Objective·To investigate the regulatory effects and underlying mechanisms of mouse mandibular osteoblasts on B cell differentiation and development.Methods·Single-cell suspensions from mouse mandibular bone were prepared using an optimized enzymatic digestion method and induced to differentiate into osteoblasts in vitro.Osteogenic potential was validated by real-time quantitative PCR(RT-qPCR),alkaline phosphatase(ALP)staining,and alizarin red S(ARS)staining.The spatial localization relationship between osteoblasts and B cells in mandibular tissues was examined via immunofluorescence staining.High-purity hematopoietic progenitor cells were isolated using fluorescence-activated cell sorting.A Transwell co-culture system was established to assess the regulatory effects of different osteoblast concentrations(5×104,2.5×105,and 5×105 cells/well)on B cell differentiation(5×104 cells/well).Flow cytometry and RT-qPCR were employed to evaluate B cell viability and differentiation.Additionally,RT-qPCR was used to analyze the expression of osteoblast-secreted factors associated with B cell development during osteogenic differentiation.Results·Mandibular osteoblasts exhibited robust osteogenic potential,as confirmed by ALP/ARS staining and high expression of osteogenic markers(Runx2,Osx,Ocn,and Alp)via RT-qPCR.Immunofluorescence revealed close spatial proximity between osteoblasts and B cells in mandibular tissues.In the co-culture system,osteoblasts promoted B cell differentiation in a concentration-dependent manner.RT-qPCR and immunofluorescence demonstrated that osteoblasts significantly upregulated key genes involved in B cell development(Ebf1,Rag1,Il7r,and Pax5;all P<0.001).Furthermore,osteoblast-derived factors(Il7,Baff,and Flt3l)were markedly elevated during osteogenic differentiation(all P<0.05).Conclusion·Mandibular osteoblasts enhance B cell differentiation and development in a concentration-dependent manner,likely through secreting growth factors that upregulate critical B cell differentiation genes.

Objective·To investigate the regulatory effects and underlying mechanisms of mouse mandibular osteoblasts on B cell differentiation and development.Methods·Single-cell suspensions from mouse mandibular bone were prepared using an optimized enzymatic digestion method and induced to differentiate into osteoblasts in vitro.Osteogenic potential was validated by real-time quantitative PCR(RT-qPCR),alkaline phosphatase(ALP)staining,and alizarin red S(ARS)staining.The spatial localization relationship between osteoblasts and B cells in mandibular tissues was examined via immunofluorescence staining.High-purity hematopoietic progenitor cells were isolated using fluorescence-activated cell sorting.A Transwell co-culture system was established to assess the regulatory effects of different osteoblast concentrations(5×104,2.5×105,and 5×105 cells/well)on B cell differentiation(5×104 cells/well).Flow cytometry and RT-qPCR were employed to evaluate B cell viability and differentiation.Additionally,RT-qPCR was used to analyze the expression of osteoblast-secreted factors associated with B cell development during osteogenic differentiation.Results·Mandibular osteoblasts exhibited robust osteogenic potential,as confirmed by ALP/ARS staining and high expression of osteogenic markers(Runx2,Osx,Ocn,and Alp)via RT-qPCR.Immunofluorescence revealed close spatial proximity between osteoblasts and B cells in mandibular tissues.In the co-culture system,osteoblasts promoted B cell differentiation in a concentration-dependent manner.RT-qPCR and immunofluorescence demonstrated that osteoblasts significantly upregulated key genes involved in B cell development(Ebf1,Rag1,Il7r,and Pax5;all P<0.001).Furthermore,osteoblast-derived factors(Il7,Baff,and Flt3l)were markedly elevated during osteogenic differentiation(all P<0.05).Conclusion·Mandibular osteoblasts enhance B cell differentiation and development in a concentration-dependent manner,likely through secreting growth factors that upregulate critical B cell differentiation genes.

Objective·To construct and verify the mouse model that can mimic the vitamin A deficiency(VAD)-like craniofacial skeletal deformity and do not cause embryonic death.Methods·Based on the Cre-LoxP system,the OsxCre;Rosa26dn/dn mice expressing osteoblast-specific dominant-negative retinoid acid receptor α(dnRARα)mutation were obtained by hybridization through OsxCre and Rosa26dnRARa/ddnRARa mice,to achieve the conditional inhibition of retinoic acid signaling to simulate VAD disease.Femur bone mesenchymal stem cells(BMSCs)and parietal bone cells of OsxCre;Rosa26dn/dn mice and their control littermates were isolated and underwent osteogenic induction,to assess the expression of retinoid acid receptor α(RARα)protein through Western blotting.Osteoblasts induced from parietal bone cells of OsxCre;Rosa26dn/dn mice and their control littermates were isolated and the effect of retinoic acid signaling inhibition was verified through dual luciferase gene reporter assay.Meanwhile,Ad-eGFP or Ad-Cre adenovirus-infected femur BMSCs and parietal bone cells of Rosa26dn/dnmice underwent osteogenic induction to assess the expression of dominant-negative mutant protein and the inhibition of the retinoic acid signaling pathway in vitro by Western blotting and dual luciferase gene reporter assay.Moreover,the skulls of 6-week-old OsxCre;Rosa26dn/dn mice were collected,and Micro-CT scanning and three-dimensional(3D)reconstruction were performed to verify the craniofacial skeletal deformities of the mouse model.Results·Western blotting results demonstrated that the level of RARα protein increased in the femur and parietal osteoblasts of OsxCre;Rosa26dn/dn mice compared to that of their control littermates,and also increased in the Ad-Cre-infected femur and parietal osteoblasts of Rosa26dn/dn mice compared to that in the Ad-eGFP-infected group(P＜0.05).Dualluciferase gene reporter assay results indicated that the activity of retinoid acid response element(RARE)was inhibited in the osteoblasts of OsxCre;Rosa26dn/dn mice compared to their control littermates,and was also inhibited in the Ad-Cre-infected group compared to the Ad-eGFP-infected group(P＜0.05).Micro-CT and 3D reconstruction suggested that the skull of 6-week-old OsxCre;Rosa26dn/dn mice exhibited VAD-like craniofacial skeletal deformities,including smaller size of the skull and osteogenesis imperfecta compared to their control littermates.Conclusion·An osteoblast-specific dnRARα expressing mouse model that can mimic VAD-like craniofacial skeletal deformity is successfully constructed,therefore providing a new model for exploring the pathogenesis and therapeutic targets of VAD-like craniofacial skeletal deformity in the future.

The temporomandibular joint is the only joint structure within the craniofacial skeletal system,responsible for performing functions related to opening and closing mouth movements,such as chewing,speaking,and facial expression in daily life.The condyle of the mandible,as a vital component of the temporomandibular joint,originates from the mandibular process formed by the first gill arch and is the key growth center at the end of the mandibular ramus.Condyle is composed of a layer of cartilage as its surface and subchondral bone below,exhibiting unique biological processes during its growth and development.In the articular fossa,the functional movement of the condyle depends on its normal physiological and anatomical structure,which plays a crucial role in establishing occlusion and shaping facial features.Abnormal growth and development can lead to the occurrence of condylar deformities,which affect the vertical height of the patient's maxillofacial region and ultimately lead to secondary skeletal class Ⅱ or Ⅲ craniofacial deformities.During the process of growth and development,the condyle is subject to complex signal regulation.In recent years,with in-depth research on the temporomandibular joint,researchers have begun to discuss the regulatory mechanisms of condyle growth and development from the perspectives of gene expression and molecular level,in order to explain the causes of temporomandibular joint diseases and condylar deformities.This article provides a review on the growth process and structure of condyle,classification and pathological manifestations of condylar deformities,and related regulatory mechanisms of the growth and development of condyle,as well as pathogenesis of condylar deformities.The aim of this article is to provide research ideas for temporomandibular joint diseases and craniofacial malformations caused by abnormal development of the mandibular condyle in clinical practice.

Objective·To explore the effect of all-trans retinoic acid(ATRA)of different concentrations on osteogenic differentiation of jaw bone mesenchymal stem cells(jBMSCs)in rats.Methods·jBMSCs from 4-week-old Sprague-Dawley(SD)rats were isolated and cultured with whole bone marrow adherence method.The surface antigens were identified by using flow cytometry.Alkaline phosphatase(ALP)staining/alizarin red staining,oil red O staining and alcian blue staining were used to prove the multilineage differentiation potential of jBMSCs after osteogenic,adipogenic and chondrogenic induction respectively.jBMSCs were induced in osteogenic medium with ATRA of concentration of 0.01,0.1,1,5,10,20 μmol/L in vitro,and dimethyl sulfoxide(DMSO)was used as control group.Cell viability of jBMSCs in different groups were determined by CCK8.ALP staining and alizarin red staining were used to investigate the osteogenic ability of jBMSCs in each group and screened the concentrations for subsequent experiments.Quantitative real-time polymerase chain reaction(qPCR)and immunofluorescence staining were used to analyze the expressions of osteogenesis-related genes and proteins in jBMSCs of different concentrations.Results·The flow cytometry analysis showed that more than 98%of P1 jBMSCs were positive for CD29+CD90+CD31-CD45-,which was congruent with the characteristics of bone mesenchymal stem cells.The results of ALP staining/alizarin red staining,oil red O staining and alcian blue staining indicated that the P1 jBMSCs had the multilineage differentiation potential of osteogenesis,adipogenesis and chondrogenesis.The results of ALP staining/alizarin red staining showed that the osteogenic activity and mineralization ability of jBMSCs in 0.01,0.1 and 1 μmol/L ATRA groups were increased compared with those in the control group,while the osteogenic activity and mineralization ability were decreased when the concentration of ATRA increased,especially higher than 5 μmol/L(all P<0.05).qPCR analysis showed that the mRNA expression levels of osteogenesis-related genes such as Alp,bone sialoprotein(Bsp),collagen type Ⅰ α1(Col1a1)and osteocalcin(Ocn)were higher in the 0.1 and 1 μmol/L ATRA groups compared to the control group.However,further increasing the concentration of ATRA led to a decrease in gene expression levels,and when the concentration exceeded 5 μmol/L,it began to be lower than the control group level(all P<0.05).The immunofluorescence staining showed that the expression of osteogenic related proteins SP7,ALP and OCN in the 0.1 and 1 μmol/L ATRA groups were increased compared to the control group,while further increasing the concentration of ATRA led to a decrease in protein expression.When the concentration was higher than 5 μmol/L,it began to be lower than the control group level(all P<0.05).Conclusion·Lower concentrations(0.1,1 μmol/L)of ATRA can promote the osteogenic differentiation of rat jBMSCs,and the promoting effect reaches its peak at 0.1 μmol/L,while the effect can be weakened by further increasing the concentration.Higher concentrations(5,10,20 μmol/L)of ATRA could inhibit the osteogenic differentiation of rat jBMSCs,showing an inhibitory effect.In this study,the dual-directional effect of retinoic acid on osteogenic differentiation of jBMSCs was demonstrated in vitro,and 0.1 μmol/L ATRA was identified as the optimal concentration for osteogenic differentiation of jBMSCs in rats,which provided a reference basis for the development of in vivo studies and clinical application of ATRA.

OBJECTIVE To determine the prevalence of frontal recess cells variations in patients with frontal sinus associated headache according to the International Frontal Sinus Anatomy Classification(IFAC).METHODS A retrospective study was conducted on the CT scans of sinuses in patients with frontal sinus associated headache.We reviewed 46 patients with frontal sinus-related headache who had clinical symptoms and were relieved after nasal endoscopic surgery.The development of frontal recess cells in the frontal recess drainage area was analyzed,and the variation of middle meatus and sinus involvement were analyzed in the same time.The Anatomical variations and imaging characteristics of frontal recess cells development in patients with frontal sinus associated headache were analyzed.RESULTS A total of 92 sinus CT profiles were analyzed in 46 patients.The most common cells were agger nasi cell(ANC)(100%,92/92),followed by supra bulla cell(SBC)(78.3%,72/92),supra agger cell(SAC)(67.4%,62/92),supra bulla frontal cell(SBFC)(27.2%,25/92),supra agger frontal cell(SAFC)(20.7%,19/92),frontal septal cell(FSC)(8.7%,8/92)and supraorbital ethmoid cell(SOEC)(0%,0/92).In the conventional frontal sinus drainage area,SAFC(P=0.0108),SAC(P=0.0104)and SAFC(P=0.0088)in the IFAC classification were significantly associated with the occurrence of frontal sinus associated headache.At the same time,the middle concha bullosa also showed a significant correlation with the occurrence of frontal sinus associated headache in the lower segment of the frontal recess drainage channel(P=0.0390).CONCLUSION In the frontal recess drainage channel,the abnormal development of SAC,SAFC,SBFC and the middle concha bullosa are significantly correlated with frontal sinus associated headache.

Objective:To investigate the feasibility of simultaneous arteriovenous enhancement of neck CT with two-stage injection of contrast agent and its effect on image quality and radiation dose.Methods:A total of 30 patients undergoing neck CT enhancement scan due to space-occupying lesions in Beijing Tongren Hospital, Capital Medical University from February to April 2022 were prospectively included as the experimental group. The neck CT enhancement scan was performed with two-stage injection of contrast agent and arteriovenous simultaneous enhancement. The dosage of contrast agent was calculated according to the patient′s body weight, and the method of two-stage injection was adopted. The dosage of contrast agent in the first stage was 0.7 ml/kg, with normal saline in the middle stage, and the second stage (began at 35 s) was 0.3 ml/kg. A total of 30 patients with gender and age matching with the experimental group from December 2021 to January 2022 were retrospectively collected as the control group. The control group was treated with the traditional arterial phase and venous phase scanning method with the dosage of 1.0 ml/kg contrast agent. The arterial phase was scanned at the 30 s and the venous phase was scanned at the 60 s. The CT values of bilateral carotid arteries and jugular veins in the experimental group were measured, the CT values of bilateral carotid arteries in the arterial phase were measured in the control group, and the CT values of bilateral carotid arteries and jugular veins in the venous phase were measured. Carotid artery enhancement score was performed for images of experimental group and control group in arterial and venous phase, and jugular vein and lesion enhancement score was performed for images of experimental group and control group in venous phase. The effective dose was calculated for both groups. The difference of carotid artery CT values between images was compared by one-way analysis of variance, and LSD method was used for pairwise comparison. The CT values of jugular vein were compared using independent sample t test. Kruskal-Wallis test was used to compare carotid artery enhancement scores, and Nemenyi method was used for pairwise comparison. Jugular vein and lesion enhancement scores and effective dose were compared by Mann-Whitney U test. Results:The CT value of carotid artery of experimental group [left (276±24) HU, right (273±25) HU] was lower than that of control group in arterial phase [left (329±33) HU, right (327±32) HU], and higher than that in the venous phase [left (147±15) HU, right (148±16) HU]. All the differences were statistically significant ( P<0.001). The CT value of jugular vein of experimental group [left (206±18) HU, right (203±19)] was higher than that of control group in the venous phase [left (154±15) HU, right (151±15)], the difference was statistically significant ( t=11.88, 11.76, both P<0.001). There was no significant difference in carotid artery enhancement score between experimental group and control group in arterial phase ( P=0.624), but the carotid artery enhancement score of the experimental group was higher than that of the control group in the venous phase, and the difference was statistically significant ( P<0.001). The scores of jugular vein and lesion enhancement in experimental group were higher than those of control group in venous phase, and the difference was statistically significant ( Z=5.01, P<0.001). The effective dose of the experimental group [2.41(2.04, 2.72) mSv] was decreased by 52.2% compared with the control group [5.04(4.18, 5.44) mSv], and the difference was statistically significant ( Z=-6.24, P<0.001). Conclusions:The neck CT enhanced scan with two-stage injection of contrast agent and arteriovenous simultaneous enhancement method can obtain comprehensive images of arterial and venous phases, and realize simultaneous enhancement of carotid artery, jugular vein and lesions, and reduce radiation dose.

Heme, which exists widely in living organisms, is a porphyrin compound with a variety of physiological functions. Bacillus amyloliquefaciens is an important industrial strain with the characteristics of easy cultivation and strong ability for expression and secretion of proteins. In order to screen the optimal starting strain for heme synthesis, the laboratory preserved strains were screened with and without addition of 5-aminolevulinic acid (ALA). There was no significant difference in the heme production of strains BA, BAΔ6 and BAΔ6ΔsigF. However, upon addition of ALA, the heme titer and specific heme production of strain BAΔ6ΔsigF were the highest, reaching 200.77 μmol/L and 615.70 μmol/(L·g DCW), respectively. Subsequently, the hemX gene (encoding the cytochrome assembly protein HemX) of strain BAΔ6ΔsigF was knocked out to explore its role in heme synthesis. It was found that the fermentation broth of the knockout strain turned red, while the growth was not significantly affected. The highest ALA concentration in flask fermentation reached 82.13 mg/L at 12 h, which was slightly higher than that of the control 75.11 mg/L. When ALA was not added, the heme titer and specific heme production were 1.99 times and 1.45 times that of the control, respectively. After adding ALA, the heme titer and specific heme production were 2.08 times and 1.72 times higher than that of the control, respectively. Real-time quantitative fluorescent PCR showed that the expressions of hemA, hemL, hemB, hemC, hemD, and hemQ genes at transcription level were up-regulated. We demonstrated that deletion of hemX gene can improve the production of heme, which may facilitate future development of heme-producing strain.
Gene Deletion ; Bacillus amyloliquefaciens/metabolism* ; Aminolevulinic Acid/metabolism* ; Heme/metabolism* ; Fermentation

Purpose: To explore the effect of intravesical electrical stimulation (IVES) on urinary adenosine triphosphate (ATP) and nitric oxide (NO) in rats with detrusor underactivity (DU) induced by bilateral pelvic nerve crush (bPNC), and to determine the underlying peripheral mechanism. Methods: Twenty-four female Sprague-Dawley rats were equally divided into 3 groups: sham; bPNC; and IVES. Rats in the IVES group began to receive IVES treatment 10 days after bPNC (20 minutes per day for 14 consecutive days). After the 14th IVES, rat urine was collected and cystometry was performed. The serum creatinine, blood urea nitrogen, and urinary ATP and NO levels were measured, and a routine urinalysis was performed. Results: The maximum cystometric capacity (MCC), maximum changes in bladder pressure during filling (∆FP), and postvoid residual urine (PVR) in the IVES group were significantly lower than the bPNC group, and the maximum changes in bladder pressure during voiding (∆VP) was significantly higher than the bPNC group. Compared with the sham group, the MCC, ∆FP and PVR were significantly increased, and the maximum voiding pressure (MVP) and ∆VP were significantly decreased in the bPNC group. After bPNC, urinary ATP was significantly decreased, and urinary NO was significantly increased. In IVES-treated rats, urinary ATP was significantly higher than the bPNC group, and NO was significantly lower than the bPNC group. In addition, the ATP-to-NO ratio of the rats in the bPNC group was significantly lower than the sham and IVES groups. Correlation analysis showed that the ATP and NO were not correlated with the MCC, ∆FP, MVP, ∆VP, and PVR. Conclusions Promoting the release of urothelial ATP and inhibiting the release of urothelial NO may be one of the peripheral mechanisms underlying IVES in the treatment of DU. Specifically, IVES may shift the balance between excitation and inhibition toward excitation.

Botulinum neurotoxin subtype A (BoNT-A) has been part of the urology treatment arsenal since it was first used in the treatment of detrusor-sphincter dyssynergia more than 30 years ago. BoNT-A has been recommended as an effective treatment for neurogenic detrusor overactivity and overactive bladder. However, direct intradetrusor injection of BoNT-A using cystoscopy after anesthesia may cause hematuria, pain, and infection; these adverse events have motivated urologists to find less invasive and more convenient ways to administer BoNT-A. The development of nanotechnology has led to the advancement of intravesical drug delivery. Using versatile nanocarriers to transport BoNT-A across the impermeable urothelium is a promising therapeutic option. In this review, we discuss the effectiveness and feasibility of liposomes, thermosensitive polymeric hydrogels, and hyaluronan-phosphatidylethanolamine as carriers of BoNT-A for intravesical instillation. To date, these carriers have not reached a similar efficacy as intradetrusor injections in long-term observations. Hopefully, researchers will make a breakthrough with new nanomaterials to develop clinical applications in the future.