OBJECTIVE: To investigate the antioxidant and osteogenic induction capabilities of calcium phosphate nanoflowers (hereinafter referred to as nanoflowers) in vitro at different concentrations. METHODS: Nanoflowers were prepared using gelatin, tripolyphosphate, and calcium chloride. Their morphology, microstructure, elemental composition and distribution, diameter, and molecular constitution were characterized using scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and energy-dispersive spectroscopy. Femurs and tibias were harvested from twelve 4-week-old Sprague Dawley rats, and bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured using the whole bone marrow adherent method, followed by passaging. The third passage cells were identified as stem cells by flow cytometry and then co-cultured with nanoflowers at concentrations of 0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, and 3.6 mg/mL. Cell counting kit 8 (CCK-8) assay was performed to screen for the optimal concentration that demonstrated the best cell viability, which was subsequently used as the experimental concentration for further studies. After co-culturing BMSCs with the screened concentration of nanoflowers, the biocompatibility of the nanoflowers was verified through live/dead cell staining, scratch assay, and cytoskeleton staining. The antioxidant capacity was assessed by using reactive oxygen species (ROS) fluorescence staining. The in vitro osteoinductive ability was evaluated via alkaline phosphatase (ALP) staining, alizarin red staining, and immunofluorescence staining of osteocalcin (OCN) and Runt-related transcription factor 2 (RUNX2). All the above indicators were compared with the control group of normally cultured BMSCs without the addition of nanoflowers. RESULTS: Scanning electron microscopy revealed that the prepared nanoflowers exhibited a flower-like structure; transmission electron microscopy scans discovered that the nanoflowers possessed a multi-layered structure, and high-magnification images displayed continuous atomic arrangements, with the nanoflower diameter measuring (2.00±0.25) μm; energy-dispersive spectroscopy indicated that the nanoflowers contained elements such as C, N, O, P, and Ca, which were uniformly distributed across the flower region; Fourier transform infrared spectroscopy analyzed the absorption peaks of each component, demonstrating the successful preparation of the nanoflowers. Through CCK-8 screening, the concentrations of 0.8, 1.2, and 1.6 mg/mL were selected for subsequent experiments. The live/dead cell staining showed that nanoflowers at different concentrations exhibited good cell compatibility, with the 1.2 mg/mL concentration being the best (P<0.05). The scratch assay results indicated that the cell migration ability in the 1.2 mg/mL group was superior to the other groups (P<0.05). The cytoskeleton staining revealed that the cell morphology was well-extended in all concentration groups, with no significant difference compared to the control group. The ROS fluorescence staining demonstrated that the ROS fluorescence in all concentration groups decreased compared to the control group after lipopolysaccharide induction (P<0.05), with the 1.2 mg/mL group showing the weakest fluorescence. The ALP staining showed blue-purple nodular deposits around the cells in all groups, with the 1.2 mg/mL group being significantly more prominent. The alizarin red staining displayed orange-red mineralized nodules around the cells in all groups, with the 1.2 mg/mL group having more and denser nodules. The immunofluorescence staining revealed that the expressions of RUNX2 and OCN proteins in all concentration groups increased compared to the control group, with the 1.2 mg/mL group showing the strongest protein expression (P<0.05). CONCLUSION The study successfully prepares nanoflowers, among which the 1.2 mg/mL nanoflowers exhibits excellent cell compatibility, antioxidant properties, and osteogenic induction capability, demonstrating their potential as an artificial bone substitute material.
Animals ; Osteogenesis/drug effects* ; Mesenchymal Stem Cells/drug effects* ; Calcium Phosphates/pharmacology* ; Rats, Sprague-Dawley ; Rats ; Antioxidants/chemistry* ; Cells, Cultured ; Cell Differentiation/drug effects* ; Nanostructures/chemistry* ; Tissue Engineering/methods* ; Bone Marrow Cells/cytology* ; Coculture Techniques ; Tissue Scaffolds/chemistry* ; Male ; Biocompatible Materials/chemistry* ; Cell Survival ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Cell Proliferation

OBJECTIVE: To explore the mechanism by which ω-3 polyunsaturated fatty acids (hereinafter referred to as "ω-3") exert antioxidant stress protection and promote osteogenic differentiation in MC3T3-E1 cells, and to reveal the relationship between ω-3 and the key antioxidant stress pathway involving nuclear factor E2-related factor 2 (Nrf2) and NAD (P) H quinone oxidoreductase 1 (NQO1) in MC3T3-E1 cells. METHODS: The optimal concentration of H ₂O ₂ (used to establish the oxidative stress model of MC3T3-E1 cells in vitro) and the optimal intervention concentrations of ω-3 were screened by cell counting kit 8. MC3T3-E1 cells were divided into blank control group, oxidative stress group (H ₂O ₂), low-dose ω-3 group (H ₂O ₂+low-dose ω-3), and high-dose ω-3 group (H ₂O ₂+high-dose ω-3). After osteoblastic differentiation for 7 or 14 days, the intracellular reactive oxygen species (ROS) level was measured by fluorescence staining and flow cytometry, and the mitochondrial morphological changes were observed by biological transmission electron microscope; the expression levels of Nrf2, NQO1, heme oxygenase 1 (HO-1), Mitofusin 1 (Mfn1), and Mfn2 were detected by Western blot to evaluate the cells' antioxidant stress capacity; the expression levels of Runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) were detected by immunofluorescence staining and Western blot; osteogenic potential of MC3T3-E1 cells was evaluated by alkaline phosphatase (ALP) staining and alizarin red staining. RESULTS: Compared with the oxidative stress group, the content of ROS in the low and high dose ω-3 groups significantly decreased, and the protein expressions of Nrf2, NQO1, and HO-1 significantly increased ( P<0.05). At the same time, the mitochondrial morphology of MC3T3-E1 cells improved, and the expressions of mitochondrial morphology-related proteins Mfn1 and Mfn2 significantly increased ( P<0.05). ALP staining and alizarin red staining showed that the low-dose and high-dose ω-3 groups showed stronger osteogenic ability, and the expressions of osteogenesis-related proteins RUNX2 and OCN significantly increased ( P<0.05). And the above results showed a dose-dependence in the two ω-3 treatment groups ( P<0.05). CONCLUSION ω-3 can enhance the antioxidant capacity of MC3T3-E1 cells under oxidative stress conditions and upregulate their osteogenic activity, possibly through the Nrf2/NQO1 signaling pathway.
Oxidative Stress/drug effects* ; NF-E2-Related Factor 2/metabolism* ; NAD(P)H Dehydrogenase (Quinone)/metabolism* ; Animals ; Mice ; Osteogenesis/drug effects* ; Cell Differentiation/drug effects* ; Fatty Acids, Omega-3/pharmacology* ; Signal Transduction/drug effects* ; Osteoblasts/drug effects* ; Reactive Oxygen Species/metabolism* ; Cell Line ; Hydrogen Peroxide/pharmacology* ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Antioxidants/pharmacology* ; Heme Oxygenase-1/metabolism*

OBJECTIVE: To initially investigate the function of neuronal pentraxin 1 (NPTX1) gene on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). METHODS: hBMSCs were induced to undergo osteogenic differentiation, and then RNA was collected at different time points, namely 0, 3, 7, 10 and 14 d. The mRNA expression levels of key genes related with osteogenic differentiation, including runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), and NPTX1, were detected on the basis of quantitative real-time polymerase chain reaction (qPCR) technology. In order to establish a stable NPTX1-knockdown hBMSCs cell line, NPTX1 shRNA lentivirus was constructed and used to infect hBMSCs. ALP staining, alizarin red (AR) staining, and qPCR were employed to assess the impact of NPTX1-knockdown on the osteogenic differentiation ability of hBMSCs. RESULTS: The results showed that during the osteogenic differentiation of hBMSCs in vitro, the mRNA expression levels of osteogenic genes RUNX2, ALP and OCN significantly increased compared with 0 d, while NPTX1 expression decreased markedly (P < 0.01) as the osteogenic induction period exten-ded. At 72 h post-infection with lentivirus, the result of qPCR indicated that the knockdown efficiency of NPTX1 was over 60%. After knocking down NPTX1 in hBMSCs, RNA was extracted from both the NPTX1-knockdown group (sh NPTX1 group) and the control group (shNC group) cultured in regular proliferation medium. The results of qPCR showed that the expression levels of osteogenic-related genes RUNX2 and osterix (OSX) were significantly higher in the sh NPTX1 group compared with the shNC group (P < 0.01). ALP staining revealed a significantly deeper coloration in the sh NPTX1 group than in the shNC group at the end of 7 d of osteogenic induction. AR staining demonstrated a marked increase in mineralized nodules in the sh NPTX1 group compared with the shNC group at the end of 14 d of osteogenic induction. CONCLUSION NPTX1 exerts a modulatory role in the osteogenic differentiation of hBMSCs, and its knockdown has been found to enhance the osteogenic differentiation of hBMSCs. This finding implies that NPTX1 could potentially serve as a therapeutic target for the treatment of osteogenic abnormalities, including osteoporosis.
Humans ; Mesenchymal Stem Cells/cytology* ; Osteogenesis/genetics* ; Cell Differentiation/genetics* ; Nerve Tissue Proteins/genetics* ; Cells, Cultured ; C-Reactive Protein/genetics* ; RNA, Small Interfering/genetics* ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Bone Marrow Cells/cytology* ; Gene Knockdown Techniques ; Osteocalcin/metabolism* ; Alkaline Phosphatase/metabolism* ; RNA, Messenger/metabolism*

Loss-of-function variants of low-density lipoprotein receptor-related protein 5 (LRP5) can lead to reduced bone formation, culminating in diminished bone mass. Our previous study reported transcription factor osterix (SP7)‍-binding sites on the LRP5 promoter and its pivotal role in upregulating LRP5 expression during implant osseointegration. However, the potential role of SP7 in ameliorating LRP5-dependent osteoporosis remained unknown. In this study, we used mice with a conditional knockout (cKO) of LRP5 in mature osteoblasts, which presented decreased osteogenesis. The in vitro experimental results showed that SP7 could promote LRP5 expression, thereby upregulating the osteogenic markers such as alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2), and β‍-catenin (P<0.05). For the in vivo experiment, the SP7 overexpression virus was injected into a bone defect model of LRP5 cKO mice, resulting in increased bone mineral density (BMD) (P<0.001) and volumetric density (bone volume (BV)/total volume (TV)) (P<0.001), and decreased trabecular separation (Tb.‍Sp) (P<0.05). These data suggested that SP7 could ameliorate bone defect healing in LRP5 cKO mice. Our study provides new insights into potential therapeutic opportunities for ameliorating LRP5-dependent osteoporosis.
Animals ; Low Density Lipoprotein Receptor-Related Protein-5/metabolism* ; Osteoporosis/genetics* ; Mice ; Mice, Knockout ; Sp7 Transcription Factor/physiology* ; Osteogenesis ; Bone Density ; Osteoblasts/metabolism* ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Mice, Inbred C57BL ; beta Catenin/metabolism*

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

OBJECTIVES: To explore the promoting effect of ginsenoside Rb3 (Rb3) on osteogenesis in periodontitis environment, and to explain its mechanism. METHODS: Human periodontal ligament stem cells (hPDLSCs) were cultured by tissue block method and identified by flow cytometry. Cell counting kit-8 (CCK8) method and calcein acetoxymethyl ester/propidium iodide staining were used to detect the effect of Rb3 on the viability of hPDLSCs cells. In vitro cell experiments were divided into control group, 10 μg/mL lipopolysaccharides (LPS) group, 10 μg/mL LPS+100 μmol/L Rb3 group and 10 μg/mL LPS+200 μmol/L Rb3 group. Alkaline phosphatase (ALP) staining was used to detect the ALP activity of hPDLSCs in each group after osteogenesis induction. The expression of hPDLSCs interleukin-6 (IL-6), interleukin-8 (IL-8), runt-related transcription factor 2 (RUNX2) and transforming growth factor-β (TGF-β)genes in each group after osteogenesis was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) method. Western blot was used to detect the protein expression of hPDLSCs phosphorrylated extracellular signal-regulated kinase (p-ERK) in each group. Sprague-Dawley rats were randomly divided into the control group, ligation group and ligation+Rb3 group. The left molar-maxillary tissue was subjected to micro-computed tomography (micro-CT) scanning. After the scanning, the left molar-maxilla was made into periodontal tissue sections. Hematoxylin-eosin (HE) staining was used to detect the infiltration and loss of adhesion of inflammatory cells. Masson staining was used to detect the destruction of gingival collagen fibers. Immunofluorescence staining was used to detect the protein expression of RUNX2 and p-ERK. The expression of TGF-β in rat gingival tissue was detected by qRT-PCR. The protein expression of IL-6 in peripheral serum of rats was detected by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used to detect the proportion of Treg cells in rat heart blood. The experimental data were statistically analyzed by Graph Pad Prism10.1.2 software. RESULTS: Rb3 had no effect on the cell activity of hPDLSCs. The results of qRT-PCR and ALP staining showed that Rb3 could inhibit the gene expression of IL-6 and IL-8 in inflammatory hPDLSCs, promote TGF-β gene and promote the osteogenic differentiation of inflammatory hPDLSCs. Western blot showed that Rb3 inhibited the protein expression of inflammatory hPDLSCs p-ERK. The results from micro-CT, Masson staining, and HE staining demonstrated that Rb3 promotes alveolar bone formation in rats with periodontitis, while simultaneously inhibiting the destruction of periodontal fibrous tissue, reducing attachment loss, and suppressing inflammatory cell infiltration. The results of flow cytometry showed that Rb3 could promote the differentiation of Treg cells in peripheral blood of periodontitis rats. The results of ELISA and qRT-PCR showed that Rb3 could inhibit the protein expression of IL-6 and promote the gene expression of TGF-β in periodontitis rats. Immunofluorescence results showed that Rb3 could promote the protein expression of RUNX2 and inhibit the protein expression of p-ERK in periodontitis rats. CONCLUSIONS Rb3 can reduce the inflammatory reaction of periodontal tissues in periodontitis rats, and promote the osteogenic differentiation of hPDLSCs by regulating p-ERK pathways.
Animals ; Ginsenosides/pharmacology* ; Osteogenesis/drug effects* ; Periodontitis/metabolism* ; Rats ; Periodontal Ligament/cytology* ; Humans ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Stem Cells/drug effects* ; Interleukin-6/metabolism* ; Rats, Sprague-Dawley ; Interleukin-8/metabolism* ; Cells, Cultured ; MAP Kinase Signaling System/drug effects* ; Transforming Growth Factor beta/metabolism* ; Signal Transduction ; Male ; Phosphorylation ; Lipopolysaccharides ; Extracellular Signal-Regulated MAP Kinases/metabolism* ; Alkaline Phosphatase/metabolism*

OBJECTIVES: Investigating the protective effect of naringenin (NAR) on the osteogenic potential of human periodontal ligament stem cells (hPDLSCs) under oxidative stress and its related mechanisms. METHODS: The oxidative damage model of hPDLSCs was established using hydrogen peroxide (H₂O₂) andthe hPDLSCs were treated with different concentrations of NAR and 0.5 μmol/L forkhead box protein O-1 (FOXO1) inhibitor AS1842856. After that, the cell counting kit-8 (CCK8) was used to determine the optimal concentrations of H₂O₂ and NAR. The alkaline phosphatase (ALP) staining and real time fluorescent quantitative reverse transcription polymerase chain reaction (qRT-PCR) were employed to assess the expression of ALP, runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) in hPDLSCs of each group. The enzyme-linked immunosorbent assay (ELISA) and 2',7'-dichlorofluorescin diacetate (DCFH-DA) staining were utilized to evaluate the expression of reactive oxygen species (ROS), malondialdehyde (MDA) and lactate dehydrogenase (LDH) in hPDLSCs. Meanwhile, qRT-PCR and western blot were used to detect the expression levels of FOXO1 and β-catenin, both are pathway related genes and proteins. RESULTS: H₂O₂ exposure led to an increase in oxidative damage in hPDLSCs, characterized by a rise in intracellular ROS levels and increased expression of MDA and LDH (P<0.05). At the same time, the osteogenic differentiation ability of hPDLSCs decreased, as evidenced by lighter ALP staining and reduced expression levels of osteogenic differentiation-related genes ALP, RUNX2 and OCN (P<0.05). Co-treatment with NAR alleviated the oxidative damage in hPDLSCs, enhanced their antioxidant capacity, and restored their osteogenic ability. The FOXO1 inhibitor AS1842856 downregulated the expression of β-catenin (P<0.05) and significantly diminished both the antioxidant effect of NAR and its ability to restore osteogenesis (P<0.05). CONCLUSIONS NAR can enhance the antioxidant capacity of hPDLSCs by activating the FOXO1/β-catenin signaling pathway within hPDLSCs, thereby mitigating oxidative stress damage and alleviating the loss of osteogenic capacity.
Humans ; Oxidative Stress/drug effects* ; Periodontal Ligament/cytology* ; Hydrogen Peroxide ; Forkhead Box Protein O1/metabolism* ; Stem Cells/cytology* ; Flavanones/pharmacology* ; beta Catenin/metabolism* ; Osteogenesis/drug effects* ; Signal Transduction ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Alkaline Phosphatase/metabolism* ; Osteocalcin/metabolism* ; Cells, Cultured ; Cell Differentiation/drug effects*

OBJECTIVES: This study investigated the effects of a polycaprolactone (PCL)-polyethylene glycol (PEG) scaffold incorporated with concentrated growth factor (CGF) on the adhesion, proliferation, and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). METHODS: The PCL-PEG-CGF composite scaffold was fabricated using an immersion and freeze-drying technique. Its microstructure, mechanical properties, and biocompatibility were systematically characterized. The hPDLSCs were isolated through enzymatic digestion, and the hPDLSCs were identified through flow cytometry. Third-passage hPDLSCs were seeded onto the composite scaffolds, and their adhesion, proliferation and osteogenic differentiation were assessed using CCK-8 assays, 4',6-diamidino-2-phenylindole (DAPI) staining, alkaline phosphatase (ALP) staining, alizarin red staining, and Western blot analysis of osteogenesis-related proteins [Runt-related transcription factor 2 (Runx2), ALP, and morphogenetic protein 2 (BMP2)]. RESULTS: Scanning electron microscopy revealed that the PCL-PEG-CGF composite scaffold exhibited a honeycomb-like structure with heterogeneous pore sizes. The composite scaffold exhibited excellent hydrophilicity, as evidenced by a contact angle (θ) approaching 0° within 6 s. Its elastic modulus was measured at (4.590 0±0.149 3) MPa, with comparable hydrophilicity, fracture tensile strength, and fracture elongation to PCL-PEG scaffold. The hPDLSCs exhibited significantly improved adhesion to the PCL-PEG-CGF composite scaffold compared with the PCL-PEG scaffold (P<0.01). Additionally, cell proliferation was markedly improved in all the experimental groups on days 3, 5, and 7 (P<0.01), and statistically significant differences were found between the PCL-PEG-CGF group and other groups (P<0.01). The PCL-PEG-CGF group showed significantly elevated ALP activity (P<0.05), increased mineralization nodule formation, and upregulated expression of osteogenic-related proteins (Runx2, BMP2 and ALP; P<0.05). CONCLUSIONS The PCL-PEG-CGF composite scaffold exhibited excellent mechanical properties and biocompatibility, enhancing the adhesion and proliferation of hPDLSCs and promoting their osteogenic differentiation by upregulating osteogenic-related proteins.
Humans ; Polyesters/chemistry* ; Periodontal Ligament/cytology* ; Polyethylene Glycols/chemistry* ; Stem Cells/cytology* ; Tissue Scaffolds ; Cell Proliferation ; Osteogenesis ; Cell Differentiation ; Cell Adhesion ; Bone Morphogenetic Protein 2/metabolism* ; Cells, Cultured ; Alkaline Phosphatase/metabolism* ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Intercellular Signaling Peptides and Proteins/pharmacology* ; Tissue Engineering/methods*

OBJECTIVE: To Investigate the effects of lithocholic acid (LCA) on the balance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). METHODS: Twelve 10-week-old SPF C57BL/6J female mice were randomly divided into an experimental group (undergoing bilateral ovariectomy) and a control group (only removing the same volume of adipose tissue around the ovaries), with 6 mice in each group. The body mass was measured every week after operation. After 4 weeks post-surgery, the weight of mouse uterus was measured, femur specimens of the mice were taken for micro-CT scanning and three-dimensional reconstruction to analyze changes in bone mass. Tibia specimens were taken for HE staining to calculate the number and area of bone marrow adipocytes in the marrow cavity area. ELISA was used to detect the expression of bone turnover markers in the serum. Liver samples were subjected to real-time fluorescence quantitative PCR (RT-qPCR) to detect the expression of key genes related to bile acid metabolism, including cyp7a1, cyp7b1, cyp8b1, and cyp27a1. BMSCs were isolated by centrifugation from 2 C57BL/6J female mice (10-week-old). The third-generation cells were exposed to 0, 1, 10, and 100 μmol/L LCA, following which cell viability was evaluated using the cell counting kit 8 assay. Subsequently, alkaline phosphatase (ALP) staining and oil red O staining were conducted after 7 days of osteogenic and adipogenic induction. RT-qPCR was employed to analyze the expressions of osteogenic-related genes, namely ALP, Runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), as well as adipogenic-related genes including Adiponectin (Adipoq), fatty acid binding protein 4 (FABP4), and peroxisome proliferator-activated receptor γ (PPARγ). RESULTS: Compared with the control group, the body mass of the mice in the experimental group increased, the uterus atrophied, the bone mass decreased, the bone marrow fat expanded, and the bone metabolism showed a high bone turnover state. RT-qPCR showed that the expressions of cyp7a1, cyp8b1, and cyp27a1, which were related to the key enzymes of bile acid metabolism in the liver, decreased significantly ( P<0.05), while the expression of cyp7b1 had no significant difference ( P>0.05). Intervention with LCA at concentrations of 1, 10, and 100 μmol/L did not demonstrate any apparent toxic effects on BMSCs. Furthermore, LCA inhibited the expressions of osteogenic-related genes (ALP, Runx2, and OCN) in a dose-dependent manner, resulting in a reduction in ALP staining positive area. Concurrently, LCA promoted the expressions of adipogenic-related genes (Adipoq, FABP4, and PPARγ), and an increase in oil red O staining positive area. CONCLUSION After menopause, the metabolism of bile acids is altered, and secondary bile acid LCA interferes with the balance of osteogenic and adipogenic differentiation of BMSCs, thereby affecting bone remodelling.
Female ; Mice ; Animals ; Core Binding Factor Alpha 1 Subunit/pharmacology* ; PPAR gamma/metabolism* ; Steroid 12-alpha-Hydroxylase/metabolism* ; Mice, Inbred C57BL ; Cell Differentiation ; Osteogenesis ; Mesenchymal Stem Cells ; Bile Acids and Salts/pharmacology* ; Bone Marrow Cells ; Cells, Cultured ; Azo Compounds

OBJECTIVE: To explore the clinical phenotypes and genetic diagnosis of 2 sporadic cases for cleidocranial dysplasia. METHODS: The clinical data of two cases of CCD admitted to the Third Affiliated Hospital of Zhengzhou University on December 16, 2021 and December 9, 2021 were analyzed retrospectively, and the whole exome sequencing (WES), chromosome microarray analysis and copy number variation sequencing were performed. RESULTS: The main ultrasonographic findings of the fetus had included poorly calcified skull bones, budging of parieto-occipital area, compression and deformation of skull, and loss of nasal bone. The infant's clinical phenotypes included delayed closure of anterior fontanelle, recurrent respiratory tract infection, growth retardation, and clavicular hypoplasia. By WES analysis, the fetus was found to harbor a heterozygous c.911_914delinsTTT variant of the RUNX2 gene, whilst the infant was found to harbor a heterozygous c.1008delT variant of the RUNX2 gene. Both variants were verified by Sanger sequencing to have occurred de novo. CONCLUSION For sporadic cases featuring cleidocranial dysplasia, prenatal ultrasonography is particularly important. Hypoplastic clavicle, skull calcification and nasal bone absence are the main features. Diagnosis should also be suspected for infants featuring growth retardation, recurrent respiratory tract infections and clavicular dysplasia. The identification of the c.911_914delinsTTT and c.1008delT variants of the RUNX2 gene has facilitated genetic counseling and prenatal diagnosis, and also expanded the mutational spectrum of the RUNX2 gene.
Female ; Humans ; Pregnancy ; Cleidocranial Dysplasia/genetics* ; Core Binding Factor Alpha 1 Subunit ; DNA Copy Number Variations ; Growth Disorders ; Retrospective Studies