Objective:To identify tooth number abnormalities on pediatric panoramic radiographs based on deep learning.Methods:Eight hundred panoramic radiographs of children aged 4 to 11 years meeting the inclusion and exclusion criteria were selected and randomly assigned by writing programs in Python (version 3.9) to the training set (480 images), verification set (160 images) and internal test set (160 images), taken in Department of Pediatric Dentistry, Peking University School and Hospital of Stomatology between November 2012 to August 2020. And all panoramic radiographs of children aged 4 to 11 years taken in the First Outpatient Department of Peking University School and Hospital of Stomatology from June 2022 to December 2022 were collected as the external test set (907 images). All of the 1 707 images were obtained by operators to determine the outline and to label the tooth position of each deciduous tooth, permanent tooth, permanent tooth germ and additional tooth. The deep learning model with ResNet-50 as the backbone network was trained on the training set, validated on the verification set, tested on the internal test set and external test set. The images of test sets were divided into two categories according to whether there was abnormality of tooth number, to calculate sensitivity, specificity, positive predictive value and negative predictive value, and then divided into four types of extra teeth and missing permanent teeth both existed, extra teeth existed only, missing permanent teeth existed only, and normal teeth number, to calculate Kappa values. Results:The sensitivity, specificity, positive predictive value and negative predictive value were 98.0%, 98.3%, 99.0% and 96.7% in the internal test set, and 97.1%, 98.4%, 91.9% and 99.5% in the external test set respectively, according to whether there was abnormality of tooth number. While images were divided into four types, the Kappa value obtained in the internal test set was 0.886, and that in the external test set was 0.912. Conclusions:In this study, a deep learning-based model for identifying abnormal tooth number of children was developed, which could identify the position of additional teeth and output the position of missing permanent teeth on the basis of identifying normal deciduous and permanent teeth and permanent tooth germs on panoramic radiographs, so as to assist in diagnosing tooth number abnormalities.

Objective: To explore the effect and possible mechanisms of intermittent alkaline on rat vascular smooth muscle cells (VSMCs) calcification induced by high phosphorus. Methods: VSMCs were isolated from rat thoracic aorta and cultured in vitro. The fourth generation VSMCs were randomly divided into control group, high phosphorus+ pH7.4, high phosphorus+ pH7.5, high phosphorus+ pH7.6 and high phosphorus+ pH7.7 group with random number table. The control group was cultured in DMEM with 10% fetal bovine serum. Other groups were cultured in DMEM with 10 mmol/L β-glycerophosphate and alkalized by 7.4% NaHCO₃ to adjust the pH respectively. After the intervention of 4 hours, the control group was replaced with the normal medium containing 10% fetal bovine serum, the other 4 groups were replaced with high phosphorus based on the pH value of the culture medium, and then replaced the culture medium every other day. After 4 days intervention, the mRNA and protein expression of L type calcium channel β₃ subunit(LTCC β₃) and Runt related transcription factor 2 (Runx2) were detected by RT-PCR and Western blot. After 4 days intervention, the level of VSMC calcium ion was detected by Fluo-3/AM. After 14 days intervention, alkaline phosphatase (ALP) activity was measured by enzyme linked immunosorbent assay (ELISA) and the calcification was observed by measuring calcium content. Results: (1) Compared with control group, the gene and protein expressions of LTCC β₃ were higher in high phosphorus+ pH7.4 group (0.49±0.03 vs. 0.23±0.02 and 0.45±0.03 vs. 0.26±0.02 respectively, all P<0.05). Compared with high phosphorus+ pH7.4 group, the mRNA(0.86±0.05) and protein(0.62±0.04) expressions of LTCC β₃ were higher in high phosphorus+ pH7.5 group (P<0.05). Compared with high phosphorus+ pH7.5 group, the mRNA(0.99±0.05) and protein(0.80±0.03) expressions of LTCC β₃ were higher in high phosphorus+ pH7.5 group (all P<0.05). Compared with high phosphorus+ pH7.6 group, the mRNA(1.16±0.05) and protein(0.93±0.03) expressions of LTCC β₃ were higher in high phosphorus+ pH7.7 group (all P<0.05). (2) Compared with control group, calcium ion influx were higher in high phosphorus+ pH7.4 group (124.61±6.06 vs. 75.68±7.82, P<0.05). Compared with high phosphorus+ pH7.4 group, calcium ion influx was higher in high phosphorus+ pH7.5 group(210.85±9.75, P<0.05). Compared with high phosphorus+ pH7.5 group, calcium ion influx was higher in high phosphorus+ pH7.6 group(298.44±11.42, P<0.05). Compared with high phosphorus+ pH7.6 group, calcium ion influx was higher in high phosphorus+ pH7.7 group(401.13±11.41, P<0.05). (3) Compared with control group, the mRNA and protein expressions of Runx2 and ALP were higher in high phosphorus+ pH7.4 group (0.60±0.04 vs. 0.34±0.03, 0.42±0.04 vs. 0.21±0.02, 67.2±4.3 vs. 23.2±2.3 respectively, all P<0.05). Compared with high phosphorus+ pH7.4 group, the mRNA(0.76±0.05) and protein(0.68±0.03) expressions of Runx2 and ALP(102.1±5.4) were higher in high phosphorus+ pH7.5 group (all P<0.05). Compared with high phosphorus+ pH7.5 group, the mRNA(0.90±0.05) and protein(0.90±0.05) expressions of Runx2 and ALP(139.3±4.9) were higher in high phosphorus+ pH7.6 group (all P<0.05). Compared with high phosphorus+ pH7.6 group, the mRNA(1.11±0.05) and protein(1.08±0.06) expressions of Runx2 and ALP(197.0±6.7) were higher in high phosphorus+ pH7.7 group (all P<0.05). (4) Compared with control group, the calcium content were higher in high phosphorus+ pH7.4 group ((75.4±4.3)mg/g pro vs.(25.2±2.1)mg/g pro, P<0.05). Compared with high phosphorus+ pH7.4 group, the calcium content were higher in high phosphorus+ pH7.5 group ((100.8±5.7) mg/g pro, P<0.05). Compared with high phosphorus+ pH7.5 group, the calcium content were higher in high phosphorus+ pH7.6 group ((143.5±6.1) mg/g pro, P<0.05). Compared with high phosphorus+ pH7.6 group, the calcium content were higher in high phosphorus+ pH7.7 group ((205.1±8.2) mg/g pro, P<0.05). Conclusion Intermittent alkaline stimulation can promote high phosphorus induced rat VSMCs calcification possibly through upregulating LTCC β₃ subunit gene and protein expression, increasing calcium ion influx and enhancing VSMCs phenotypic transformation.

Objective To explore the effect of different pH values on calcification of rat vascular smooth muscle cells (VSMCs) through bone morphogenetic protein (BMP)-2 signaling pathway. Methods Healthy male SD rats aged 5-8 weeks were selected in the study. VSMCs from rat thoracic aorta were cultured in vitro, and then identified by immunocytochemistry. The VSMCs were randomly divided into 4 groups by random sampling method:normal group (pH 7.4), pH7.4+high phosphorus group, pH 7.1+high phosphorus group, and pH 7.7+high phosphorus group. Calcium deposition and alkaline phosphatase (AKP) activity were measured by alizarin red staining and enzyme linked immunosorbent assay. The expressions of BMP-2, Smad1 and Runx2 mRNA were detected by RT-PCR. Results Compared with the control group, the calcification staining was increased in pH 7.4+high phosphorus group, calcium content was increased and expressions of BMP-2, Smad1, Runx2 mRNA and AKP activity were also increased (P<0.05). While compared with the pH 7.4+high phosphorus group, calcification staining, calcium content, expressions of BMP-2, Smad1, Runx2 mRNA and AKP activity were decreased in pH 7.1+high phosphorus group (P<0.05). The calcification staining, calcium content, expressions of BMP-2, Smad1, Runx2 mRNA and AKP activity were increased in pH 7.7+high phosphorus group (P<0.05). Conclusion The extracellular acidic environment (pH 7.1) can inhibit high-phosphotus-induced VSMCs calcification, whereas extracellular alkaline environment (pH 7.7) induce high-phosphotus-induced VSMCs calcification. The mechanism is presumably that VSMCs calcification is induced by influencing BMP-2 pathway, which may be mediated by VSMCs phenotype transdifferentiation of BMP-2 signaling pathway.

Objective To investigate surgical outcomes of treatment of pilon fractures of Ruedi -Allgower types Ⅱ and Ⅲ by raft screws fixation combined with bone grafting.Methods 21 patients with pilon fractures of Ruedi -Allgower types II and Ⅲ received surgery were selected.They were 15 men and 6 women,23 to 68 years of age(average 38.2 years).16 cases were closed and 5 cases open.12 cases were type II and 9 cases type III.Intervals between injury and operation ranged from 7 to 14 days,with 8.5 days on average.All of them were treated with raft screws fixation by plate type L combined with bone grafting.Results All cases were followed up for an average of 12 months(range from 8 to 30 months).Fractures healed from 16 to 24 weeks,with 18.2 weeks on average.According to the Burwell -Charnley radiological criteria,anatomical reduction was achieved in 17 cases,and fair reduction in 4.According to the Mazur clinical criteria,ankle functions were rated as excellent in 8 cases,good in 10 cases,fair in 2 cases and poor in 1 case.The rate of excellent and good was 85.7%.Conclusion Raft screws fixation combined with bone grafting are effective for the pilon fractures of Ruedi -Allgower types II and Ⅲ,preventing articular surface collapse and leading to anatomic reduction,and has stable fixation and early functional rehabilitation.

Objective To explore the effects of L?type calcium channel (LTCC) α1C and β3 subunits on that magnesium inhibited thoracic aortic calcification induced by β?glycerophosphate (β?GP). Methods Vascular smooth muscle cells (VSMCs) and aortic rings from rat aortic were cultured, then divided into control group, high phosphorus group (10 mmol/L β?GP), magnesium group (10 mmol/L β?GP+3 mmol/L MgSO4) and 2?APB (an inhibitor of magnesium transporter) group (10 mmol/L β?GP+3 mmol/L MgSO4+0.1 mmol/L 2?APB). Calcium deposition of VSMCs and aortic rings were respectively measured by alizarin red staining and Von Kossa staining, meanwhile the quantification of their calcium was tested by OCPC. The mRNA expressions of Runx2, LTCCα1C andβ3 in VSMCs were detected by RT?PCR, and their protein expressions were detected by Western blotting. Intracellular calcium ion of VSMCs was tested by fluorescence probe and alkaline phosphatase (ALP)activity was measured by ELISA. The Runx2 expression of aortic rings was detected by immunohistochemistry. Results After VSMCs stimulated for 7 days, calcium, ALP, mRNA and protein expressions of LTCCα1C, LTCCβ3 and Runx2, and intracellular calcium ion in high phosphorus group were higher than those in control group (all P<0.05). Moreover, calcium, ALP, mRNA and protein expressions of LTCCα1C, LTCCβ3 and Runx2, and intracellular calcium ion were decreased in magnesium group as compared with those in high phosphorus group (all P<0.05). In aortic rings, magnesium group had lower calcium and protein expression of Runx2 than high phosphorus group. No statistical difference between 2?APB group and high phosphorus group was observed in above indexes (all P>0.05). Conclusion Magnesium may down?regulate expressions of LTCCα1C andβ3 subunit, prevent calcium influx and then inhibit osteogenic differentiation so as to reduce β?glycerophosphate?induced VSMCs calcification.

Objective To observe the role of intermediate conductance calcium?activated potassium channels (KCa3.1) in alkalinization and β?glycerophosphate induced vascular calcification. Methods Vascular smooth muscle cells (VSMCs) and aortic rings were obtained from rat thoracic aorta, and then randomly divided into control group (pH was provided into 7.4, 8.0), high phosphorus groups (pH was provided into 7.4, 7.7 and 8.0, VSMCs in three groups were treated with 10 mmol/L β?glycerophosphate; HCl and NaHCO3 were used to adjust the pH) and TRAM?34 group (20 nmol/L was added into pH8.0 high phosphorus dulbecco's modified eagle's medium). Calcium deposition and alkaline phosphatase (ALP) activity were measured by Alizarin red staining, calcium content and enzyme linked immunosorbent assay after cells were simulated for 12 days. Intracellular free Ca2 + was measured by ELISA. The expression of KCa3.1, runt?related transcription factor 2 (Runx2) were detected by RT?PCR and Western blotting 4 days after cells were stimulated. Calcium deposition was measured by von Kossa staining and calcium content after aortic rings were cultured for 12 days. The expressions of KCa3.1 and Runx2 were detected by immunohistochemistry after aortic rings were cultured for 4 days. Results Compared with control group, calcification in VSMCs and aortic rings were significantly increased in high phosphorus group (P<0.05) while decreased in TRAM?34 group (P<0.05). Compared with control group, the expressions of KCa3.1, Runx2 and the activity of ALP in high phosphorus groups were increased (P<0.05) while decreased in TRAM?34 group (P<0.05). Besides, expressions of Runx2 and KCa3.1 were augmented as the pH was higher (P<0.05). The expression of Runx2 in aortic rings was the same situation. Besides, the Ca2+ influx was blocked by TRAM?34 (P<0.05). Conclusions Alkalinization contributes to β?glycerophosphate induced VSMCs calcification through increase of Ca2 + influx, up?regulation of KCa3.1 and promotion of osteogenic/chondrogenic differentiation.

Objective To investigate the relationship between single nucleotide polymorphism of SET8 gene and the risk of clear cell renal cell carcinoma (CCRCC). Methods We selected 140 CCRCC patients and 130 healthy controls in this case?control study. Genotype of single nucleotide polymorphism ( rs16917496) at the miR?502 binding site in the 3′UTR of SET8 mRNA in the CCRCC patients and healthy controls was tested and the association between genotype and risk of cancer was assessed. The expression of SET8 was determined by immunohistochemistry and the relationship between expression of SET8 and genotype of rs16917496 was analyzed. Results In the control group, CC, CT and TT genotypes were found in 30, 32 and 68 persons, respectively, while in the CCRCC patients, CC, CT and TT genotypes were found in 14 , 47 and 79 cases, respectively. The frequencies of rs16917496 CT and TT genotypes in the CCRCC group were significantly higher than those in the control group ( P<0.05) . Compared with the CC genotype, patients with CT and TT genotypes were more susceptible to develop CCRCC ( P<0. 05 ) . CT and TT genotypes of rs16917496 at the miR?502 binding site of the SET8 gene were associated with expression of SET8. Conclusions Genotype of the SNP rs16917496 at the miR?502 binding site in the 3′ untranslated region of the SET8 gene is associated with the expression of SET8 protein. Analysis of genetic polymorphisms in miRNA binding sites may help to identify the subgroups of population susceptible to CCRCC.

Objective To investigate the relationship between single nucleotide polymorphism of SET8 gene and the risk of clear cell renal cell carcinoma (CCRCC). Methods We selected 140 CCRCC patients and 130 healthy controls in this case?control study. Genotype of single nucleotide polymorphism ( rs16917496) at the miR?502 binding site in the 3′UTR of SET8 mRNA in the CCRCC patients and healthy controls was tested and the association between genotype and risk of cancer was assessed. The expression of SET8 was determined by immunohistochemistry and the relationship between expression of SET8 and genotype of rs16917496 was analyzed. Results In the control group, CC, CT and TT genotypes were found in 30, 32 and 68 persons, respectively, while in the CCRCC patients, CC, CT and TT genotypes were found in 14 , 47 and 79 cases, respectively. The frequencies of rs16917496 CT and TT genotypes in the CCRCC group were significantly higher than those in the control group ( P<0.05) . Compared with the CC genotype, patients with CT and TT genotypes were more susceptible to develop CCRCC ( P<0. 05 ) . CT and TT genotypes of rs16917496 at the miR?502 binding site of the SET8 gene were associated with expression of SET8. Conclusions Genotype of the SNP rs16917496 at the miR?502 binding site in the 3′ untranslated region of the SET8 gene is associated with the expression of SET8 protein. Analysis of genetic polymorphisms in miRNA binding sites may help to identify the subgroups of population susceptible to CCRCC.

Objective To observe the role of TRAM-34 (1-((2-chlorophenyl) diphenylmethyl)-1H-pyrazole),the blocker of intermediate conductance calcium-activated potassium channel (KCa3.1),on β-glycerophosphate induced vascular calcification in vitro.Methods Vascular smooth muscle cells (VSMCs) were obtained from rat thoracic aorta,and VSMCs after the fourth passage and aortic rings were divided into control group (cultured in DMEM with 10％ fetal bovine serum),high phosphorus group (cultured in DMEM with 10％ fetal bovine serum and 10％ β-glycerophosphate) and TRAM-34 group (20 nmol/L TRAM-34 was added into high phosphorus DMEM).Calcium deposition of VSMCs and aortic rings were measured by o-cresolphthalein complexone method.Calcium influx of VSMCs was measured by immunofluorescence probe Fluo-3 AM.The expression of runt-related transcription factor 2 (Runx2) was detected by RT-PCR and Western blot for cells and immunohistochemistry for aortic rings.ALP activity was measured by alkaline phosphatase activity detection kit.Results (1) Compared with control group,calcification was significantly increased in high phosphorus group ((121.67 ± 6.17) mg/g vs.(84.38 ±8.17) mg/g,P ＜0.05) and this effect could be attenuated by TRAM-34 ((93.31 ± 11.36) mg/g,P ＜0.05 vs.high phosphorus group) after 12 days culture.Similar results were found in aortic rings cultured for 12 days—high phosphorus group:(7.17 ± 0.57) mg/g vs.control:(1.18 ± 0.13) mg/g (P ＜ 0.05) and TRAM-34:(4.71 ±0.42) mg/g,P ＜0.05 vs.high phosphorus group.(2) Compared with control group,the calcium influx was higher in high phosphorus group (349.22 ±40.47 vs.151.67 ± 16.94,P ＜0.05)and reduced in TRAM-34 group (194.67 ± 22.21,P ＜ 0.05 vs.high phosphorus group) in VSMCs simulated for 4 days.(3) Both mRNA and protein expressions of Runx2 in high phosphorus groups were higher than in control group (0.630 ±0.033 vs.0.340 ±0.058 and 0.865 ±0.031 vs.0.414 ±0.011,both P ＜ 0.05) and lower in TRAM-34 group (0.399 ± 0.023 and 0.575 ± 0.014,both P ＜ 0.05 vs.high phosphorus group) in VSMCs simulated for 4 days.Besides,compared with high phosphorus group,the expression of Runx2 was decreased in control group (0.113 ± 0.010 vs.0.067 ± 0.008,P ＜ 0.05) and TRAM-34 group (0.069±0.006,P＜0.05) after aortic rings were cultured for 4 days.(4) Compared with control group,the activity of ALP was significantly increased in high phosphorus group (96.56 ± 9.84vs.46.92 ± 4.60,P ＜0.05) and decreased in TRAM-34 group (70.20 ± 8.41,P ＜ 0.05 vs.high phosphorus group) in VSMCs simulated for 12 days.Conclusion KCa3.1 blocker TRAM-34 can inhibit β3-glycerophosphate induced VSMCs and aortic ring calcification through inhibiting calcium influx,downregulating Runx2 expression and attenuating osteogenic differentiation.

Objective To explore the effect and mechanism of magnesium on calcification induced by hyperphosphate.Methods Vascular smooth muscle cells (VSMCs) were primarily cultured in vitro and induced calcification by β-glycerophosphate (β-GP).VSMCs were randomly divided into control group,high phosphorus group (10 mmol/L β-GP),magnesium intervèntion group(10 mmol/L β-GP + 3 mmol/L MgSO4) and 2-aminoethoxy-diphenylborate (2-APB,an inhibitor of magnesium transporter) intervention group(10 mmol/L β-GP+3 mmol/L MgSO4+ 10-4 mol/L 2-APB).Calcium deposition and alkaline phosphatase (ALP) activity were measured by alizarin red staining,quantification of calcium and euzyme linked immunosorbent assay.RT-PCR and Western blotting were used to observe the expression of core binding factor α-1 (Cbfα-1) mRNA and protein,respectively.In vivo,male Sprague-Dawley rats (n=24) were randomly divided into control group (methylcellulose+high phosphorous diet),vascular calcification group (adenine suspension + high phosphorous diet),high magnesium intervention group(adenine suspension+high phosphorous and magnesium diet).The aortic pulse wave velocity (PWV) was measured,and vascular calcification was determined by von Kossa stain and quantification of calcium.Cbfα-1 in aortic was measured by immunohistochemistry.Results In vitro,compared with high phosphorus group,calcification,ALP activity (P ＜ 0.05) and Cbfα-1expression in VSMCs were significantly decreased in magnesium intervention group after incubation for 14 days,but the addition of 2-APB might inhibit the protective effect of magnesium on VSMCs.Dynamic observation of Cbfα-1 showed that magnesium significantly inhibited the expression of Cbfα-1 (P ＜ 0.05) on the third day and the inhibitory role was obviously increased in a time-dependent manner.Consistent with the findings in vitro,the aortic PWV,calcification were all significantly reduced (P ＜ 0.05) in high magnesium intervention group with high serum magnesium level,when compared with vascular calcification group.Immunohistochemistry showed that hypermagnesemia downregulated obviously the expression of Cbfα-1 induced by hyperphosphatemia(P ＜ 0.05).Conclusion Magnesium protects against vascular calcification by inhibiting osteogenic differentiation of VSMCs.