OBJECTIVES: The mechanism of the odontogenic differentiation of apical papillary cells (APCs) stimulated by bioactive glass 45S5 is still unclear. This study aims to investigate the effect of autophagy on the odontogenic differentiation of APCs stimulated by bioactive glass 45S5. METHODS: APCs were isolated and cultured in vitro, and the cell origin was identified by flow cytometry. The culture medium was prepared with 1 mg/mL 45S5, and its pH and ion concentration were determined. The experiments were divided into control, 45S5, and 3-methyladenine (3-MA) 45S5 groups. In the 45S5 group, APCs were induced to culture with 1 mg/mL 45S5. In the 3-MA 45S5 group, the autophagy inhibitor 3-MA was added to 1 mg/mL 45S5. Protein immunoblotting assay (Western blot) was used to detect the expression of autophagy-associated proteins of microtubule-associated protein 1 light-chain 3β (LC3B) and P62 after 24 h of induction culture in each group. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of bone sialoprotein (BSP), Runt-related transcription factor 2 (Runx2), dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) after 7 d of induction culture. Cellular alkaline phosphatase (ALP) staining analyzed cellular ALP activity at 7 d of induction, and alizarin red staining evaluated the formation of mineralized nodules at 21 d of induction. RESULTS: The pH of the 45S5 extract culture medium was 8.65±0.01, which was not significantly different from that of the control group (P>0.05). The silicon ion concentration of the 45S5 induction culture medium was (1.56±0.07) mmol/L, which was higher than that of the control group (0.08±0.01) mmol/L (P<0.05). The calcium ion concentration of the 45S5 induction culture was (1.57±0.15) mmol/L, which was not significantly different from that of the control group (P>0.05). Western blot results showed that LC3B-Ⅱ/Ⅰ ratio increased and P62 expression decreased in the 45S5 group compared with those in the control group (P<0.05). By contrast, the ratio decreased and the expression increased in the 3-MA 45S5 group compared with those in the 45S5 group (P<0.05). RT-qPCR results showed that the expression of BSP, Runx2, DMP-1, and DSPP enhanced in the 45S5 group compared with that in the control group (P<0.05), but the expression decreased in the 3-MA 45S5 group compared with that in the 45S5 group (P<0.05). Semi-quantitative analysis of ALP staining and alizarin red staining showed that the ALP activity was enhanced, and the formation mineralized nodule increased in the 45S5 group compared with those in the control group. The ALP activity weakened, and the formation mineralized nodules were reduced in the 3-MA 45S5 group compared with that those in the 45S5 group. CONCLUSIONS Cell autophagy participates in the odontogenic differentiation of APCs induced by 1 mg/mL 45S5 in vitro.
Autophagy/drug effects* ; Cell Differentiation/drug effects* ; Odontogenesis/drug effects* ; Dental Papilla/cytology* ; Humans ; Microtubule-Associated Proteins/metabolism* ; Glass/chemistry* ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Extracellular Matrix Proteins/metabolism* ; Ceramics/pharmacology* ; Adenine/pharmacology* ; Sialoglycoproteins/metabolism* ; Phosphoproteins/metabolism* ; Integrin-Binding Sialoprotein/metabolism* ; Alkaline Phosphatase/metabolism* ; RNA-Binding Proteins

OBJECTIVE: To investigate the clinical characteristics and genetic etiology of a Chinese pedigree affected with Hereditary dentin dysplasia type II (DD-II) due to variant of dentin sialophosphoprotein (DSPP) gene. METHODS: A child diagnosed with DD- II at the Third Clinical Division of Peking University Hospital of Stomatology in December 2021 and her family members were selected as study subjects. Clinical data were retrospectively analyzed. Saliva samples were collected from the proband, her parents and sister for genomic DNA extraction. Whole exome sequencing (WES) was carried out. Candidate variant was verified by Sanger sequencing and TOPO-TA cloning sequencing. The candidate variant was also subjected to bioinformatics analysis using Mutation Taster v2021. Secondary and tertiary structures of the wild-type and variant DSPP proteins were predicted with psipred v4.0 and PyMOL v2.3 software, respectively. The pathogenicity of the variant was classified based on the guidelines from American College of Medical Genetics and Genomics (ACMG). This study was approved by the Medical Ethics Committee of Peking University Hospital of Stomatology (Ethics No.: PKUSSIRB-202162021). RESULTS: The proband and her mother and sister had all exhibited typical clinical manifestations of hereditary DD-II. The primary dentition of the proband displayed yellowish brown discoloration, wear, and obliteration in the chamber and root canal, while the permanent teeth of the proband's sister and mother appeared nearly normal in both color and appearance, though with obliteration in the chamber and root canal. Her father showed normal dentition. WES identified a heterozygous c.1915_1918delAAGT, p.(Lys639Glnfs*674) frameshift variant in the DSPP gene. Sanger sequencing and TOPO-TA cloning sequencing confirmed the presence of this variant in the proband, the proband's sister, and the mother, while the proband's father was negative for the variant, indicating an autosomal dominant inheritance pattern. The variant was predicted to be pathogenic by Mutation Taster v2021. Prediction of the secondary structure of the DSPP protein showed that the variant has changed it from coil to helix. The tertiary structure prediction of the DSPP protein showed change of the spatial structure of the variant DSPP, with the loops in the variant region replaced by helices at multiple sites. Based on the guidelines from the ACMG, the variant was classified as pathogenic (PVS1+PM2_Supporting+PP1+PP4). CONCLUSION Phenotypic analysis and genetic testing of this family has clarified the clinical diagnosis of hereditary DD- II. The c.1915_1918delAAGT variant probably underlay the pathogenesis of DD-II in this family. Above results have expanded the phenotypic spectrum of the disease and may contribute to further clinical and genetic research on this disease.
Humans ; Pedigree ; Female ; Extracellular Matrix Proteins/chemistry* ; Male ; Sialoglycoproteins/chemistry* ; Dentin Dysplasia/genetics* ; Asian People/genetics* ; Phosphoproteins/chemistry* ; Child ; Mutation ; China ; Exome Sequencing ; Adult ; East Asian People

OBJECTIVETo regenerate dentin-pulp complex by tissue engineering with human stem cells from apical papilla cells (SCAP) as the seed cells.

METHODSSCAP was separated from from normal human impacted third molars with immature roots by outgrowth culture. The cells were then cultured in the differentiation medium for 3 weeks or in normal medium for 60 days, and analyzed for mineralization potential by Alizarin red staining. The osteo/odontogenic markers including alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OC) and dentin sialoprotein (DSP) were investigated by immunofluorescence staining and reverse transcription-polymerase chain reaction. The co-cultured mixture of SCAP and HA/TCP, or HA/TCP alone was implanted subcutaneously on the back of nude mice for 8 weeks, and the implants were collected and examined by HE and immunohistochemical staining.

RESULTSRound alizarin red-positive nodules formed in the isolated cells after cell culture in the differentiation medium for 3 weeks or in normal medium for 60 days with positive staining for osteo/odontogenic markers. SCAP with HA/TCP could regenerate pulp-dentin complex-like tissue in nude mice. The cells near the dentin-like tissue were positive for DSP. No mineral tissue was found in mice receiving HA/TCP implantation.

CONCLUSIONSSCAP may serve as a promising seed cell for dentin-pulp complex tissue engineering.

Adolescent ; Adult ; Alkaline Phosphatase ; analysis ; Animals ; Cell Culture Techniques ; Cell Differentiation ; Coculture Techniques ; Dental Papilla ; cytology ; Dental Pulp ; cytology ; Extracellular Matrix Proteins ; analysis ; Female ; Humans ; Integrin-Binding Sialoprotein ; analysis ; Mice ; Mice, Nude ; Odontogenesis ; physiology ; Osteocalcin ; analysis ; Phosphoproteins ; analysis ; Sialoglycoproteins ; analysis ; Stem Cells ; chemistry ; physiology ; Tissue Engineering ; methods ; Young Adult

AIMTo evaluate the effect of interleukin-1 receptor antagonist(IL-1ra) on apoptosis and associated mechanism of eosinophil in guinea pig with asthma.

METHODSA model of guinea pig with asthma was established. After inhalation of different concentrations of IL-1ra, asthma was induced in the guinea pig for 8 days, the concentration of eosinophil cationic protein (ECP) in serum and bronchoalveolar lavage fluid (BALF), IL-5 in serum, the eosinophil counts and apoptosis were assayed by radioimmunology, enzyme-linked immunosorbent assay(ELISA), fluoromicroscope and light microscope.

RESULTSIL-1ra indirectly decreased the level of IL-5 in serum, improved the apoptosis of eosinophil(EOS) in lung, then decreased the level of ECP in serum and BALF.

CONCLUSIONInhalation of nebulized IL-1ra showed protective effect against asthma through change of the activity and infiltration of EOS in lung.

Animals ; Apoptosis ; Asthma ; blood ; chemically induced ; pathology ; Blood Proteins ; metabolism ; Bronchoalveolar Lavage Fluid ; chemistry ; Eosinophil Granule Proteins ; Eosinophils ; drug effects ; pathology ; Female ; Guinea Pigs ; Interleukin 1 Receptor Antagonist Protein ; Interleukin-5 ; blood ; Leukocyte Count ; Male ; Ovalbumin ; Random Allocation ; Receptors, Interleukin-1 ; antagonists & inhibitors ; Ribonucleases ; blood ; metabolism ; Sialoglycoproteins ; pharmacology

OBJECTIVETo test the bovine cementoblasts (CBs) cementum-forming ability in vivo.

METHODSRoot fragments of newborn bovine freshly extracted mandibular incisor were cultured routinely and 4th-5th passages of CBs were harvested. CBs were then cultured in the medium supplemented with 50 mg/L alpha-ascorbic acid and 10 mmol/l beta-glycerolphosphate to form a thick layer as tissue engineering scaffold for cementum formation. Collagen membrane was used as control scaffold. 2 x 10(6) cells were attached to the CBs-made carrier as well as collagen membrane scaffolds and transplanted subcutaneously into immunodeficient mice. Transplants were harvested at 7th week. Histological sections were stained with HE, alizarin red S and van Kossa methods as well as monoclonal Ab against bovine cementum attachment protein (CAP).

RESULTSCBs-made scaffold supported more cementum-like tissue (CLT) formation than collagen-made scaffold. The CLT formed on CBs scaffold was partly calcified with embedded cells. Uncalcified cementoid-like material could be seen on the surface and was encircled by cubical CB-like cells. The CLT was also positive to CAP and van Kossa staining.

CONCLUSIONSThese results suggest that the bovine CBs can form cementum-like tissue. The cell-made carrier is a better scaffold than collagen membrane.

Alkaline Phosphatase ; analysis ; Animals ; Bone Transplantation ; methods ; Cattle ; Cell Adhesion Molecules ; analysis ; Cells, Cultured ; Dental Cementum ; chemistry ; cytology ; transplantation ; Immunohistochemistry ; Integrin-Binding Sialoprotein ; Male ; Mice ; Mice, Nude ; Osteocalcin ; analysis ; Osteonectin ; analysis ; Sialoglycoproteins ; analysis ; Tissue Engineering ; methods ; Transplantation, Heterologous

OBJECTIVETo investigate the timing and location of the expression of bone sialoprotein (BSP) and osteopontin (OPN) in developing dental tissues of rats.

METHODSEvery three neonatal rats were sacrificed at day 1, weeks 1, 2, 3, 5 and 8. The mandibles were dissected and the first molar and the surrounding tissue were fixed, then demineralized with 15% EDTA. Immunohistochemical technique was used to determine the expression of BSP and OPN in dental tissues and the surrounding bone at different time points.

RESULTSImmunoreactivitis of BSP and OPN were present in matured ameloblasts, dentinoblasts, cementoblasts, osteoblasts, and the matrix. The expression of BSP and OPN in cementum and alveolar bone was stronger than that in enamel and dentine. In cementum and alveolar bone, BSP appeared to be concentrated in unmineralized and mineralized tissues, but OPN was concentrated in the mineralizing frontier and reversal line.

CONCLUSIONSBSP and OPN play an important role in the development and mineralization of rat mineralized tissues. The expression of BSP was different from OPN, indicating their different functions.

Animals ; Immunohistochemistry ; Integrin-Binding Sialoprotein ; Male ; Osteopontin ; Rats ; Rats, Sprague-Dawley ; Sialoglycoproteins ; analysis ; Tooth ; chemistry

Advanced atherosclerosis is often associated with dystrophic calcification and remodeling of extracellular matrix of vascular wall. Recently many studies have documented a general relationship between calcification and severity of coronary disease, and discussed the feasibility of electron beam computed tomography for detecting and quantifying the coronary artery calcification in the patients. The present study investigated the expression and the localization of osteopontin, one of noncollagenous bone matrix protein, within the calcified coronary arteries. Autopsy-derived coronary artery specimens were scanned and reconstructed to visualize the pattern of coronary calcification using a novel microscopic computed tomography technique. The localization of the osteopontin were evaluated by immunohistochemial stain with LF7. The present study showed that the pattern of coronary calcification is variable and the expression of osteopontin is localized mainly to calcified lesion. The smooth muscle cells in addition to macrophage expressed osteopontin protein in human coronary atherosclerotic plaques. Soluble osteopontin released near to the sites of vascular calcification may represent an adaptive mechanism aimed at regulating the process of vascular calcification.
Aged ; Calcinosis/metabolism ; Coronary Arteriosclerosis/pathology* ; Coronary Arteriosclerosis/metabolism* ; Coronary Vessels/pathology* ; Coronary Vessels/metabolism ; Coronary Vessels/chemistry* ; Female ; Human ; Immunohistochemistry ; Male ; Middle Age ; Sialoglycoproteins/biosynthesis ; Sialoglycoproteins/analysis*

Sialic acid residues are constant constituents of the glycoproteins of the airways in all species. Sialoglycoproteins are the main acidic glycoprotein and their functions are to mediate cell adherence, to control the viscoelasticity of mucus and to serve as receptor sites for the binding of exogenous macromolecules. The purpose of this study was to investigate the differences in the distribution of sialoglycoproteins as a terminal sugar and in the composition of the penultimate sugar according to aging in the murine nasal respiratory and olfactory mucosa. Nasal cavities of mice (BALB/c) were fixed by intracardiac perfusion with 2.0% glutaraldehyde and embedded in Epon 812. First, the serial sections were stained with Maackia amurensis agglutinin (MAA) and Sambucus nigra agglutinin (SNA). Then, the adjacent sections were stained with DBA and PNA before and after neuraminidase digestion in all experimental groups. Apical cell surfaces of olfactory mucosa and cilia on a few ciliated cells in the mucosa of the septum and nasal floor were labelled with MAA, but cell surfaces of respiratory mucosa, Bowman's glands and goblet cells were not labelled with MAA, irrespective of aging. Apical cell surfaces of both olfactory and respiratory mucosa and Bowman's glands were stained with SNA, however, goblet cells were not labelled with SNA. After neuraminidase digestion to remove terminal sialic acid residues of sialoglycoproteins, only cell surfaces of respiratory mucosa were labelled with PNA, but goblet cells, cell surfaces of olfactory mucosa and Bowman's glands were not labelled with PNA. Cell surfaces and Bowman's glands of olfactory mucosa were labelled with DBA after neuraminidase digestion, but cell surfaces of respiratory mucosa and goblet cells were not labelled with DBA. Our results indicate that there were different carbohydrate structures of sialoglycoconjugates in olfactory and respiratory mucosa, and it was not influenced by aging.
Aging/metabolism* ; Animal ; Carbohydrates/analysis* ; Mice ; Mice, Inbred BALB C ; Nasal Mucosa/chemistry* ; Olfactory Mucosa/chemistry* ; Sialoglycoproteins/analysis*