BACKGROUND:Gradient artificial bone repair scaffolds can mimic unique anatomical features in musculoskeletal tissues,showing great potential for repairing injured musculoskeletal tissues. OBJECTIVE:To review the latest research advances in gradient artificial bone repair scaffolds for tissue engineering in the musculoskeletal system and describe their advantages and fabrication strategies. METHODS:The first author of the article searched the Web of Science and PubMed databases for articles published from 2000 to 2023 with search terms"gradient,bone regeneration,scaffold".Finally,76 papers were analyzed and summarized after the screening. RESULTS AND CONCLUSION:(1)As an important means of efficient and high-quality repair of skeletal system tissues,gradient artificial bone repair scaffolds are currently designed bionically for the natural gradient characteristics of bone tissue,bone-cartilage,and tendon-bone tissue.These scaffolds can mimic the extracellular matrix of native tissues to a certain extent in terms of structure and composition,thus promoting cell adhesion,migration,proliferation,differentiation,and regenerative recovery of damaged tissues to their native state.(2)Advanced manufacturing technology provides more possibilities for gradient artificial bone repair scaffold preparation:Gradient electrospun fiber scaffolds constructed by spatially differentiated fiber arrangement and loading of biologically active substances have been developed;gradient 3D printed scaffolds fabricated by layered stacking,graded porosity,and bio-3D printing technology;gradient hydrogel scaffolds fabricated by in-situ layered injections,simple layer-by-layer stacking,and freeze-drying method;and in addition,there are also scaffolds made by other modalities or multi-method coupling.These scaffolds have demonstrated good biocompatibility in vitro experiments,were able to accelerate tissue regeneration in small animal tests,and were observed to have significantly improved histological structure.(3)The currently developed gradient artificial bone repair scaffolds have problems such as mismatch of gradient scales,unclear material-tissue interactions,and side effects caused by degradation products,which need to be further optimized by combining the strengths of related disciplines and clinical needs in the future.

Objective:To investigate the expression of squamous cell heat shock protein 53(CRNN)in esophageal squamous cell carcinoma(ESCC),and toevaluate its impact on the biological behavior of ESCC cells Eca9706.Methods:Immunohistochemical method was used to detect the expression of CRNN protein in 93 ESCC tissues and 101 normal esophageal epithelial tissues adjacent to cancer,and the associations of CRNN expression levels with the clinical pathological characteristics and survival prognosis of ESCC patients were analyzed.Receiver operating characteristic(ROC)curve was used to analyze the predictive performance of CRNN expression level on ESCC.The Eca9706 cells were divided into control group and CRNN group(overexpression of CRNN).Cell counting kit-8(CCK-8)assay was used to detect the proliferation activities of Eca9706 cells in two groups;Transwell chamber assay was used to detect the numbers of migration cells of Eca9706 cells in two groups;plate clone formation assay was used to assess the numbers of clone formation of Eca9706 cells in two groups;flow cytometry was used to detect the apoptotic rates of Eca9706 cells in two groups.Results:Compared with adjacent normal esophageal epithelial tissue,the expression intensity of CRNN protein in ESCC tissue was significantly decreased(x2=23.476,P＜0.001).The downregulation of CRNN protein expression in ESCC patients was associated with tumor location(x2=5.353,P=0.021)and histological grade(x2=4.434,P=0.035),but not with age(x2=0.102,P=0.750),gender(x2=0.050,P=0.822),tumor stage(x2=0.047,P=0.828)or lymph node metastasis(x2=0.553,P=0.457).Survival analysis showed that ESCC patients in high expression of CRNN protein group had better prognosis than those in low expression of CRNN protein group(P=0.013).Univariable Cox proportional hazards regression analysis showed the associations between overall survival rate in ESCC patients and the expression level of CRNN protein[hazard ratio(HR)=0.198,95％confidence interval(CI):0.047-0.842,P=0.028]and tumor stage(HR=2.479,95％CI:1.247-4.929,P=0.010).Multivariable Cox regression analysis showed that the expression level of CRNN protein(HR=0.213,95％CI:0.050-0.895,P=0.035)and tumor stage(HR=2.391,95％CI:1.198-4.772,P=0.013)were independent factors for the prognosis of ESCC.Compared with control group,the proliferation activity of cells in CRNN group was significantly decreased(P=0.004),the number of clone formation was decreased(P=0.002),the number of migration cells was decreased(P=0.002),and the apoptotic rate was significantly increased(P=0.006).Conclusion:Low expression level of CRNN protein suggests poor prognosis for the ESCC patients.Overexpression of CRNN may inhibit the proliferation,migration and invasion abilities of ESCC cells,and promote their apoptosis.

Objective:To discuss the expression of Rh family C glycoprotein(RHCG)in the esophageal squamous cell carcinoma(ESCC)tissue and its effect on the malignant biological behavior of ESCC cells,and to clarify the value of RHCG as a diagnostic and prognostic marker for the ESCC patients.Methods:A total of 143 ESCC tissue samples and 105 adjacent normal tissue samples were collected.Using immunohistochemical staining method,141 ESCC samples were divided into two groups:RHCG low expression group(immunohistochemistry score≤6)and RHCG high expression group(immunohistochemistry score>6).Immunohistochemical method was used to detect the RHCG protein expression in 143 ESCC tissues and 105 normal tissues,and the relationship between the clinicopathological characteristics of the ESCC patients was analyzed.Receiver operating characteristic(ROC)curve and Kaplan-Meier survival analysis were used to evaluate the value of RHCG in diagnosis and prognosis of the ESCC patients;univariate and multivariate COX regression analysis were used to determine the independent risk factors affecting the prognosis of the ESCC patients.Gene Expression Profiling Interactive Analysis(GEPIA2)database was used to analyze the expression of RHCG mRNA in various tumor tissues.The ESCC TE-1 cells were cultured and transfected in to 6-well cell culture plates with different Lipofectamine2000∶RHCG ratios;the cells in RHCG transfection group were transfected with weights of 2.0,2.5,and 3.0 μg for 24 and 48 h,respectively,and the cells in NC group transfected with empty vector as control.Western blotting method was used to detect the RHCG protein expression level in the TE-1 cells in various groups after transfection at different concentrations and verify the optimal transfection conditions;cell counting kit-8(CCK-8)assay was used to detect the proliferation activities of the TE-1 cells;plate clone formation assay was used to detect the colony formation numbers of the TE-1 cells;Transwell chamber assay was used to detect the numbers of migrating TE-1 cells.Results:Compared with adjacent normal tissue,the RHCG gene expression level in various cancer tissues including ESCC,glioblastoma multiforme,and head and neck squamous cell carcinoma was significantly decreased(P<0.05).RHCG protein was mainly located on the cell membrane of normal esophageal squamous epithelial cells;the RHCG protein expression intensity in ESCC tissues was lower than that in adjacent normal esophageal tissue(χ2=109.373,P<0.001),and the patients in RHCG low expression group had poorer differentiation than those in RHCG high expression group(P=0.041).The area under the curve(AUC)value of RHCG for diagnosing ESCC was 0.86,with sensitivity and specificity of 95.1%and 75.0%,respectively;the Kaplan-Meier survival analysis results showed that compared with high RHCG expression group,the patients in low RHCG expression group had shorter survival time and poorer prognosis[harard ratio(HR)=0.269,95%confidence interval(CI):0.113-0.639,P=0.020];the COX regression analysis results showed that low RHCG expression could serve as an independent risk factor affecting the prognosis of ESCC[HR=4.569,95%CI=1.315-15.877,P=0.017)].The Western blotting results verified that the optimal transfection condition was 3.0 μg RHCG plasmid for 48 h,at which time RHCG overexpression was optimal and RHCG protein expression level was highest.The CCK-8 assay results showed that compared with control group,the proliferation activity in RHCG overexpression group was decreased on the 4th day after cell seeding(P<0.001).In the TE-1 cells,the colony formation number of the TE-1 cells in RHCG over-expression group was lower than that in control group(t=17.70,P<0.001).The Transwell chamber assay results showed that compared with control group,the number of migrating cells in RHCG over-expression group was decreased(t=23.74,P<0.001).Conclusion:RHCG expression is decreased in ESCC tissues and associated with poor prognosis in ESCC patients;overexpression of RHCG can inhibit the proliferation and migration of the TE-1 cells,providing a theoretical basis for RHCG as a novel diagnostic and prognostic marker and therapeutic target for ESCC.

Objective To analyze and identify the volatile constituents in different parts(flowers,stems and leaves)of Huai chrysanthemumin,and to lay a theoretical foundation for the comprehensive utilization for it.Methods The volatile oil in different parts of Huai chrysanthemumin were extracted by hydrodistillation,respectively.Their constituents were analyzed by gas chromatography-mass spectrometry(GC-MS).The compounds were identified by library search and literature screening.The relative percentage of each compound was obtained by the area normalization method.The differences in their chemical compositions were analyzed by Venn diagram,principal component analysis(PCA)and cluster heat map analysis.Results A total of 62 volatile chemical components were identified from different parts of Huai chrysanthemumin,including monoterpenes,sesquiterpenes,and their derivatives,as well as a small amount of aliphatic compounds.32,42 and 40 volatile components were detected from the flowers,stems and flowers,respectively.Furthermore 17 volatile components were shared by three parts,whereas 5,6 and 16 volatile components were unique to the flowers,stems and leaves,respectively.The results of stoichiometric analysis showed that both PCA and cluster heat map analysis could separate the flowers,stems and leaves,and their volatile components were different.Conclusion The types and contents of the volatile oil in the stems,leaves and flowers of Huai chrysanthemumin have certain variability,which provide a scientific basis for the further medicinal or industrial exploitation of different parts of Huai chrysanthemumin.

Objective:Diabetic retinopathy (DR) has been reported as the leading cause of blindness among diabetic adults, which is closely related to poor quality of life and increased burden of disability. This study aimed to aggregate the optimally available evidence on modifiable risks of DR.Methods:Until June 2023, PubMed, Cochrane Library, CNKI, and Wanfang databases were used to retrieve Meta-analysis about various risk factors for DR, and Meta-analysis were analyzed and summarized. R 4.3.2 software was used for each Meta-analytic association to calculate the effect size, 95% CI, heterogeneity, small-study effects, excess significance bias, and 95% prediction intervals. The credibility of significant evidence was graded. Results:We captured 23 eligible papers (72 associations) covering a wide range of medication use, concomitant diseases, daily intervention, biomarkers, lifestyle, and physical measurement index. Among them, higher HbA1c variability ( RR=1.45, 95% CI: 1.26-1.66) and urine microalbumin positive ( OR=2.44, 95% CI: 1.99-2.97) were convincing (grade Ⅰ) evidence, and insulin use ( RR=3.48, 95% CI: 2.14-5.67) was highly suggestive (grade Ⅱ) evidence. Moreover, hypertension ( OR=2.03, 95% CI: 1.06-3.97), poor glycemic control ( OR=4.35, 95% CI: 1.47-12.85), positive macroalbuminuria ( OR=8.42, 95% CI: 3.52-20.15), long sleep duration ( OR=2.05, 95% CI: 1.37-3.05), vitamin D deficiency ( OR=2.02, 95% CI: 1.17-3.50), periodontitis ( OR=4.51, 95% CI: 1.76-11.55) were the main risk factors for DR. Intensive blood pressure intervention ( RR=0.78, 95% CI: 0.65-0.94), dietary control ( OR=0.64, 95% CI: 0.47-0.89) and moderate intensity physical activity ( RR=0.76, 95% CI: 0.59-0.97) yielded significant protective associations with DR. Conclusions:Intensive blood pressure glycemic control, and a healthy lifestyle pattern could reduce the risk of DR. This study provides the evidence to identify high-risk populations and recommends rational treatment options and healthy living interventions.

Background Bacteria are the most diverse and widely sourced microorganisms in the indoor air of subway stations, where pathogenic bacteria can spread through the air, leading to increased health risks. Objective To understand the status and distribution characteristics of indoor air bacterial pollution in subway stations and compartments in a city of Central South China, and to provide a scientific basis for formulating intervention measures to address indoor air bacteria pollution in subways. Methods Three subway stations and the compartments of trains parking there in a city in Central South China were selected according to passenger flow for synchronous air sampling and monitoring. Temperature, humidity, wind speed, carbon dioxide (CO₂), fine particulate matter (PM_2.5), and inhalable particulate matter (PM₁₀) were measured by direct reading method. In accordance with the requirements of Examination methods for public places-Part 3: Airborne microorganisms (GB/T 18204.3-2013), air samples were collected at a flow rate of 28.3 L·min⁻¹, and total bacterial count was estimated. Bacterial microbial species were identified with a mass spectrometer and pathogenic bacteria were distinguished from non-pathogenic bacteria according to the Catalogue of pathogenic microorganisms transmitted to human beings issued by National Health Commission. Kruskal-Wallis H test was used to compare the subway hygiene indicators in different regions and time periods, and Bonferroni test was used for pairwise comparison. Spearman correlation test was used to evaluate the correlation between CO₂ concentration and total bacterial count. Results The pass rates were 100.0% for airborne total bacteria count, PM_2.5, and PM₁₀ in the subway stations and train compartments, 94.4% for temperature and wind speed, 98.6% for CO₂, but 0% for humidity. The overall median (P₂₅, P₇₅) total bacteria count was 177 (138,262) CFU·m⁻³. Specifically, the total bacteria count was higher in station halls than in platforms, and higher during morning peak hours than during evening peak hours (P<0.05). A total of 874 strains and 82 species were identified by automatic microbial mass spectrometry. The results of identification were all over 9 points, and the predominant bacteria in the air were Micrococcus luteus (52.2%) and Staphylococcus hominis (9.8%). Three pathogens, Acinetobacter baumannii (0.3%), Corynebacterium striatum (0.1%), and Staphylococcus epidermidis bacilli (2.2%) were detected in 23 samples (2.6%), and the associated locations were mainly distributed in train compartments during evening rush hours. Conclusion The total bacteria count in indoor air varies by monitoring sites of subway stations and time periods, and there is a risk of opportunistic bacterial infection. Attention should be paid to cleaning and disinfection during peak passenger flow hours in all areas.

Objective:To analyze the application value of color Doppler ultrasound,microribonucleic acid-19a(miR-19a)and miR-210 in carotid artery stenosis.Methods:A total of 102 patients with carotid artery stenosis who were diagnosed and treated in Cangzhou People's Hospital from October 2020 to October 2022 were selected as the research group,and 102 health persons who underwent color Doppler ultrasound of physical examination in hospital at the same period were selected as the health control group.Both two groups underwent color Doppler ultrasound examination and digital subtraction angiography(DSA)within one week after admitted to hospital.The miR-19a and miR-210 between the two groups were compared,and the miR-19a and miR-210 of patients with different stenosis degrees of research group also were compared.And then,the diagnostic values of color Doppler,miR-19a,miR-210 and the combination of them were analyzed in diagnosing carotid artery stenosis.Results:The results of color Doppler ultrasound in detecting common carotid artery 102 patients of research group indicated that 15 cases were normal,and 15 cases occurred mild stenosis,and 21 cases occurred moderate stenosis,and 21 cases occurred severe stenosis and 33 cases occurred complete occlusion.The miR-19a of the research group was significantly higher than that of the health control group,and the miR-210 of the research group was significantly lower than that of the health control group(t=10.755,-5.903,P<0.05).The miR-19a results of the research group indicated that the miR-19a of mild stenosisthat of moderate stenosis>severe stenosis>complete occlusion,and the differences were statistical significant(F=160.399,147.462,P<0.05),respectively.The receiver operating characteristic(ROC)curve analysis showed that the area under curve(AUC)values of color Doppler ultrasound,miR-19a,miR-210 and combined diagnosis of them for carotid artery stenosis were respectively 0.892,0.842,0.729 and 0.978,and the sensitivity and specificity of the combined diagnosis were respectively 97.1%and 95.1%.Conclusion:The combined diagnosis of color Doppler ultrasound,miR-19a and miR-210 has higher diagnostic value for carotid artery stenosis.

Objective To investigate the role of histone deacetylase 6(HDAC6)in podocyte injury in diabetic kidney disease(DKD)in mice.Methods 1)The 8-week-old male CD-1 mice were selected to construct the model of DKD with streptozocin(STZ).After the model was established,the mice were intraperitoneally injected with HDAC6 inhibitor CAY10603(5mg/kg/daily)or same volume vehicle as control.The mice were divided into four groups,control(CTL)+vehicle(Veh)(n=5),CLT+CAY10603(n=3),STZ+Veh(n=9),and STZ+CAY10603(n=7).Mice in STZ+Veh and STZ+CAY10603 groups developed DKD,while mice in the CTL+Veh and CTL+CAY10603 groups were served as normal controls.The therapeutic effect was evaluated through urine albumin-to-creatinine ratio(uACR)and renal pathology after the 2-week treatment with CAY10603.2)Human podocytes were cultured in vitro and were divided into four groups as follows:CTL,transforming growth factor-β(TGFβ),TGFβ+CAY10603(250 nmol/L),and TGFβ+CAY10603(500 nmol/L)groups.The control group did not receive any treatment,the last three groups were given 36-h TGFβtreatment at 5 ng/μL,with or without CAY10603 as indicated for an additional 12 h.Western blot was performed to determine the inhibitory effect of CAY10603 on NLRP3 inflammasome.3)HDAC6 knockout(KO)mice were generated and used to create STZ-induced model of DKD.The mice were divided into four groups:C57BL/6J wild type(WT)(n=6),HDAC6 KO(n=6),WT+STZ(n=10),and HDAC6 KO+STZ(n=9).Samples were collected 16 weeks after successful modeling and changes in uACR and renal pathology were evaluated accordingly.Results After 2 weeks of treatment,mice in the STZ+CAY10603 group exhibited reduction in uACR(P＜0.05)and inhibition of glomerular mesangium expansion(P＜0.05)compared with those of the mice in the STZ+Veh group.There was no statistically significant difference in the indicators between the CTL+Veh group and the CTL+CAY10603 group.In vitro cultured podocytes,compared with the control group,NLRP3 inflammasome activation was seen in the TGFβ group.CAY10603 treatment significantly inhibited the activation of NLRP3 in the dosage-dependent manner(P＜0.05).Compared with those of the WT group,the WT+STZ group showed increased uACR(P＜0.05),obvious glomerulosclerosis and loss of podocytes numbers.Compared with those of the WT+STZ group,the HDAC6 KO+STZ group showed effectively reduction of uACR(P＜0.05)and improvement in the renal pathological changes in mice.There was no significant difference in these aspects between the WT and HDAC6 KO groups.Conclusion Inhibition of HDAC6 alleviates proteinuria and podocyte injury in the mouse model of DKD by suppressing the activation of NLRP3 inflammasome.

Objective:To explore the clinical and electrophysiological characteristics of flail arm syndrome (FAS).Methods:Clinical and electrophysiological data were collected on 13 FAS patients and 31 persons with upper limb onset amyotropic lateral sclerosis (UL-ALS), including the amplitude of compound muscle action potential (CMAP) related to median nerve, ulnar nerve, and axillary nerve motor conduction. A split-hand index (SI) was calculated by dividing the CMAP amplitude of abductor pollicis brevis by that of the abductor digiti minimi. Clinical features, the CMAP amplitudes and SIs were compared between the FAS and UL-ALS patients.Results:Compared with UL-ALS patients, the age at onset among the FAS patients was older (averaging 60.9 years). The development to the second stage was longer (24±6 months). The upper limb reflexes of 15% of the FAS patients had disappeared and those of 77% were weakened, while the lower limb reflexes of 54% of the FAS patients were active and 38% were weakened, significantly different from the UL-ALS patients. However, there were no significant differences in the CMAP amplitudes of the median and ulnar nerves, nor in SI between the FAS and UL-ALS patients. The SIs of the FAS patients with upper motor neuron signs were significantly lower than those of FAS patients without such signs. Among the FAS patients, the average CMAP amplitude of the ulnar nerve was the highest, followed by those of the median and axillary nerves. Among the UL-ALS patients, however, the average CMAP amplitude of the ulnar nerve was not significantly different from that of the axillary nerve.Conclusions:FAS patients with upper motor neuron signs are more likely to have slip hand. The CMAP amplitude of their axillary nerve tends to be lower than that of their median and ulnar nerves. FAS seems to be a special type of ALS.

Objective To report the clinical features, myopathological changes, and gene mutations in five Chinese patients with mitochondrial diseases caused by POLG gene mutations. Methods Clinical materials of five unrelated patients who were referred to Department of Neurology, Peking University First Hospital from April 2012 to January 2018, carrying POLG gene mutations, were retrospectively analyzed. Muscle/nerve biopsies and targeted second-generation gene sequencing were performed on the patients. Results Among the five patients, three were male and two were female. Two cases were dominant inheritance and three were sporadic or recessive inheritance. The ages of onset were from 15 to 40 years with disease course of one to 26 years. One of them showed atypical SANDO (sensory ataxic neuropathy, dysarthria, and ophthalmoparesis) syndrome accompanied by cardiac preexcitation syndrome. There were two cases with autosomal dominant and one case with recessive progressive external ophthalmoplegia plus syndrome. One case presented with cognitive delay and sensory neuropathy. The pathological changes of mitochondrial myopathy were observed in all four patients with muscle involvement. Sural nerve biopsy in the patient with cognitive delay and sensory ataxia revealed chronic axonal pathological changes. POLG gene mutations were found in all five patients by targeted next generation sequencing, including single heterozygous mutations in two dominant inherited patients (c. 914 G＞A and c. 2864A＞G, respectively), and compound heterozygous POLG gene mutations in the other three sporadic/recessive inherited patients (c. 2591 A＞G/c. 1790 G＞A, c. 924G＞T/c. 3002delG and c. 1613A＞T/c. 1612 G＞T, respectively). There were six novel mutations not reported before, i.e., c.914G＞A(p.S305N), c.924G＞T(p.Q308H), c.1613A＞T(p.E538V), c.1612G＞T(p.E538*), c.1790 G＞A(p.R597Q) and c.3002delG. Conclusions POLG gene mutations can lead to different clinical spectrums. Progressive external ophthalmoplegia, limb weakness and axonal sensory neuropathy are common presentations in this group of patients with POLG gene related mitochondrial neuromuscular diseases. Novel mutations found in this study expand the mutational spectrum of POLG gene.