A Library of Promoter-gfp Fusion Reporters for Studying Systematic Expression Pattern of Cyclic-di-GMP Metabolism-Related Genes in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa is an environmental microorganism and is a model organism for biofilm research. Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that plays critical roles in biofilm formation. P. aeruginosa contains approximately 40 genes that encode enzymes that participate in the metabolism of c-di-GMP (biosynthesis or degradation), yet it lacks tools that aid investigation of the systematic expression pattern of those genes. In this study, we constructed a promoter-gfp fusion reporter library that consists of 41 reporter plasmids. Each plasmid contains a promoter of corresponding c-di-GMP metabolism-related (CMR) genes from P. aeruginosa reference strain PAO1; thus, each promoter-gfp fusion reporter can be used to detect the promoter activity as well as the transcription of corresponding gene. The promoter activity was tested in P. aeruginosa and Escherichia coli. Among the 41 genes, the promoters of 26 genes showed activity in both P. aeruginosa and E. coli. The library was applied to determine the influence of different temperatures, growth media, and subinhibitory concentrations of antibiotics on the transcriptional profile of the 41 CMR genes in P. aeruginosa. The results showed that different growth conditions did affect the transcription of different genes, while the promoter activity of a few genes was kept at the same level under several different growth conditions. In summary, we provide a promoter-gfp fusion reporter library for systematic monitoring or study of the regulation of CMR genes in P. aeruginosa. In addition, the functional promoters can also be used as a biobrick for synthetic biology studies. IMPORTANCE The opportunistic pathogen P. aeruginosa can cause acute and chronic infections in humans, and it is one of the main pathogens in nosocomial infections. Biofilm formation is one of the most important causes for P. aeruginosa persistence in hosts and evasion of immune and antibiotic attacks. c-di-GMP is a critical second messenger to control biofilm formation. In P. aeruginosa reference strain PAO1, 41 genes are predicted to participate in the making and breaking of this dinucleotide. A major missing piece of information in this field is the systematic expression profile of those genes in response to changing environment. Toward this goal, we constructed a promoter-gfp transcriptional fusion reporter library that consists of 41 reporter plasmids, each of which contains a promoter of corresponding c-di-GMP metabolism-related genes in P. aeruginosa. This library provides a helpful tool to understand the complex regulation network related to c-di-GMP and to discover potential therapeutic targets.

A novel method to establish the rabbit model of knee osteoarthritis: intra-articular injection of SDF-1 induces OA.

BACKGROUND: Animal model of Knee Osteoarthritis (OA) is the primary testing methodology for studies on pathogenic mechanisms and therapies of human OA disease. Recent major modeling methods are divided into artificially induced and spontaneous. However, these methods have some disadvantages of slow progression, high cost and no correlation with the pathogenesis of OA. METHODS: Our studies attempted to find a rapid, easy, and consistent with the natural pathological process of OA modeling method by intra-articular injection of stromal cell-derived factor 1 (SDF-1) in the rabbit knee. After induction we collected cartilage specimens from the medial femoral condyle to undergo macroscopic, histological, immunohistochemical, and biochemical evaluations. Meanwhile, compared with Hulth surgical method to evaluate its efficacy. RESULTS: Macroscopic observation and modified Mankin score of histological staining exhibited typical features of middle stage OA cartilage in SDF-1 injected groups. Immunohistochemically, the positive expression of interleukin-1 (IL-1) and tumor necrosis factor α(TNF-α) was earlier and higher in high dose SDF-1 group than the surgical group. The matrix metalloproteinases (MMPs) in synovial fluid and chondrocytes significantly increased, but type II collagen (COLII) and aggrecan (ACAN) protein expressions decreased in SDF-1 injected group following the extension of time and increase of SDF-1 concentration. CONCLUSIONS: Our data indicated intra-articular injection of SDF-1 (40µg/kg, three times for 12 weeks) can induce rabbit knee OA model successfully more rapidly and easily than traditional surgical modeling. The study provided a further option for the establishment of knee OA animal model.

Genomic characterization and pathogenicity of porcine deltacoronavirus strain CHN-HG-2017 from China.

Porcine deltacoronavirus (PDCoV) was first detected in Hong Kong and has recently spread to many countries around the world. PDCoV causes acute diarrhea and vomiting in pigs, resulting in significant economic losses in the global pork industry. In this study, a Chinese PDCoV strain, designated CHN-HG-2017, was isolated from feces of a suckling piglet with severe watery diarrhea on a farm located in central China. Subsequently, the virus was identified by an indirect immunofluorescence assay and electron microscopy. A nucleotide sequence alignment showed that the whole genome of CHN-HG-2017 is 97.6%-99.1% identical to other PDCoV strains. Analysis of potential recombination sites showed that CHN-HG-2017 is a possible recombinant originating from the strains CH/SXD1/2015 and Vietnam/HaNoi6/2015. Furthermore, the pathogenicity of this recombinant PDCoV strain was investigated in 5-day-old piglets by oral inoculation. The challenged piglets developed typical symptoms, such as vomiting, anorexia, diarrhea and lethargy, from 1 to 7 days post-inoculation (DPI). Viral shedding was detected in rectal swabs until 14 DPI in the challenged piglets. Interestingly, high titers of virus-neutralizing antibodies in sera were detected at 21 DPI. Tissues of small intestines from CHN-HG-2017-infected piglets at 4 DPI displayed significant macroscopic and microscopic lesions with clear viral antigen expression. Our analysis of the full genome sequence of a recombinant PDCoV and its virulence in suckling piglets might provide new insights into the pathogenesis of PDCoV and facilitate further investigation of this newly emerged pathogen.

Controlled Growth of Ultra-Thick Polymer Brushes via Surface-Initiated Atom Transfer Radical Polymerization with Active Polymers as Initiators.

Polymer brushes exhibit functionalities useful for a large number of applications. Often these functionalities only emerge when the polymer brushes have a desired thickness. Here, a significant breakthrough is achieved in the synthesis of ultra-thick polymer brushes using polymer initiators in the approach of surface-initiated atom transfer radical polymerization, yielding polymer brushes with a controllable thickness up to 15.1 µm. This is reportedly the thickest polymer brush ever synthesized. This approach is applicable for several monomers such as acrylonitrile, methyl acrylate, and styrene, and for other types of polymer substrates such as fibers.

Cloning, expression, and characterization of a novel nitrilase, PaCNit, from Pannonibacter carbonis Q4.6.

OBJECTIVE: Identification of a heavy metal ion-stimulated nitrilase with broad-spectrum substrate specificity. RESULTS: A novel nitrilase, PaCNit, was identified from Pannonibacter carbonis Q4.6 and its enzymatic properties were investigated. The maximum activity of PaCNit was observed at 65 °C and pH 7.0. PaCNit showed broad substrate specificity towards aliphatic, aromatic, and heterocyclic nitriles, and was tolerant to different organic solvents. Remarkably, PaCNit activity was highly stimulated by metal ions, particularly by Ag+ and Hg2+. CONCLUSION: PaCNit nitrilase has a broad range of substrate specificity and can be activated by heavy metal ions. This specific characteristic makes it have a great potential for industrial application.

Pannonibacter carbonis sp. nov., isolated from coal mine water.

Two bacterial strains were isolated from coal mine water in China. Isolates were facultatively anaerobic, Gram-stain-negative, rod-shaped, motile by means of a single polar flagellum, and they did not produce bacteriochlorophyll α. Cells grew in tryptic soy broth with 0-5.5 % (w/v) NaCl, at 4-55 °C and pH 3.5-10.5. Isolates were positive for catalase, oxidase, urease, Voges-Proskauer test, gelatin hydrolysis and H2S production. Analysis of 16S rRNA gene sequences indicated that the closest relatives of strains Q4.6T and Q2.11 were the type strains Labrenzia suaedae DSM 22153T (97.4 %), Pannonibacter phragmitetus DSM 14782T (96.9 and 97.0 %) and Pannonibacter indicus DSM 23407T (96.8 %). The genomic average nucleotide identity (ANI) value for Q4.6T and Q2.11 was 100 %; however, this value was less than 77.7 % for the type strains P. phragmitetus and P. indicus, and less than 74.0 % for the type strain L. suaedae. The cellular fatty acid profile of strains Q4.6T and Q2.11 consisted primarily of C18 : 1ω7c. The principal quinone of the isolates was Q-10. The polar lipid profile consisted of diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidyl ethanolamine and phosphatidyl choline. On the basis of phylogenetic analysis, genomic ANI analysis, DNA-DNA hybridization results, as well as phenotypic and chemotaxonomic data, strains Q4.6T and Q2.11 are assigned as a novel species within the genus Pannonibacter. The type strain is Pannonibacter carbonis Q4.6T (=CGMCC 1.15703T=KCTC 52466T).

Thauera sinica sp. nov., a phenol derivative-degrading bacterium isolated from activated sludge.

A bacterial strain, K11T, capable of degrading phenol derivatives was isolated from activated sludge of a sewage treatment plant in China. This strain, which can degrade more than ten phenol derivatives, was identified as a Gram-stain negative, rod-shaped, asporogenous, facultative anaerobic bacterium with a polar flagellum. The strain was found to grow in tryptic soy broth in the presence of 0-2.5% (w/v) NaCl (optimum 0-1%), at 4-43 °C (optimum 30-35 °C) and pH 4.5-10.5 (optimum 7.5-8). Comparative analysis of nearly full-length 16S rRNA gene sequences showed that this strain belongs to the genus Thauera. The 16S rRNA gene sequence was found to show high similarity (97.5%) to that of Thauera chlorobenzoica 3CB-1T, with lesser similarity to other recognised Thauera strains. The G+C content of the DNA of the strain was determined to be 67.8 mol%. The DNA-DNA hybridization value between K11T and Thauera aromatica DSM6984T was 10.4 ± 4.5%. The genomic OrthoANI values of K11T with the other nine type strains of genus Thauera were less than 81.1%. Chemotaxonomic analysis of strain K11T revealed that Q-8 is the predominant quinone; the polar lipids contain phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids and five uncharacterised lipids; the major cellular fatty acid was identified as summed feature 3 (C16:1 ω7c and/or iso-C15:0 2-OH; 45.9%), followed by C16:0 (20.5%) and C18:1 ω7c (15.8%). Based on the phenotypic and phylogenetic evidence, DNA-DNA hybridisation, OrthoANI, chemotaxonomic analysis and results of the physiological and biochemical tests, a new species named Thauera sinica sp. nov. is proposed with strain K11T (= CGMCC 1.15731T = KACC 19216T) designated as the type strain.

Deinococcus petrolearius sp. nov. isolated from crude oil recovery water in China.

A Gram-stain positive, non-motile, spherical, red-pigmented and facultatively anaerobic bacterium, designated strain 6.1T, was isolated from a crude oil recovery water sample from the Huabei oil field in China. The novel strain exhibited tolerance of UV irradiation (> 1000 J m-2). Based on 16S rRNA gene sequence comparisons, strain 6.1T shows high similarity to Deinococcus citri DSM 24791T (98.1%) and Deinococcus gobiensis I-0T (97.8%), with less than 93.5% similarity to other closely related taxa. The major cellular fatty acids were identified as summed feature 3 (C16:1 ω7c and/or iso-C15:0 2-OH), followed by iso-C17:1 ω9c and C16:0. The polar lipid profile was found to contain phospholipids, glycolipids, phosphoglycolipids and aminophospholipids. The predominant respiratory quinone was identified as MK-8. The DNA G + C content was determined to be 68.3 mol %. DNA-DNA hybridization between strain 6.1T and D. citri DSM 24791T was 45.6 ± 7.1% and with D. gobiensis I-OT was 36.6 ± 4.7%. On the basis of phylogenetic, chemotaxonomic and phenotypic data, we conclude strain 6.1T represents a novel species of the genus Deinococcus, for which we propose the name Deinococcus petrolearius sp. nov. The type strain is 6.1T (= CGMCC 1.15053T = KCTC 33744T).

Catechol 2,3-dioxygenase from a new phenolic compound degrader Thauera sp. K11: purification and biochemical characterization.

Catechol 2,3-dioxygenase (C23O) from a new phenolic compound degrader Thauera sp. K11 was purified and characterized. The native form of the enzyme was determined as a homotetramer with a molecular weight of 140 kDa, and its isoelectric point was close to 6.4. One iron per enzyme subunit was detected using atom absorption spectroscopy, and the effective size of C23O in its dilute solution (0.2 g L-1 , pH 8.0) was 14.5 nm. The optimal pH and temperature were 8.4 and 45 °C, respectively. The addition of Mg2+ , Cu2+ , Fe2+ , and Mn2+ could improve the enzyme activity, while Ag+ was found to be a strong inhibitor. C23O was stable in alkali conditions (pH 7.6-11.0) and thermostable below 50 °C. The final purified C23O had a sheet content of 53%, consistent with the theoretical value. This showed that the purified catechol 2,3-dioxygenase folded with a reasonable secondary structure.

Sequential Immunizations with Influenza Neuraminidase Protein Followed by Peptide Nanoclusters Induce Heterologous Protection.

Enhancing cross-protections against diverse influenza viruses is desired for influenza vaccinations. Neuraminidase (NA)-specific antibody responses have been found to independently correlate with a broader influenza protection spectrum. Here, we report a sequential immunization regimen that includes priming with NA protein followed by boosting with peptide nanoclusters, with which targeted enhancement of antibody responses in BALB/c mice to certain cross-protective B-cell epitopes of NA was achieved. The nanoclusters were fabricated via desolvation with absolute ethanol and were only composed of composite peptides. Unlike KLH conjugates, peptide nanoclusters would not induce influenza-unrelated immunity. We found that the incorporation of a hemagglutinin peptide of H2-d class II restriction into the composite peptides could be beneficial in enhancing the NA peptide-specific antibody response. Of note, boosters with N2 peptide nanoclusters induced stronger serum cross-reactivities to heterologous N2 and even heterosubtypic N7 and N9 than triple immunizations with the prototype recombinant tetrameric (rt) N2. The mouse challenge experiments with HK68 H3N2 also demonstrated the strong effectiveness of the peptide nanocluster boosters in conferring heterologous protection.

Fully Automatic Deep Learning Model for Spine Refracture in Patients with OVCF: A Multi-Center Study.

BACKGROUND: The reaserch of artificial intelligence (AI) model for predicting spinal refracture is limited to bone mineral density, X-ray and some conventional laboratory indicators, which has its own limitations. Besides, it lacks specific indicators related to osteoporosis and imaging factors that can better reflect bone quality, such as computed tomography (CT). OBJECTIVE: To construct a novel predicting model based on bone turn-over markers and CT to identify patients who were more inclined to suffer spine refracture. METHODS: CT images and clinical information of 383 patients (training set = 240 cases of osteoporotic vertebral compression fractures (OVCF), validation set = 63, test set = 80) were retrospectively collected from January 2015 to October 2022 at three medical centers. The U-net model was adopted to automatically segment ROI. Three-dimensional (3D) cropping of all spine regions was used to achieve the final ROI regions including 3D_Full and 3D_RoiOnly. We used the Densenet 121-3D model to model the cropped region and simultaneously build a T-NIPT prediction model. Diagnostics of deep learning models were assessed by constructing ROC curves. We generated calibration curves to assess the calibration performance. Additionally, decision curve analysis (DCA) was used to assess the clinical utility of the predictive models. RESULTS: The performance of the test model is comparable to its performance on the training set (dice coefficients of 0.798, an mIOU of 0.755, an SA of 0.767, and an OS of 0.017). Univariable and multivariable analysis indicate that T_P1NT was an independent risk factor for refracture. The performance of predicting refractures in different ROI regions showed that 3D_Full model exhibits the highest calibration performance, with a Hosmer-Lemeshow goodness-of-fit (HL) test statistic exceeding 0.05. The analysis of the training and test sets showed that the 3D_Full model, which integrates clinical and deep learning results, demonstrated superior performance with significant improvement (p-value < 0.05) compared to using clinical features independently or using only 3D_RoiOnly. CONCLUSION: T_P1NT was an independent risk factor of refracture. Our 3D-FULL model showed better performance in predicting high-risk population of spine refracture than other models and junior doctors do. This model can be applicable to real-world translation due to its automatic segmentation and detection.

Disulfide-stabilized trimeric hemagglutinin ectodomains provide enhanced heterologous influenza protection.

Influenza virus infection poses a continual menace to public health. Here, we developed soluble trimeric HA ectodomain vaccines by establishing interprotomer disulfide bonds in the stem region, which effectively preserve the native antigenicity of stem epitopes. The stable trimeric H1 ectodomain proteins exhibited higher thermal stabilities in comparison with unmodified HAs and showed strong binding activities towards a panel of anti-stem cross-reactive antibodies that recognize either interprotomer or intraprotomer epitopes. Negative stain transmission electron microscopy (TEM) analysis revealed the stable trimer architecture of the interprotomer disulfide-stapled WA11#5, NC99#2, and FLD#1 proteins as well as the irregular aggregation of unmodified HA molecules. Immunizations of mice with those trimeric HA ectodomain vaccines formulated with incomplete Freund's adjuvant elicited significantly more potent cross-neutralizing antibody responses and offered broader immuno-protection against lethal infections with heterologous influenza strains compared to unmodified HA proteins. Additionally, the findings of our study indicate that elevated levels of HA stem-specific antibody responses correlate with strengthened cross-protections. Our design strategy has proven effective in trimerizing HA ectodomains derived from both influenza A and B viruses, thereby providing a valuable reference for designing future influenza HA immunogens.

Antiviral effects of duck type I and type III interferons against Duck Tembusu virus in vitro and in vivo.

Duck Tembusu Virus (DTMUV) is a newly emerging avian flavivirus that causes substantial economic losses to the duck industry in Asia by causing severe egg drop syndrome and fatal encephalitis in domestic ducks. During viral replication, host cells recognize the RNA structures produced by DTMUV, which triggers the production of interferons (IFNs) to inhibit viral replication. However, the function of duck type I and type III IFNs in inhibiting DTMUV infection remains largely unknown. In this study, we expressed and purified recombinant duck IFN-ß (duIFN-ß) and IFN-λ (duIFN-λ) in Escherichia coli and evaluated their antiviral activity against vesicular stomatitis virus (VSV). Furthermore, we found that both duIFN-ß and duIFN-λ activated the ISRE promoter and induced the expression of ZAP, OAS, and RNaseL in duck embryo fibroblasts (DEFs). Notably, duIFN-ß showed faster and more potent induction of ISGs in vitro and in vivo compared to duIFN-λ. Moreover, both duIFN-ß and duIFN-λ showed high potential to inhibit DTMUV infection in DEFs, with duIFN-ß demonstrating better antiviral efficacy than duIFN-λ against DTMUV in ducks. In conclusion, our results revealed that both duIFN-ß and duIFN-λ can induce ISGs production and exhibit significant antiviral activity against DTMUV in vitro and in vivo, providing new insights for the development of antiviral therapeutic strategies in ducks.

Dual functions: A coumarin-chalcone conjugate inhibits cyclic-di-GMP and quorum-sensing signaling to reduce biofilm formation and virulence of pathogens.

Antibiotic resistance or tolerance of pathogens is one of the most serious global public health threats. Bacteria in biofilms show extreme tolerance to almost all antibiotic classes. Thus, use of antibiofilm drugs without bacterial-killing effects is one of the strategies to combat antibiotic tolerance. In this study, we discovered a coumarin-chalcone conjugate C9, which can inhibit the biofilm formation of three common pathogens that cause nosocomial infections, namely, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli, with the best antibiofilm activity against P. aeruginosa. Further investigations indicate that C9 decreases the synthesis of the key biofilm matrix exopolysaccharide Psl and bacterial second messenger cyclic-di-GMP. Meanwhile, C9 can interfere with the regulation of the quorum sensing (QS) system to reduce the virulence of P. aeruginosa. C9 treatment enhances the sensitivity of biofilm to several antibiotics and reduces the survival rate of P. aeruginosa under starvation or oxidative stress conditions, indicating its excellent potential for use as an antibiofilm-forming and anti-QS drug.

Boost immunizations with NA-derived peptide conjugates achieve induction of NA inhibition antibodies and heterologous influenza protections.

Neuraminidase is suggested as an important component for developing a universal influenza vaccine. Targeted induction of neuraminidase-specific broadly protective antibodies by vaccinations is challenging. To overcome this, we rationally select the highly conserved peptides from the consensus amino acid sequence of the globular head domains of neuraminidase. Inspired by the B cell receptor evolution process, a reliable sequential immunization regimen is designed to result in immuno-focusing by steering bulk immune responses to a selected region where broadly protective B lymphocyte epitopes reside. After priming neuraminidase protein-specific antibody responses in C57BL/6 or BALB/c inbred mice strains by immunization or pre-infection, boost immunizations with certain neuraminidase-derived peptide-keyhole limpet hemocyanin conjugates significantly strengthened serum neuraminidase inhibition activities and cross-protections. Overall, this study provides proof of concept for a peptide-based sequential immunization strategy for achieving targeted induction of cross-protective antibody response, which provides references for designing universal vaccines against other highly variable pathogens.

The Chinese knotting technique assist anatomical anterior cruciate ligament reconstruction for aggressive rehabilitation.

Aggressive rehabilitation after anterior cruciate ligament (ACL) reconstruction may result in better clinical outcomes and fewer complications such as knee stiffness and weakness. We explored the effect of the Chinese knotting technique (CKT) for aggressive rehabilitation after ACL reconstruction. Ninety-one anatomical ACL reconstruction cases from 2016 to 2020 were retrospectively reviewed. All patients were operated by the same senior physician and his team. According to the reconstruction with or without CKT, the patients were divided into 2 groups. Both groups received aggressive rehabilitation. The follow-up time of 91 patients was more than 2 years. In total, 43 out of the 91 patients were in the CKT group, and 48 were in the routine group. The knee joint kinematics recorded by Opti_Knee revealed no significant difference among the CKT group, the routine group, and healthy adults at 3, 6, 12, and 24 months after the operation, respectively. The internal and external rotation angle and the anteroposterior displacement at 3 and 6 months after the operation in the CKT group were smaller than in the routine group and were similar to that of the healthy adults. There was no significant difference in flexion and extension angle, varus or valgus angle, proximal-distal displacement, or the internal or external displacement between the 2 groups. In addition, there was no significant difference in 6 degrees of freedom of the knee between the 2 groups at 12 and 24 months after the operation, respectively, which was similar to healthy adults. Compared to the routine group, the International Knee Documentation Committee scores were significantly higher in the CKT group at the 3, 6, and 12 months, respectively, but no difference was observed at 24 months (P = .749). The Lysholm score was significantly higher in the CKT group at the 3 and 6 months postoperatively, while there was no difference at 12 and 24 months, respectively. In short-term observation, the ACL reconstruction with CKT, which can sustain aggressive rehabilitation and prevent the loosening of ACL graft, can lead to better clinical outcomes and kinematics recovery of the knee compared to routine technique.

Chimeric Virus-like Particles Co-Displaying Hemagglutinin Stem and the C-Terminal Fragment of DnaK Confer Heterologous Influenza Protection in Mice.

Influenza virus hemagglutinin (HA) stem is currently regarded as an extremely promising immunogen for designing universal influenza vaccines. The appropriate antigen-presenting vaccine vector would be conducive to increasing the immunogenicity of the HA stem antigen. In this study, we generated chimeric virus-like particles (cVLPs) co-displaying the truncated C-terminal of DnaK from Escherichia coli and H1 stem or full-length H1 antigen using the baculovirus expression system. Transmission electronic micrography revealed the expression and presentation of H1 stem antigens on the surface of VLPs. Vaccinations of mice with the H1 stem cVLPs induced H1-specific immune responses and provided heterologous immune protection in vivo, which was more effective than vaccinations with VLPs displaying H1 stem alone in protecting mice against weight loss as well as increasing survival rates after lethal influenza viral challenge. The results indicate that the incorporation of the truncated C-terminal of DnaK as an adjuvant protein into the cVLPs significantly enhances the H1-specific immunity and immune protection. We have explicitly identified the VLP platform as an effective way of expressing HA stem antigen and revealed that chimeric VLP is an vaccine vector for developing HA stem-based universal influenza vaccines.

[Research progress of internal tension relieving technique in assisting anterior cruciate ligament reconstruction].

OBJECTIVE: To review the research progress of internal tension relieving technique in assisting anterior cruciate ligament (ACL) reconstruction with tendon grafts. METHODS: The in vivo and in vitro biomechanical tests, animal experiments, and clinical studies on the use of internal tensioning relieving technique assisted ACL reconstruction in recent years were extensively reviewed, the impact of this technology on the biomechanics, histological changes of grafts, and the clinical effectiveness were analyzed and summarized. RESULTS: The internal tensioning relieving technique based on non-absorbable high-strength sutures can reduce the risk of relaxation and rupture by enhancing the biomechanical strength of tendon grafts in vitro and in vivo, it shows good biocompatibility and support for the ligamentation of the tendon grafts and the establishment of the direct tendon-bone interface in terms of histology. This technique improves postoperative initial joint stability, range of motion, and functional scores in clinical practic, when combining with the enhanced recovery after surgery can effectively promote patients to return to pre-injury exercise level without serious complications. CONCLUSION: The preliminary research results have confirmed the efficacy and safety of the internal tension relieving technique on assisting ACL reconstruction, then showes some degree of significance and prospect, but more research is needed to further optimize tension-relieving devices and related surgical techniques, and clarify the specific effects of this technique on graft's structure remodeling, biomechanical function, and long-term clinical results.

[Research progress in femoral tunnel positioning points of medial patellofemoral ligament reconstruction].

OBJECTIVE: To review the research progress of location methods and the best femoral insertion position of medial patellofemoral ligament (MPFL) reconstruction of femoral tunnel, and provide reference for surgical treatment. METHODS: The literature about femoral insertion position of the MPFL reconstruction in recent years was extensively reviewed, and the anatomical and biomechanical characteristics of MPFL, as well as the advantages and disadvantages of femoral tunnel positioning methods were summarized. RESULTS: The accurate establishment of the femoral anatomical tunnel is crucial to the success of MPFL reconstruction. At present, there are mainly two kinds of methods for femoral insertion: radiographic landmark positioning method and anatomical landmark positioning method. Radiographic landmark positioning method has such advantages as small incision and simple operation, but it can not be accurately positioned for patients with severe femoral trochlear dysplasia. It is suggested to combine with the anatomical landmark positioning method. These methods have their own advantages and disadvantages, and there is no unified positioning standard. In recent years, the use of three-dimensional design software can accurately assist in the MPFL reconstruction, which has become a new trend. CONCLUSION: Femoral tunnel positioning of the MPFL reconstruction is very important. The current positioning methods have their own advantages and disadvantages. Personalized positioning is a new trend and has not been widely used in clinic, its effectiveness needs further research and clinical practice and verification.

Construction of tissue engineering bone with the co­culture system of ADSCs and VECs on partially deproteinized biologic bone in vitro: A preliminary study.

Scaffold­based bone tissue engineering has therapeutic potential in the regeneration of osseous defects. The present study aimed to explore the adhesion and cell viability of a co­culture system composed of vascular endothelial cells PI­/Annexin V+ represents early apoptotic cells, and PI+/Annexin V+ represents late apoptotic cells (VECs) and adipose­derived stem cells (ADSCs) on partially deproteinized biologic bone (PDPBB) in vitro, and determine the optimum time period for maximum cell viability that could possibly be used for standardizing the scaffold transplant into the in vivo system. VECs and ADSCs were isolated from pregnant Sprague­Dawley rats and confirmed by immunostaining with von Willebrand factor and CD90, respectively. PDPBB was prepared using standardized protocols involving coating partially deproteinized bone with fibronectin. PDPBB was incubated in a mono­culture with VECs or ADSCs, or in a co­culture with both of these cells at a ratio of 1:1. An MTT assay was used to assess the adhesion and cell viability of VECs and ADSCs on PDPBB in the three different cultures. Scanning electron microscopy was used to observe the adhesion, cell viability and morphology of the different types of cells on PDPBB. It was observed that the absorbance of each group increased gradually and peaked on the 10th day; the highest absorbance was found for the co­cultured cells group. The difference of cell viability between each cell group was statistically significant. On the 10th day, in the co­cultured cells group, several cells adhered on the PDPBB material and a nest­like distribution morphology was observed. Therefore, the adhesion and cell viability of the co­cultured cells was higher compared with the mono­cultures of VECs or ADSCs. As cell viability was highest on the 10th day, this could be the optimal length of time for incubation and therefore could be used for in vivo experiments.