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Traditional bone tissue engineering techniques require the extraction and proliferation of seed cells, followed by prolonged in vitro culture to form bone tissue constructs. In contrast, in situ mineralization bone tissue engineering utilizes alkaline phosphatase within the body's microenvironment to induce scaffold mineralization. This approach promotes further proliferation and differentiation of osteoblasts and the formation of bone tissue constructs, thereby simplifying the traditional bone tissue engineering process. This study uses electrospinning technology to prepare a novel biologically active scaffold for bone tissue engineering using poly(lactic-co-glycolic acid) (PLGA) and calcium glycerophosphate. The morphology and composition of the scaffolds were characterized using SEM, EDS, and XRD, revealing well-defined fibrous structures and the successful incorporation of calcium glycerophosphate into the PLGA fibers. In vitro simulation of the bone microenvironment using alkaline phosphatase effectively catalyzed the in situ mineralization of calcium glycerophosphate within the scaffold. SEM observations showed substantial mineral aggregation on the surface of the fibrous membranes, and XRD characterization confirmed that the diffraction peaks of the minerals correspond to hydroxyapatite. The cytotoxicity, cell proliferation, and osteogenic differentiation assessments on MC3T3-E1 pre-osteoblasts cultured on the prepared scaffolds indicate that the scaffolds are non-toxic to cells and possess good osteogenic differentiation ability, enabling in situ mineralization. This suggests that the scaffolds have broad prospects for application in bone defect repair.
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Objective: TPMS porous structures have adjustable stiffness, a smooth surface, and highly connected pores, which help avoid stress concentration within the dot-matrix structure and promote cell adhesion and proliferation. A cervical interbody cage based on this type of porous structure was designed and fabricated, and its mechanical properties and biocompatibility were evaluated. Methods: TPMS porous structures have adjustable stiffness, a smooth surface, and highly connected pores, which help avoid stress concentration within the dot-matrix structure and promote cell adhesion and proliferation. A cervical interbody cage based on this type of porous structure was designed and fabricated, and its mechanical properties and biocompatibility were evaluated. Results: The volume fraction of the 3D-printed TC4-based Tubular-G structure was linearly related to compressive strength. Adjusting the volume fraction resulted in a Tubular-G structure with a modulus and yield strength similar to human bone, without stress concentration within the structure. The designed and fabricated TC4-based Tubular-G porous cervical interbody cage demonstrated excellent anti-sagging properties and biocompatibility. Conclusions: The volume fraction of the 3D-printed TC4-based Tubular-G structure was linearly related to compressive strength. Adjusting the volume fraction resulted in a Tubular-G structure with a modulus and yield strength similar to human bone, without stress concentration within the structure. The designed and fabricated TC4-based Tubular-G porous cervical interbody cage demonstrated excellent anti-sagging properties and biocompatibility.
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BACKGROUND: Shigella is among the most important human pathogenic microorganisms, infecting both humans and nonhuman and causing clinically severe diarrhea. Shigella must be enriched before detection, which is time-consuming. OBJECTIVES: To develop a sensitive, rapid, and specific method for Shigella detection. MATERIALS AND METHODS: Shigella was used as an antigen to generate monoclonal antibodies (mAbs). mAbs were screened via indirect enzyme-linked immunosorbent assay (ELISA) and western blot, and two mAbs were selected. The mAb A3 showed high affinity and specificity and was used to develop immune magnetic beads (IMBs) for Shigella enrichment. An immunocapture (IC)-PCR primer was designed from the ipaH gene, and IC-PCR was developed based on the IMBs and PCR. RESULTS: This system shortened the Shigella detection time to 70 min. The sensitivity of the IC-PCR was 9 colony-forming units.mL-1 in artificial milk. The accuracy of the IC-PCR was confirmed using 46 clinical samples collected from monkeys. The IC-PCR results were consistent with the serological and biochemical assays. CONCLUSION: The IC-PCR described herein accurately detected Shigella from milk samples, monkeys and can thus be used to complement classical detection methods.
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Shigella is an important human food-borne zoonosis bacterial pathogen, and can cause clinically severe diarrhea. There is an urgent need to develop a specific, sensitive, and rapid methodology for detection of this pathogen. In this study, loop-mediated isothermal amplification (LAMP) combined with magnetic immunocapture assay (IC-LAMP) was first developed for the detection of Shigella in pure culture, artificial milk, and clinical stool samples. This method exhibited a detection limit of 8.7 CFU/mL. Compared with polymerase chain reaction, IC-LAMP is sensitive, specific, and reliable for monitoring Shigella. Additionally, IC-LAMP is more convenient, efficient, and rapid than ordinary LAMP, as it is more efficiently enriches pathogen cells without extraction of genomic DNA. Under isothermal conditions, the amplification curves and the green fluorescence were detected within 30 min in the presence of genomic DNA template. The overall analysis time was approximately 1 h, including the enrichment and lysis of the bacterial cells, a significantly short detection time. Therefore, the IC-LAMP methodology described here is potentially useful for the efficient detection of Shigella in various samples.
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BACKGROUND: Herpes B virus (BV) is a zoonotic disease caused by double-stranded enveloped DNA virus with cercopithecidae as its natural host. The mortality rate of infected people could be up to 70% with fatal encephalitis and encephalomyelitis. Up to now, there are no effective treatments for BV infection. Among the various proteins encoded by monkey B virus, gD, a conserved structural protein, harbors important application value for serological diagnosis of frequent variations of the monkey B virus. OBJECTIVES: This study aimed to expressed the gD protein of BV in Escherichia coli by a recombinant vector, and prepare specific monoclonal antibodies against gD of BV to pave the way for effective and quick diagnosis reagent research. MATERIALS AND METHODS: The gD gene of BV was optimized by OptimWiz to improve codon usage bias and synthesis, and the recombinant plasmid, pET32a/gD, was constructed and expressed in E. coli Rosetta (DE3). The expressed fusion protein, His-gD, was purified and the BALB/c mice were immunized by this protein. Spleen cells from the immunized mice and SP2/0 myeloma cells were fused together, and the monoclonal cell strains were obtained by indirect enzyme-linked immunosorbent assay (ELISA) screening, followed by preparation of monoclonal antibody ascetic fluid. RESULTS: The optimized gD protein was highly expressed in E. coli and successfully purified. Five monoclonal antibodies (mAbs) against BV were obtained and named as 4E3, 3F8, 3E7, 1H3 and 4B6, and with ascetic fluid titers of 2 × 10(6), 2 × 10(5), 2 × 10(5), 2 × 10(3) and 2 × 10(2), respectively. The 1H3 and 4E3 belonged to the IgG2b subclass, while 3E7, 3F8 and 4B6 belonged to the IgG1 subclass. CONCLUSIONS: The cell lines obtained in this work secreted potent, stable and specific anti-BV mAbs, which were suitable for the development of herpes B virus diagnosis reagents.