BACKGROUND:The micro/nanostructured gradient biomimetic surface of implant materials can simulate the structure of the extracellular environment in human bone tissue,thereby achieving perfect bone integration function.However,further research is needed on the mechanisms by which the surface microstructure of bone implant materials regulates cell function and promotes osteogenesis. OBJECTIVE:To analyze the effect of titanium sheet microstructure surface on osteogenic differentiation of MC3T3-E1 osteoblasts. METHODS:(1)At a constant voltage of 5 V or 20 V,nanotube arrays of different diameters were prepared on the surface of titanium sheets by acid etching and anodic oxidation techniques,and were recorded as group R5 and group R20,respectively.The surface morphology,roughness,and hydrophilicity of pure titanium sheet(without acid etching or anodizing treatment)were measured in group R5 and group R20.(2)MC3T3-E1 osteoblasts of logarithmic growth stage were inoculated on the surface of pure titanium sheets,R5 group and R20 group respectively.After 24 hours of osteogenic induction culture,the expression of mechanical sensitive channel protein 1 was analyzed by RT-PCR and immunofluorescence staining.Osteoblast inducible base with or without the mechanosensitive channel protein 1 activator Yada1 was added,and alkaline phosphatase staining was performed after 7 days of culture.Alizarin red staining was performed after 14 days of culture. RESULTS AND CONCLUSION:(1)The surface of pure titanium sheets was smooth under scanning electron microscope.Relatively uniform and orderly nanotube arrays with average diameters of about 30 nm and 100 nm were observed on the surface of titanium sheets of groups R5 and R20,respectively.The results of scanning electron microscope were further verified by atomic force microscopy.The surface roughness of titanium sheet of group R5 was higher than that of pure titanium(P<0.05),and the water contact angle was lower than that of pure titanium(P<0.05).The surface roughness of titanium sheet in group R20 was higher than that in group R5(P<0.05),and the water contact angle was lower than that in group R5(P<0.05).(2)RT-PCR and immunofluorescence staining showed that the expression of mechanosensitive channel protein 1 in group R5 was higher than that in pure titanium group(P<0.05),and the expression of mechanosensitive channel protein 1 in group R20 was higher than that in group R5(P<0.05).Under the osteogenic induction,compared with the condition without Yada1,there were no significant changes in the activity of alkaline phosphatase and the deposition of calcified nodules in pure titanium group after Yada1 addition,while the activity of alkaline phosphatase and the deposition of calcified nodules in groups R5 and R20 after Yada1 addition were significantly increased(P<0.05).With or without Yada1,the alkaline phosphatase activity and calcified nodule deposition in group R5 were higher than those in pure titanium group(P<0.05),and the alkaline phosphatase activity and calcified nodule deposition in group R20 were higher than those in group R5(P<0.05).(3)The results show that the surface microstructure of titanium sheet can promote the osteogenic differentiation of osteoblast MC3T3-E1 by activating mechanosensitive channel protein 1.

This work aims to explore the effect of glycolysis on the replication of porcine reproductive and respiratory syndrome virus (PRRSV) in porcine alveolar macrophages (PAMs). The changes of glucose metabolism, PRRSV protein levels, PRRSV titers, and the relative expression levels of genes and proteins in PAMs were analyzed by ELISA, qPCR, virus titration, and Western blotting after PRRSV infection. The effect of hypoxia-inducible factor-1α (HIF-1α) on PRRSV replication was subsequently assessed by specific siRNAs targeting to HIF-1α. The results showed that PRRSV infection enhanced glycolysis, elevated the levels of glucose uptake and lactate in the supernatant (P<0.05 and 0.01, respectively), reduced ATP production (P<0.05), and up-regulated the expression of hexokinase 2 (HK2), 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3), and pyruvate kinase isozyme type M2 (PKM2) in PAMs (P<0.05 and 0.01, respectively). Glycolysis inhibitors down-regulated the expression of PRRSV proteins and reduced virus titers (P<0.01). The knockdown of HIF-1α by siRNAs inhibited glycolysis and lowered PRRSV titers (P<0.05). In addition, the interferon pathways inhibited by PRRSV infection were reversed by the inhibition of glycolysis. These findings may facilitate further investigation of the role of glycolysis in PRRSV replication.
Porcine respiratory and reproductive syndrome virus/physiology* ; Glycolysis ; Swine ; Animals ; Virus Replication ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Macrophages, Alveolar/metabolism* ; Porcine Reproductive and Respiratory Syndrome/virology* ; Cells, Cultured ; RNA, Small Interfering/genetics*

Objective:To investigate the genetic safety of allogeneic bone marrow mesenchymal stem cells (BMSCs) transplantation in traumatic brain injury (TBI).Methods:(1) In vivo experiment: BMSCs from male SD rats were isolated and cultured. Moderate TBI models were prepared by implanting and fixing micro-drug injection cannula into the left ventricle of 12 female SD rats, and 3 d after that, striking the right cerebral cortex of the rats with pneumatic precision percussion device was performed. Four h, and 3, 6, 9, and 12 d after modeling, TBI rats were given a single/multiple BMSCs infusion (2.5×10 5/time, total volume 10 μL) by cannula; 48 and 72 h, and 10 and 14 d after modeling, brain tissues of TBI rats (3 at each time point) were prepared into paraffin specimens. Immunofluorescent staining was used to detect the microglia activation, and RNAscope ? technology was used to detect the co-localization of astrocytes, neurons, microglia and transplanted BMSCs to observe whether the allogeneic BMSCs were integrated with the host brain cells after transplantation into TBI host. (2) In vitro experiment: the frozen and revived microglial cell line BV2 was transfected with green fluorescent protein (GFP)-positive lentiviral particles, and then, BMSCs prelabeled with pHrodo RED probe and BV2 cells pretreated with lipopolysaccharide were co-cultured in a certain ratio (BV2:BMSCs=1:1, 1:2, 2:1); after 36 and 72 h of co-culture, the phagocytosis between the 2 kinds of cells was observed under confocal fluorescence inverted microscope to observe the specific action forms of microglia on BMSCs. Results:(1) In vivo experiment: 48 and 72 h, and 10 and 14 d after modeling, no colocalization of transplanted BMSCs with astrocytes or neurons was found in paraffin sections of brain tissue in TBI rats; however, 10 and 14 d after modeling, microglia in TBI rats were obviously activated and migrated to the left lateral ventricle and choroid plexus, and co-localization of microglia with transplanted BMSCs was observed. (2) In vitro experiment: phagocytosis occurred after co-culture of BV2 cells at different proportions with BMSCs for 36 and 72 h. Conclusion:After transplantation, allogeneic BMSCs do not integrate with astrocytes or neurons of the TBI host, but they could be phagocytosed by microglia, indicating that allogeneic BMSCs transplantation for TBI is genetically safe.

Objective To analyze the characteristics of the muscle strength around the knee joint of chondromalacia patellae patients, and to explore the difference with normal people.Methods In March, 2021, 70 knee-onset chondromalacia patellae patients (experimental group) and 35 normal people (control group) were measured isokinetic muscle strength of flexion and extension of knee in angular velocities of 60°/s and 180°/s.Results At 60°/s and 180°/s, the peak torque, the peak torque-to-weight ratio and the total work of the flexor and extensor muscles on the affected side in the experimental group were lower than that of the control group (U > 1097.0, P<0.001). The peak torque, the peak torque-to-weight ratio and the total work of the flexor and extensor muscles at 60°/s and extensor muscles at 180°/s were lower on the affected side than on the healthy side in the experimental group (|Z| > 2.121, P<0.05). The peak torque ratios at 60°/s and 180°/s were more in the affected knees than in the healthy knees of experimental group and in the control group (U > 1810.0, |Z| >3.691, P<0.01).Conclusion The explosive force and endurance of the knee flexor and extensor has weakened in patients with chondromalacia patellae, and there is imbalance in knee joint muscle strength.

Objective:To develop a mesenteric creeping fat index (MCFI) based on CT enterography (CTE) to characterize the degree of creeping fat wrapping around the inflamed gut in Crohn disease (CD), and to assess the relationship between MCFI and the inflammatory intestinal stricture.Methods:From December 2018 to July 2019, the patients with CD who underwent surgery in the First Affiliated Hospital of Sun Yat-Sen University were prospectively collected. The extent of perienteric mesenteric vessels wrapping around the gut was reconstructed to develop MCFI based on CTE images. The intestinal stricture index was obtained by calculating the ratio of the maximal upstream luminal diameter divided by the minimum luminal diameter apparent within the stricturing region. Using region-by-region correlation between CTE and surgical specimen, creeping fat score in intestinal specimen was obtained by assessing the extent of creeping fat wrapping around the resected bowel segment, and HE staining was performed on the bowel specimen corresponding to creeping fat to obtain the pathological inflammatory score. The Spearman correlation analysis was used to evaluate the correlation between MCFI, creeping fat score in intestinal specimen, and inflammatory score, intestinal stricture index. The ROC curve analysis was used to assess the accuracy of MCFI in distinguishing moderate-severe and mild inflammatory bowel walls.Results:Totally 30 CD patients were enrolled. The creeping fat score in intestinal specimen positively correlated with pathological inflammatory score ( r s=0.403, P=0.027) and with intestinal stricture index ( r s=0.642, P<0.001). MCFI positively correlated with creeping fat score in intestinal specimen ( r s=0.840, P<0.001), with pathological inflammatory score ( r s=0.497, P=0.005), and with intestinal stricture index ( r s=0.599, P<0.001). ROC analysis showed that the area under the curve of MCFI for differentiating moderate-severely from mildly inflammatory bowel walls was 0.718 (95%CI 0.522-0.913). Using MCFI≥4 as a cutoff value, the sensitivity and specificity were 81.8% and 47.4%, respectively. Conclusions:There was a correlation between creeping fat and inflammatory intestinal strictures in CD. MCFI can non-invasively depict the degree of creeping fat wrapping around the gut and assess the inflammatory intestinal stricture.

Objective:To explore the diagnostic efficacy of nomogram based on multi-parameter MRI for assessment of bowel fibrosis in patients with Crohn disease（CD).Methods:The clinical and imaging data of CD patients diagnosed by surgical histopathology in the First Affiliated Hospital of Sun Yat-sen University from June 2015 to March 2018 were prospectively collected. All the patients underwent conventional MRI and diffusion kurtosis imaging（DKI) within 2 weeks before surgery. Patients who underwent surgery between June 2015 and September 2017 were included in the model building group, and those who underwent surgery between October 2017 and March 2018 were included in the model validation group. We measured the apparent diffusion coefficient（ADC) from monoexponential model of diffusion-weighted imaging（DWI), apparent diffusional kurtosis（K app), and apparent diffusion for non-Gaussian distribution（D app) from non-Gaussian DKI model, and observed T 2WI signal intensity and enhancement pattern of the same segment. One to three intestinal specimens per patient were stained with Masson′s trichrome for the histological grading of fibrosis. Correlations between qualitative/quantitative MRI indexes and histological grades were evaluated using the Spearman rank test. Multivariate logistic regression analysis was performed to identify independent factors to be included into the nomogram for predicting the degree of bowel fibrosis and its diagnostic performance was assessed by internal and external validation. Results:A total of 40 CD patients were included, including 31 in the model construction group and 9 in the model verification group. A total of 81 intestinal specimens from 31 patients were graded as none-to-mild bowel fibrosis（ n=32) and moderate-to-severe bowel fibrosis（ n=49) according to a scoring system of fibrosis. In the training cohort, the K app value of moderate-to-severely fibrotic bowel walls was significantly higher than that of none-to-mildly fibrotic bowel walls, and the D appand ADC values of moderate-to-severely fibrotic bowel walls were significantly lower than those of none-to-mildly fibrotic bowel walls（ Z=-5.999, -4.521 and -3.893; P<0.001). There was no significant difference in T 2WI signal intensity or enhancement pattern between these two groups（χ2=1.571 and 0.103; P>0.05). Moderate and mild correlations of histological fibrosis grades with K appand D app( r=0.721 and -0.483; P<0.001), and a mild correlation with ADC（ r=-0.445, P<0.001) were found. Independent factors derived from multivariate logistic regression analysis to predict the degree of bowel fibrosis were K app and D app. Internal and external validation revealed good performance of the nomogram with concordance index of 0.901（95% confidence interval, 0.824-0.978) and 1.000, respectively, for differentiating none-to-mild from moderate-to-severe fibrosis. Conclusion:The DKI-based nomogram can be used to evaluate the bowel fibrosis in CD patients and provides a visual and simple prediction method for clinic.

Objective To determine the correlation between intravoxel incoherent motion (IVIM) parameters and both histologic inflammatory and fibrotic grades of Crohn disease (CD) in adults. Methods Prospectively, 17 patients (77 lesions) with a clinical and pathological diagnosis of CD in the first affiliated hospital of sun yat-sen university from July 2015 to June 2016 underwent MRE 15 days before surgery. All patients underwent T2WI, IVIM and enhanced MRI and calculated IVIM parameters include diffusion-related coefficient (D), perfusion-related coefficient (D*) and perfusion-related fraction (f). Histological intestinal inflammation and fibrosis was scored using the surgical histopathology as reference standard and further divided into mild-moderate (score 1 to 2) and severe (score 3 to 4) groups. Intestinal microvessel density (MVD) were also analyzed. Differences in IVIM parameters among different histological inflammation and fibrosis grades were assessed with the Kruskal-Wallis test. The Wilcoxon test was used for assessing differences in f between mild-moderate and severe fibrosis. The bivariate correlations between IVIM parameters and histological inflammation and fibrosis grades were analyzed using partial correlation . The bivariate correlations between IVIM parameters and MVD were analyzed using Spearman rank correlation. The areas under the receiver operating characteristics curves (AUROC) were analyzed to evaluate the efficacy for distinguishing severe from mild-moderate fibrosis. Results Of 77 surgical specimens, there were 41 mild-moderate and 36 severe inflammatory bowel segments, along with 22 mild-moderate and 55 severe fibrotic bowel segments. Positive correlation was shown between histologic inflammatory and fibrotic scores (r=0.592, P<0.01). MVD (42.7 ± 39.9)/HP presented weak positive correlation with histologic inflammatory scores (r=0.332, P=0.003) while no correlation with histologic fibrotic scores (r=0.129, P=0.262) was presented. Neither the D nor the D* values significantly correlated with histologic inflammation or fibrosis (P>0.05) while the f value significantly correlated with both histologic inflammation and fibrosis (P<0.05). Significant correlation was present between the f value and histologic inflammatory and fibrotic scores, respectively (r=-0.280, -0.520;P<0.05). There was significant difference in the f value between mild-moderate and severe fibrosis(Z=-5.255,P<0.01). The AUROC for the f value to distinguish between patients with mild-moderate fibrosis and severe fibrosis were 0.885. Using a threshold fractional perfusion of 0.33, the sensitivity and specificity values were 95.5％ and 81.8％, respectively. No correlation between f, D and D*value with histologic fibrotic scores (r=0.129, P=0.262) was presented. Conclusion The f value derived from IVIM could help to evaluate the severity of intestinal inflammation and fibrosis CD in adults.

Objective To assess the diagnostic value of magnetization transfer MRI (MTI) for bowel inflammation and fibrosis in humans with Crohn disease (CD). Methods From July 2014 through April 2017, 31 patients with a confirmed diagnosis of CD were prospectively recruited from the First Affiliated Hospital of Sun Yat Sen University. They were scheduled for elective surgery due to bowel obstruction and other complications, and underwent preoperative MR enterography (MRE) and MTI within 15 days of surgery. All cases had available intestinal specimens identified on MRE and resected bowel segments for region by region matching. All patients underwent breath hold conventional MRE and MTI examinations, and then the magnetization transfer ratios (MTRs) of pathological bowel segments were measured. Using region by region correlation between MTI and surgical specimen, the bowel segments were resected to stain with HE for evaluating bowel inflammation, Masson for bowel fibrosis, and typeⅠcollagen staining for the deposition of typeⅠcollagen within the bowel walls. The histologic sections from the most severe areas were scored as 0 (normal), 1 (mild), 2 (moderate) and 3 (severe). The correlations between MTR and histologic scores were analyzed by using Spearman rank correlation or partial correlation. The differences in MTR among different grades of bowel fibrosis were analyzed by one way ANOVA. The efficacy of MTR for predicting bowel fibrosis was evaluated by receiver operating characteristic curves analysis. The difference in MTRs between purely inflammatory bowel walls and mixed fibrotic and inflammatory bowel walls was analyzed by Student s t test. Results Sixty two resected bowel specimens from 31 patients including 9 purely inflammatory bowel walls and 53 mixed fibrotic and inflammatory bowel walls were obtained in this study. There were significant differences in MTR among non fibrotic [(21.45 ± 2.65)%], mildly [(30.88 ± 6.14)%], moderately [(35.14 ± 4.31)%] and severely [(35.14 ± 4.31)%] fibrotic walls (F=38.397,P<0.01). MTRs strongly correlated with fibrosis scores (r=0.681, P<0.01). High accuracy of MTRs was shown (curve under area=0.905, P<0.01) for differentiating moderately severely fibrotic from non fibrotic and mildly fibrotic bowel walls. Using MTR of 31.50% as a cutoff value, the sensitivity and specificity were 93.6% and 80.0%, respectively. The MTRs of purely inflammatory bowel walls [(21.45 ± 2.65)%] were significantly higher than that of mixed fibrotic and inflammatory [(36.28±5.21)%] bowel walls (t=-13.052,P<0.01). MTRs correlated with the scores of type Ⅰ collagen (r=0.325, P=0.044) but did not correlate with inflammation scores (r=-0.024, P=0.857). Conclusions MTI enables quantitative evaluation of bowel fibrosis in patients with CD and can be used to differentiate purely inflammatory CD from mixed fibrotic and inflammatory CD.

[Objective] To analyze the image findings of MR enterography (MRE) and diffusion weighted imaging (DWI) in Crohn disease (CD) and to discuss the diagnostic value of MRI for evaluating the activity of CD.[Methods] 26 patients proved by clinical,imaging and pathological data were enrolled in this study.The intra-/extra-intestinal image features of CD were retrospectively analyzed,and the intestinal lesions were evaluated by MRE+DWI scoring system.The differences in image features of active and inactive CD were compared.[Results] 26 bowel segments were evaluated and included active (n =20) and inactive CD (n =6).The intestinal performance of CD included wall thickening,T2WI hyperintensity and hyperenhancement.The pathological changes of active CD were more serious when comparing with that of inactive CD.DWI showed hyperintensity in the affected bowel wall.ADC of active C D [(0.9 ± 0.2) × 10-3 mm2/s] was significantly lower than that of inactive CD [(1.5 ± 0.4)× 10-3 mm2/s] (P ＜ 0.001).Extra-enteric performance of active CD included comb sign,lymphadenectasis of mesentery,mesenteric exudation,fistula and abdominal abscess.[Conclusion] MRE combined with DWI can accurately assess CD activity and diagnose extra-enteric complications.

[Objective]To explore the efficacy of T2* mapping for evaluating inflammatory activity in the patients with Crohn disease(CD).[Methods]A total of 98 CD patients underwent MR enterographywith T2*WI. T2* values were measured by put-ting regions of interest on the thickening bowel wall on T2*mapping. The activity of bowel segment was scored by magnetic resonance index of activity(MaRIA),to analyze the relationship between T2*values with CD activity.[Results]A total of 160 bowel segments were evaluated and includedinactive(MaRIA<7,n=26),mild(7≤MaRIA<11,n=23),and moderately-severe(MaRIA≥11, n = 111)active lesions. The differences in T2* values amongthese three groups were significant(all P < 0.05). T2* values of 160 bowel segments correlatedclosely withMaRIA(r=0.743,P<0.05). High accuracy of T2*values was shown for differentiating inac-tive from active CD(AUC=0.877)anddifferentiating inactive-mild from moderate-severe CD(AUC=0.848). The threshold T2*value of 20 ms allowed differentiation of mild from moderate-severe CD with74.5%sensitivity and 84%specificity.[Conclusions]T2*values, as thequantitative indexof T2*mapping,correlate well with CD activity and showsatisfiedefficacy for diagnosing inflammatoryactivity.