BACKGROUND:Carnosic acid,a bioactive compound found in rosemary,has been shown to reduce inflammation and reactive oxygen species(ROS).However,its mechanism of action in osteoclast differentiation remains unclear. OBJECTIVE:To investigate the effects of carnosic acid on osteoclast activation,ROS production,and mitochondrial function. METHODS:Primary bone marrow-derived macrophages from mice were extracted and cultured in vitro.Different concentrations of carnosic acid(0,10,15,20,25 and 30 μmol/L)were tested for their effects on bone marrow-derived macrophage proliferation and toxicity using the cell counting kit-8 cell viability assay to determine a safe concentration.Bone marrow-derived macrophages were cultured in graded concentrations and induced by receptor activator of nuclear factor-κB ligand for osteoclast differentiation for 5-7 days.The effects of carnosic acid on osteoclast differentiation and function were then observed through tartrate-resistant acid phosphatase staining,F-actin staining,H2DCFDA probe and mitochondrial ROS,and Mito-Tracker fluorescence detection.Western blot and RT-PCR assays were subsequently conducted to examine the effects of carnosic acid on the upstream and downstream proteins of the receptor activator of nuclear factor-κB ligand-induced MAPK signaling pathway. RESULTS AND CONCLUSION:Tartrate-resistant acid phosphatase staining and F-actin staining showed that carnosic acid dose-dependently inhibited in vitro osteoclast differentiation and actin ring formation in the cell cytoskeleton,with the highest inhibitory effect observed in the high concentration group(30 μmol/L).Carnosic acid exhibited the most significant inhibitory effect during the early stages(days 1-3)of osteoclast differentiation compared to other intervention periods.Fluorescence imaging using the H2DCFDA probe,mitochondrial ROS,and Mito-Tracker demonstrated that carnosic acid inhibited cellular and mitochondrial ROS production while reducing mitochondrial membrane potential,thereby influencing mitochondrial function.The results of western blot and RT-PCR revealed that carnosic acid could suppress the expression of NFATc1,CTSK,MMP9,and C-fos proteins associated with osteoclast differentiation,and downregulate the expression of NFATc1,Atp6vod2,ACP5,CTSK,and C-fos genes related to osteoclast differentiation.Furthermore,carnosic acid enhanced the expression of antioxidant enzyme proteins and reduced the generation of ROS during the process of osteoclast differentiation.Overall,carnosic acid exerts its inhibitory effects on osteoclast differentiation by inhibiting the phosphorylation modification of the P38/ERK/JNK protein and activating the MAPK signaling pathway in bone marrow-derived macrophages.

Objective:To discuss the promotive effect of nerve growth factor(NGF),which is highly expressed in the adipose-derived stem cell(ADSC)-induced Schwann-like cells(SCLCs),on the growth of dorsal root ganglion(DRG)cell processes in the rats,and to clarify its mechanism.Methods:The ADSCs were extracted from the epididymal adipose tissue of the SD rats,and their multidirectional differentiation potential was identified through osteogenic,adipogenic,and chondrogenic induction.The ADSCs were induced to differentiate into the SCLCs,and the expression levels of glial fibrillary acidic protein(GFAP)and S100 calcium-binding protein β(S100β)protein in the ADSCs and SCLCs were detected by immunofluorescence staining and Western blotting methods.The DRG cells were isolated and cultured,and immunofluorescence staining was used to detect the βⅢ-tubulin expression in the DRG cells for identification.The SCLCs were co-cultured with the DRG cells(co-culture group),the single-culture DRG cells were regared as DRG group and toluidine blue staining was used to observe and measure the length of DRG cell processes under the optical microscope in co-culture group and DRG group.Small interfering RNA(siRNA)transfection was used to knock down NGF,and plasmid transfection was used to over-express NGF.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the NGF mRNA expression levels in the cells in various groups;enzyme-linked immunosorbent assay(ELISA)method was used to detect the NGF protein levels in the cell supernatants.The transfected SCLCs were co-cultured with DRG cells and divided into control group,siNC/vector group,NGF knockdown group(si-NGF group),and NGF over-expression group(oe-NGF group).The lengths of DRG cell processes in various groups were observed.Results:The primary ADSCs adhered within 24 h after seeding,with a small number of lipid droplets remaining.After 3 d of culture,the cells were mostly short spindle-shaped,fusiform,or polygonal,growing rapidly in a vortex pattern.After passaging,the cells exhibited a uniform morphology,appearing as long spindles arranged in a fish-school pattern.After 14 d of adipogenic induction,the cell morphology changed from spindle-shaped to flat-round,with translucent lipid droplets forming in the cytoplasm,which were stained red by Oil Red O.After 28 d of osteogenic induction,the cells appeared sand-like with blurred morphology,and calcified nodules were observed,which were stained red by Alizarin Red and deposited in the extracellular matrix.After 28 d of chondrogenic induction in a 3D culture system,millet-sized chondrogenic spheres formed.Frozen sections of the spheres were stained with Alcian Blue,and acidic mucopolysaccharides in the cartilage tissue were stained blue under the microscope.Under the fluorescence microscope,the third-passage purified ADSCs showed positive expression of CD29[fluorescein isothiocy anate(FITC)-labeled green fluorescence]and CD44(Cy3-labeled red fluorescence).The immunofluorescence staining results showed that GFAP was labeled with FITC(green fluorescence),and S100β was labeled with Cy3(red fluorescence).The Western blotting results showed that compared with ADSCs,the expression levels of S100β and GFAP proteins in the SCLCs were increased(P<0.05).The primary DRG cells began to adhere 6 h after conventional culture,and after 3 d,the cell bodies appeared round and bright,with two linear processes extending from them.Under fluorescence microscope,the cells positively expressed the neuron-specific marker βⅢ-tubulin,confirming that the isolated cells were DRG cells.Compared with the ADSCs,the NGF protein expression level in the SCLCs was increased(P<0.05).Compared with DRG group,the length of DRG cell processes in co-culture group was the highest when DRG cells and SCLCs were co-cultured at a 1∶2 ratio(P<0.05).The RT-qPCR results showed that compared with si-NC group,the expression levels of NGF mRNA in the cell supernatant in si-NGF-1,si-NGF-2,and si-NGF-3 groups were significantly decreased(P<0.05),with si-NGF-1 showing the highest knockdown efficiency,which was selected for subsequent experiments.The ELISA results showed that compared with si-NC group,the NGF levels in the cell supernatant of si-NGF-1,si-NGF-2,and si-NGF-3 groups were decreased(P<0.05).Compared with Vector group,the expression level of NGF mRNA and NGF protein level in the supernatant in oe-NGF group were increased(P<0.05).Compared with control group and siNC/vector group,the length of DRG cell processes in si-NGF group was decreased(P<0.05),while the length of DRG cell processes in oe-NGF group was increased(P<0.05).Conclusion:ADSCs can be directionally differentiated into SCLCs,and the differentiated cells highly express NGF.Knockdown or overexpression of NGF can affect the growth of DRG cell processes.

BACKGROUND:At present,the treatment of infected bone defects has the problems of long course of disease,poor treatment effect and high cost.The osteogenic effect of personalized bone replacement materials in clinical treatment is limited.Therefore,a 3D-printed bone graft material with both good osteogenic effect and antibacterial effect is urgently needed for clinical treatment. OBJECTIVE:To summarize the research status of 3D-printed scaffolds loaded with antimicrobial agents for the treatment of infected bone defects. METHODS:PubMed,Web of Science,Elsevier,and CNKI databases from January 2010 to June 2022 were searched for related articles.The Chinese search terms were"bone defect,3D printing,scaffold material,antibacterial,animal experiments,in vitro experiments".English search terms were"bone defect,3D printing,scaffold,antibiosis,animal experiment,in vitro".Finally,60 articles were included for review and analysis. RESULTS AND CONCLUSION:The 3D scaffolds made of titanium,magnesium,tantalum and other metals and their alloys have certain osteogenic properties,but do not have antibacterial function.Hydroxyapatite and other bioceramic materials have good biocompatibility and are prone to be degraded,whereas due to the lack of strength,they are usually combined with artificial polymer materials to form composite materials,which respectively mimic the inorganic and organic components in natural bone,and play their respective excellent functions.Antibiotics,silver/copper nanoparticles,antimicrobial peptides,gallium and other antibacterial agents play an antibacterial role by destroying bacterial cell membrane,producing reactive oxygen species to interfere with bacterial DNA replication,inhibiting iron absorption and other mechanisms.As a result,the 3D-printed scaffold has both antibacterial and osteogenic effects.However,there are still some problems such as drug resistance and difficult to control effective concentrations.3D-printed scaffolds are often loaded with antibacterial agents by loading drug-loaded microspheres on scaffolds,preparing antibacterial coating on the scaffold surface,and participating in joint 3D printing with drugs.The loading mode of antibacterial coating prepared on the scaffold surface is the most widely used,and its antibacterial effect is more stable.Nonetheless,the selection of the most suitable loading mode for antibacterial agents needs to be further discussed and summarized.It is a future research prospect to optimize the mechanical properties of composite scaffolds and prepare biomimetic bone scaffolds so that the degradation rate is consistent with the bone reconstruction rate in infected bone defects.The ideal antibacterial agents may play a role through a variety of antibacterial mechanisms,thus being expected to play a good antibacterial effect through low antibacterial concentration,which should be a hot spot of anti-bone infection research.After loading antibacterial agents on the surface of the scaffold,antibacterial agents can"intelligently"react to the local microenvironment,achieving controlled release,and regulating the osteogenesis,vascularization and immune response of the microenvironment,which is the focus of current research.

Objective: To investigate the mechanisms by which D-methionine (D-Met) eradicates Porphyromonas gingivalis biofilms by suppressing cyclic dimeric GMP (c-di-GMP) levels. Methods : Cell viability, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were measured to determine the effective concentrations of D-Met, which were subsequently used in the following experiments. During the P. gingivalis biofilm formation inhibition experiment and the mature biofilm disassembly experiment, biofilm biomass, exopolysaccharide (EPS), biofilm morphology, integrity of the cell membrane, and the level of c-di-GMP were determined. Results : D-Met < 40 mmol/L was biocompatible. During the biofilm formation inhibition and mature biofilm disassembly experiments, D-Met ≥ 20 mmol/L decreased the biofilm biomass and the production of EPS. SEM analysis showed that the extracellular matrix and bacterial density were drastically reduced by D-Met ≥ 20 mmol/L. TEM detection showed that 35 mmol/L D-Met ruptured the cell membrane during biofilm formation and increased the permeability of the cell membrane in the disassembly phase of mature biofilms. C-di-GMP levels decreased with increasing concentrations of D-Met in a concentration-dependent manner. Conclusion D-Met ≥ 20 mmol/L could eradicate P. gingivalis biofilms by suppressing c-di-GMP levels.