid wound closure, and minimizing scar formation, while simultaneously offering the convenience of application. In the current study, we adopted a mussel-inspired strategy to functionalize the GelMA hydrogels with DOPA to fabricate GelMA-DOPA hydrogel which exhibited an enhanced binding affinity for wound surfaces, AMP HHC-36 and CeONs are further encapsulated into the GelMA-DOPA hydrogel to confer the hydrogel wound dressing with antimicrobial and ROS-scavenging abilities. The GelMA-DOPA-AMP-CeONs dressing offered the benefits of sprayability, adhesiveness, antimicrobial activity, as well as ROS-scavenging and skin-remodeling ability, which might address the therapeutic and economic burdens associated with chronic wound treatment and management.

Improved Immunoregulation of Ultra-Low-Dose Silver Nanoparticle-Loaded TiO2 Nanotubes via M2 Macrophage Polarization by Regulating GLUT1 and Autophagy.

INTRODUCTION: The bone regeneration of endosseous implanted biomaterials is often impaired by the host immune response, especially macrophage-related inflammation which plays an important role in the bone healing process. Thus, it is a promising strategy to design an osteo-immunomodulatory biomaterial to take advantage of the macrophage-related immune response and improve the osseointegration performance of the implant. METHODS: In this study, we developed an antibacterial silver nanoparticle-loaded TiO2 nanotubes (Ag@TiO2-NTs) using an electrochemical anodization method to make the surface modification and investigated the influences of Ag@TiO2-NTs on the macrophage polarization, osteo-immune microenvironment as well as its potential molecular mechanisms in vitro and in vivo. RESULTS: The results showed that Ag@TiO2-NTs with controlled releasing of ultra-low-dose Ag+ ions had the excellent ability to induce the macrophage polarization towards the M2 phenotype and create a suitable osteo-immune microenvironment in vitro, via inhibiting PI3K/Akt, suppressing the downstream effector GLUT1, and activating autophagy. Moreover, Ag@TiO2-NTs surface could improve bone formation, suppress inflammation, and promote osteo-immune microenvironment compared to the TiO2-NTs and polished Ti surfaces in vivo. These findings suggested that Ag@TiO2-NTs with controlled releasing of ultra-low-dose Ag+ ions could not only inhibit the inflammation process but also promote the bone healing by inducing healing-associated M2 polarization. DISCUSSION: Using this surface modification strategy to modulate the macrophage-related immune response, rather than prevent the host response, maybe a promising strategy for implant surgeries in the future.

Pristimerin Suppresses RANKL-Induced Osteoclastogenesis and Ameliorates Ovariectomy-Induced Bone Loss.

Osteoporosis is characterized by bone loss and destruction of trabecular architecture, which greatly increases the burden on the healthcare system. Excessive activation of osteoclasts is an important cause of osteoporosis, and suppression of osteoclastogenesis is helpful for the treatment of osteoporosis. Pristimerin, a natural compound, possesses numerous pharmacological effects via inactivating the NF-κB and MAPK pathways, which are closely related to osteoclastogenesis process. However, the relationship between Pristimerin and osteoclastogenesis requires further investigation. In this research, we examined the effect of Pristimerin on osteoclastogenesis and investigated the related mechanisms. Our results showed Pristimerin inhibited RANKL-induced osteoclast differentiation and osteoclastic bone resorption in vitro, with decreased expression of osteoclastogenesis-related markers including c-Fos, NFATc1, TRAP, Cathepsin K, and MMP-9 at both mRNA and protein levels. Furthermore, Pristimerin suppressed NF-κB and MAPK signaling pathways, reduced reactive oxygen species (ROS) production and activated the nuclear factor erythroid 2-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signaling during osteoclastogenesis. Our in vivo experiments showed that Pristimerin remarkably ameliorated ovariectomy-induced bone loss, reduced serum levels of TNF-α, IL-1ß, IL-6, and RANKL, and increased serum level of osteoprotegerin (OPG). Therefore, our research indicated that Pristimerin is a potential chemical for the treatment of osteoporosis.

Enhanced performance of vertical-structured InGaN micro-pixelated light-emitting-diode array fabricated using an ion implantation process.

In this Letter, a new approach to fabricating a high-efficiency vertical-structured InGaN micro-pixelated light-emitting diode (µVLED) is presented. The high-resistivity selective areas are intentionally created in the n-GaN layer through a fluorine (F) ion-implantation process and then used as the electrical isolation regions for realizing a µVLED array consisting of 25×25 pixels with a diameter of 10 µm. The results prove that the dual-energy F- ion implantations not only can improve the uniformity of carrier distribution but also can effectively prevent current from leaking along the etched sidewalls, which in turn realize a more efficient carrier injection into the mesa area. More notably, the current-handling capability and corresponding optical output power density of the µVLED array are substantially higher than those of conventional vertical-structured broad-area LEDs. A measured output light power density of the F- ion-implanted µVLED array reaches a maximum value of 43 W cm-2 at 3.06 kA cm-2, before power saturation. The improved luminescence performances of the µVLED array can be attributed to an effective ion-induced heat relaxation and associated lower junction temperature.

Long-lasting bactericidal activity through selective physical puncture and controlled ions release of polydopamine and silver nanoparticles-loaded TiO2 nanorods in vitro and in vivo.

Background: Titanium (Ti) implant-associated infection, which is mostly caused by bacterial adhesion and biofilm formation, may result in implant failure and secondary surgery. Thus it is an urgent issue to prevent bacterial infections at the earliest step. Purpose: To develop a novel surface strategy of polydopamine (PDA) and silver (Ag) nanoparticle-loaded TiO2 nanorods (NRDs) coatings on Ti alloy. Materials and methods: Ag-TiO2@PDA NRDs was fabricated on Ti alloy by hydrothermal synthesis. The antibacterial activity of Ag-TiO2@PDA NRDs against Escherichia coli and methicillin-resistant Staphylococcus aureus were tested by FE-SEM, Live/Dead staining, zone of inhibition, bacteria counting method and protein leakage analysis in vitro. In addition, an implant infection model was conducted and the samples were tested by X-ray, Micro-CT and histological analysis in vivo. Besides, cell morphology and cytotoxicity of Mouse calvarial cells (MC3T3-E1) were characterized by FE-SEM, immunofluorescence and CCK-8 test in vitro. Results: Our study successfully developed a new surface coating of Ag-TiO2@PDA NRDs. The selective physical puncture of bacteria and controlled release of Ag+ ions of Ag-TiO2@PDA NRDs achieved a long-lasting bactericidal ability and anti-biofilm activity with satisfied biocompatibility. Conclusion: This strategy may be promising for clinical applications to reduce the occurrence of infection in the implant surgeries.

Poly(dopamine) and Ag nanoparticle-loaded TiO2 nanotubes with optimized antibacterial and ROS-scavenging bioactivities.

AIM: To create polydopamine (PDA) and Ag nanoparticle-loaded TiO2 nanotubes coating on titanium (Ti) alloy. MATERIALS & METHODS: TiO2-PDA-Ag coating was fabricated on Ti implants by electrochemical anodization. The in vitro and in vivo bactericidal and antibiofilm activities were tested. Intracellular reactive oxygen species (ROS) and antioxidative capability were measured, and cell proliferation, adhesion and cell morphology were characterized. RESULTS: TiO2-PDA-Ag coating showed satisfactory bactericidal and antibiofilm activities in vitro and in vivo, improved Ag release pattern, evident ROS scavenging properties and enhanced cell adhesion and proliferation. CONCLUSION: Our study successfully fabricated a PDA and Ag nanoparticle-loaded TiO2 nanotubes coating on Ti alloy. The improved Ag release kinetics and ROS-scavenging properties achieve an optimal balance between antibacterial ability and biocompatibility.

Silver-loaded nanotubular structures enhanced bactericidal efficiency of antibiotics with synergistic effect in vitro and in vivo.

Antibiotic-resistant bacteria have become a major issue due to the long-term use and abuse of antibiotics in treatments in clinics. The combination therapy of antibiotics and silver (Ag) nanoparticles is an effective way of both enhancing the antibacterial effect and decreasing the usage of antibiotics. Although the method has been proved to be effective in vitro, no in vivo tests have been carried out at present. Herein, we described a combination therapy of local delivery of Ag and systemic antibiotics treatment in vitro in an infection model of rat. Ag nanoparticle-loaded TiO2 nanotube (NT) arrays (Ag-NTs) were fabricated on titanium implants for a customized release of Ag ion. The antibacterial properties of silver combined with antibiotics vancomycin, rifampin, gentamicin, and levofloxacin, respectively, were tested in vitro by minimum inhibitory concentration (MIC) assay, disk diffusion assay, and antibiofilm formation test. Enhanced antibacterial activity of combination therapy was observed for all the chosen bacterial strains, including gram-negative Escherichia coli (ATCC 25922), gram-positive Staphylococcus aureus (ATCC 25923), and methicillin-resistant Staphylococcus aureus (MRSA; ATCC 33591 and ATCC 43300). Moreover, after a relative short (3 weeks) combinational treatment, animal experiments in vivo further proved the synergistic antibacterial effect by X-ray and histological and immunohistochemical analyses. These results demonstrated that the combination of Ag nanoparticles and antibiotics significantly enhanced the antibacterial effect both in vitro and in vivo through the synergistic effect. The strategy is promising for clinical application to reduce the usage of antibiotics and shorten the administration time of implant-associated infection.

Blockade of the human ether-a-go-go-related gene potassium channel by ketamine.

OBJECTIVES: The inhibition of the cardiac rapid delayed rectifier potassium current (IKr ) and its cloned equivalent human ether-a-go-go-related gene (hERG) channel illustrate QT interval prolonging effects of a wide range of clinically used drugs. In this study, the direct interaction of the intravenous anaesthetic ketamine with wild-type (WT) and mutation hERG currents (IhERG ) was investigated. METHODS: The hERG channel (WT, Y652A and F656A) was expressed in Xenopus oocytes and studied using standard two-microelectrode voltage-clamp techniques. KEY FINDINGS: WT hERG is blocked in a concentration-dependent manner with IC50 = 12.05 ± 1.38 µm by ketamine, and the steady-state inactivation curves are shifted to more negative potentials (about -27 mV). The mutation to Ala of Y652 and F656 located on the S6 domain attenuate IhERG blockade by ketamine, and produced approximately 9-fold and 2.5-fold increases in IC50 compared with that of WT hERG channel, respectively. CONCLUSIONS: Ketamine blocks WT IhERG expressed in Xenopus oocytes in a concentration-dependent manner and predominantly interacts with the open hERG channels. The interaction of ketamine with hERG channel may involve the aromatic residues Tyr652 and Phe656.