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Magnetic nanoparticles (MNP) have been widely used as biomaterials due to their unique magnetic responsiveness and biocompatibility, which also can promote osteogenic differentiation through their inherent micro-magnetic field. The MNP composite scaffold retains its superparamagnetism, which has good physical, mechanical and biological properties with significant osteogenic effects and . Magnetic field has been proved to promote bone tissue repair by affecting cell metabolic behavior. MNP composite scaffolds under magnetic field can synergically promote bone tissue repair and regeneration, which has great application potential in the field of bone tissue engineering. This article summarizes the performance of magnetic composite scaffold, the research progress on the effect of MNP composite scaffold with magnetic fields on osteogenesis, to provide reference for further research and clinical application.
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Cell Differentiation , Magnetite Nanoparticles , Osteogenesis , Tissue Engineering , Tissue ScaffoldsABSTRACT
Objective:To analyze the changes in biological characteristics including infectivity, growth and pathogenicity of Chlamydia muridarum ( Cm) after serial passage in vitro in special conditions in order to provide reference for screening attenuated live vaccines and virulence-related genes. Methods:Wild-type Cm strain (G0) was cultured for several passages using conventional cell culture method under alternate unassisted and assisted culture conditions. Then, the 28th generation (G28) of Cm was selected and compared with the parental G0 strain in terms of centrifugation dependence, attaching ability, intracellular growth curve, plaque size and fallopian tube lesions after genital tract infection in a mouse model. Results:Compared with the parental G0 strain, the G28 strain showed significantly decreased dependence on centrifugation during cell infection ( P<0.05) and increased attachment capacity to cells ( P<0.05). No significant differences were observed in the growth curves 32 h after cell infection or in the plaque sizes between the parental G0 and G28 strains. In the in vivo virulence test, fallopian tube lesions were observed in 87.5% of G0-infected mice and 37.5% of G28-infected mice ( P<0.05). Conclusions:Compared with the parental G0 strain, the G28 strain showed significantly enhanced in vitro infection ability, but decreased in vivo pathogenicity, which brought hope for further identification of virulence genes, isolation of attenuated strains with single genotype and development of live attenuated Chlamydia vaccines.
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OBJECTIVE: To investigate the effects of bio-crosslinker genipin pretreatment on type Ⅰ collagen mineralization. METHODS: Type Ⅰ collagen gels were prepared and pretreated with 0.5wt%genipin (experimental group) and deionized water (control group) for 2 h, respectively. The pretreated products were subjected to Fourier transform infrared spectroscopy (FT-IR). Reconstituted collagen fibrils were pretreated with genipin or deionized water for 2 h and were mineralized for 4 h. The collagen density and mineralization degree were examined with transmission electron microscopy (TEM) and analyzed with ImageJ software. Then scanning electron microscopy (SEM) and TEM were used to observe the mineralization of cross-linked demineralized dentin collagen. RESULTS FT-IR spectrum showed that the genipin was crosslinked with collagen. TEM observation and ImageJ results showed that after 4 h mineralization, the mineralization effect of 0.5wt% genipin group was significantly better than that of the control group[(73.3±5.3)%vs.(7.4±3.5)%,P<0.01]. TEM and SEM observation showed that the mineralization rate of type Ⅰ collagen and demineralized dentin pretreated with genipin were significantly faster than that of the control group. CONCLUSIONS The study demonstrates that 0.5 wt% concentration of genipin can significantly promote the mineralization of type Ⅰ collagen.
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Objective@#To observe the effect of adenosine triphosphate (ATP) phosphorylation on type Ⅰ collagen mineralization and explore the role of small molecule compound ATP in biomimetic mineralization.@*Methods@#Fourier transform infrared spectroscopy (FT-IR) was used to analyze the phosphorylation of collagen molecules by different concentrations (0, 25, 50, 100 mmol/L) of ATP. The concentration of 50 mmol/L ATP was chosen to construct the phosphorylated collagen mineralization model. Transmission electron microscopy (TEM) was used to observed the ultrastructure of mineralized collagen and the collagen mineralization rate was further calculated by ImageJ software. The surface morphology of the collagen gel ATP group and the control group was observed by scanning electron microscopy (SEM) and the elemental analysis was performed by using an X-ray energy spectrometer. The artificial demineralized dentin samples were mineralized for 2 days and 4 days to compare the effect of ATP on dentin remineralization by SEM.@*Results@#FT-IR analysis showed that the formation of new peaks at wavenumbers of 642, 818, and 902 cm-1 indicated that ATP can phosphorylate type Ⅰ collagen. Through TEM and SEM observation, the mineralization degree of type Ⅰ collagen and demineralized dentin pretreated with 50 mmol/L ATP were significantly higher than that of the control group. Compared with the control group [(31.65±1.62)%], the mineralization rate of collagen in the ATP group [(100±0)%] was significantly increased after 2 days of mineralization (P<0.05).@*Conclusions@#ATP phosphorylation can effectively promote the mineralization process of type Ⅰ collagen.
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Objective To analyze the protein expression profile of HeLa cells transfected with pORF5 gene of Chlamydia trachomatis. Methods A lentiviral expression vector containing pORF5 gene was constructed. The lentiviral expression vector and helper plasmids were co-transfected into 293T cells to construct the recombinant lentivirus, which was used to infect HeLa cells. HeLa cells transfected with pORF5 gene and control HeLa cells were sorted out by flow cytometry. The isobaric tags for relative and absolute quantitation ( iTRAQ) approach combined with nano-liquid chromatography-tandem mass spec-trometry ( NanoLC-MS/MS) analysis was performed to understand protein expression profiles and to iden-tify and quantify the differentially expressed proteins in the pORF5-transfected HeLa cells ( pORF5-Hela) and the control HeLa cells. Quantitative real-time PCR ( qRT-PCR ) and Western blot analysis were performed to detect the expression of some proteins at mRNA and protein levels, respectively. Results HeLa cell line stably transfected with pORF5 gene and control HeLa cell line were constructed successful-ly. Totally 314 proteins were differentially expressed between the pORF5-HeLa and control HeLa cells, 159 of which showed increased expression and the other 155 showed decreased expression in pORF5-HeLa cells. The differentially expressed proteins were involved in many processes, such as metabolic process, immune response, biological adhesion and so on. Results of qRT-PCR showed that the expression of HIST1H1C(histone H1. 2C), HBA1(hemoglobin subunit alpha), PARK7(parkinson disease protein 7), HMGB1(high mobility group protein B1) and HMGB2 at mRNA level in pORF5-HeLa cells were up-regulated, while the expression of CLIC1 ( chloride intracellular channel protein 1 ) , KRT7 ( typeⅡ cy-toskeletal 7), SFN(14-3-3 protein sigma) and CDKN2A(cyclin-dependent kinase inhibitor 2A) were down-regulated. Western blot analysis confirmed the enhanced expression of HMGB1 and PRAK7 at pro-tein level. The results of qRT-PCR and Western blot analysis were consistent with proteomic data. Con-clusion Expression profiles for differentially expressed proteins between pORF5-HeLa and control HeLa cells were established successfully. The differentially expressed proteins regulated by pORF5 gene were found to be related to cell metabolism, proliferation, adhesion and so on, suggesting that pORF5 might promote the growth and proliferation of Ct by regulating protein expression and biological behavior of host cells.
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Objective To investigate the role of Th17 cells and related cytokines in Helicobacter pylori(H.pylori) infection.Methods To establish a mouse model of H.pyloriinfectious gastritis.Meanwhile,a treatment group and control group were set up.The histological changes in the gastric mucosa were observed and scored under light microscopy;The expression of IL-17 and IL-23 mRNAs as well as protein in gastric tissue was detected by RT-PCR and ELISA,respectively;Th17 cells in mice splenocyte were evaluated using the flow cytometry (FCM) method.Results We found significantly higher levels of IL-17 and IL-23 protein and mRNA levels in supernatant of gastric tissue homogenates of infection group as compared to controls and Th17 cells in spleen from mice of H.pylori infection group were all obviously higher than that of control group;IL-17 mRNA,IL-23 mRNA and the ratio of Th17 cells in mice splenocyte of H.pylori infection group mice increased gradually with the time prolonged;The levels of both mRNA and protein levels of IL-17and IL-23 decreased significantly in the treatment group compared with pre-treatment;There was a positive correlation between IL-17 and IL-23 expression levels of mice gastric mucosa and gastric inflammation degree.Conclusion H.pylori infection induced the immune response of Th17 cells;The levels of IL-17 and IL-23 increased in gastric mucosa of mice after H.pyloriinfection;The degree of H.pylori-infected mice gastric inflammation was positive correlation with the levels of IL-17 and IL-23 of gastric mucosa.
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ObjectiveTo purify and characterize the monoclonal antibody (McAb) against Chlamydia trachomatis pORF5 plasmid protein.Methods The hybridoma cells stably secreting specific McAb against pORF5 were cultured in a large scale,and protein G purification by affinity chromatography was used to purify 2H4 McAb.ELISA was used to determine the antibody titer,and identify McAb isotype.Immunofluorescence assay (IFA) and Western blot were performed to detect McAb specificity.Results The purity of 2H4 antibody was 93％,the titer reached 1:1024,and 2H4 McAb was identified to belong to IgG2a isotype,2H4 McAb reacted strongly with the GST-pORF5 fusion protein and endogenous pORF5 protein expressed by Chlamydia trachomatis serovar A,D,L2,Chlamydia muridarum ( MoPn ),Chlamydia psittaci 6BC,but not other chlamydial plasmid proteins and Chlamydia pneumoniae(Cpn) AR39 strain.Conclusion2H4 McAb against pORF5 protein was successfully purified with a high titer and specificity which lay a foundation for further study on pORF5 protein structure and function.
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Objective To construct DNA vaccine containing MOMP gene of Chlamydia trachomatis and to observe immune response in mice. Methods Mice of 4 - 6 weeks old were immunized with pcDNA3.1-MOMP or pcDNA3.1 intramuscularly at a dose of 100 μg. Booster immunizations were employed at 2-week interval for two times. Specific antibody in the sera of mice and the level of IFN-γ in murine spleen lymphocyte supernatant were detected by ELISA. The proliferation response of spleen cells was detected by MTT assay. Results Significant specific antibody titers were observed and the highest titer was 1: 1 024 in mice after three times immunization with pcDNA3.1-MOMP. The proli-feration response of spleen cells were significantly higher than that of mice injected with pc DNA3.1. IFN-γ reached(532.0 + 45.4)pg/mL in immunized mice. Conclusion Strong responses of humoral and cellular immunity can be evoked by DNA vaccine of pcDNA3.1-MOMP in mice.
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Objective To express outer membrane protein 2(Omp2) of Chlamydia trachomatis, purify expressed products and study its immunity.Methods The target gene encoding Omp2 167—434 amino acid residues was amplified by PCR from C. trachomatis template DNA. The targeted DNA fragment was cloned into expression vector pET28b(+) and introduced into competent E. coli BL21(DE3) cell. Recombinant Omp2aa_ 167 ～aa_ 434 was expressed after induction by IPTG and analyzed by SDS-PAGE and Western blot, purified with Ni-NTA-His affinity chromatography. The rOmp2aa_ 167 ～aa_ 434 was used to immune rabbits for immunogenicity assessment.Results Restriction enzymes cleavage analysis and DNA sequencing confirmed that the plasmid pET28b(+)/Omp2aa_ 167 ～aa_ 434 was correctly constructed. The 35.0?103 molecular weight pure protein, which specifically reacted with serum from C. trachomatis infected patient by Western blot, was obtained by optimizing the conditions for both expression and purification. The titer of serum antibodies was above 1∶1 280 as detected by ELISA.Conclusion The expressed product showed good immunity.
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Objective To construct the recombinant plasmid containing the major outer membrane protein(MOMP) gene of Chlamydia trachomatis and expres s MOMP protein in E.coli BL21. Methods The MOMP gene was amplified by polymera se chain reaction from the genome of Chlamydia trachomatis serovar D. The amplif ied fragment was directly inserted into pUCm-T vector and verified by DNA sequen cing. MOMP gene was then subcloned into the prokaryotic expression vector pET-22 b(+). The recombinant protein of MOMP was purified by Ni-NTA affinity chromatogr aphy and identified by SDS-PAGE and Western blot. Results The MOMP gene, which is about 1 200 bp, was successfully amplified and cloned. The DNA sequence of t he cloned MOMP gene was the same as that published by the GenBank. SDS-PAGE anal ysis showed that the relative molecular weight of this fusion protein was about 47 kDa which was consistent with the theoretically predicted value, and the spec ificity of this recombinant protein was confirmed by Western blot. Conclusions The MOMP gene of Chlamydia trachomatis was successfully cloned and expressed in the prokaryotic expression system, which may lay the foundation for the developm ent of Chlamydia trachomatis vaccine.