Luminescence-Monitored Progressive Chemical Pressure Implementation Realized through Successive Y3+ and Mg2+ Doping into Ca10.5(PO4)7:Eu2.

Chemical pressure generated through ion doping into crystal lattices has been proven to be conducive to exploration of new matter, development of novel functionalities, and realization of unprecedented performances. However, studies are focusing on one-time doping, and there is a lack of both advanced investigations for multiple doping and sophisticated strategies to precisely and quantitatively track the gradual functionality evolution along with progressive chemical pressure implementation. Herein, high-valent Y3+ and equal-valent Mg2+ is successively doped to replace multiple Ca sites in Ca10.5(PO4)7:Eu2+. The luminescence evolution of Eu2+ serves as an optical probe, allowing step-by-step and atomic-level tracking of the site occupation of Y3+ and Mg2+, interassociation of Ca sites, and ultimately functionality improvement. The resulting Ca8MgY(PO4)7:Eu2+ displays a record-high relative sensitivity for optical thermometry. Utilization of the environment-sensitive emission of Eu2+ as a luminescent probe has offered a unique approach to monitoring structure-functionality evolution in vivo with atomic precision, which shall also be extended to optimization of other functionalities such as ferroelectricity, conductivity, thermoelectricity, and catalytic activity through precise control over atomic diffusion in other types of substances.

Biomechanical stress regulates mammalian tooth replacement via the integrin ß1-RUNX2-Wnt pathway.

Renewal of integumentary organs occurs cyclically throughout an organism's lifetime, but the mechanism that initiates each cycle remains largely unknown. In a miniature pig model of tooth development that resembles tooth development in humans, the permanent tooth did not begin transitioning from the resting to the initiation stage until the deciduous tooth began to erupt. This eruption released the accumulated mechanical stress inside the mandible. Mechanical stress prevented permanent tooth development by regulating expression and activity of the integrin ß1-ERK1-RUNX2 axis in the surrounding mesenchyme. We observed similar molecular expression patterns in human tooth germs. Importantly, the release of biomechanical stress induced downregulation of RUNX2-wingless/integrated (Wnt) signaling in the mesenchyme between the deciduous and permanent tooth and upregulation of Wnt signaling in the epithelium of the permanent tooth, triggering initiation of its development. Consequently, our findings identified biomechanical stress-associated Wnt modulation as a critical initiator of organ renewal, possibly shedding light on the mechanisms of integumentary organ regeneration.

MiR-26b-5p/TET3 regulates the osteogenic differentiation of human bone mesenchymal stem cells and bone reconstruction in female rats with calvarial defects.

BACKGROUND: MicroRNAs (miRNAs) play critical roles in the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs), but the mechanism by which miRNAs indirectly modulate osteogenesis remains unclear. Here, we explored the mechanism by which miRNAs indirectly modulate gene expression through histone demethylases to promote bone regeneration. METHODS AND RESULTS: Bioinformatics analysis was performed on hBMSCs after 7 days of osteogenic induction. The differentially expressed miRNAs were screened, and potential target mRNAs were identified. To determine the bioactivity and stemness of hBMSCs and their potential for bone repair, we performed wound healing, Cell Counting Kit-8 (CCK-8), real-time reverse transcription quantitative polymerase chain reaction (RT‒qPCR), alkaline phosphatase activity, alizarin red S (ARS) staining and radiological and histological analyses on SD rats with calvarial bone defects. Additionally, a dual-luciferase reporter assay was utilized to investigate the interaction between miR-26b-5p and ten-eleven translocation 3 (TET3) in human embryonic kidney 293T cells. The in vitro and in vivo results suggested that miR-26b-5p effectively promoted the migration, proliferation and osteogenic differentiation of hBMSCs, as well as the bone reconstruction of calvarial defects in SD rats. Mechanistically, miR-26b-5p bound to the 3' untranslated region of TET3 mRNA to mediate gene silencing. CONCLUSIONS: MiR-26b-5p downregulated the expression of TET3 to increase the osteogenic differentiation of hBMSCs and bone repair in rat calvarial defects. MiR-26b-5p/TET3 crosstalk might be useful in large-scale critical bone defects.

Experimental Research on Shipborne SINS Rapid Mooring Alignment with Variance-Constraint Kalman Filter and GNSS Position Updates.

Analytical coarse alignment and Kalman filter fine alignment based on zero-velocity are typically used to obtain initial attitude for inertial navigation systems (SINS) on a static base. However, in the shipboard mooring state, the static observation condition is corrupted. This paper presents a rapid alignment method for SINS on swaying bases. The proposed method begins with a coarse alignment technique in the inertial frame to obtain an initial rough attitude. Subsequently, a Kalman filter with position updates is employed to estimate the remaining misalignment error. To enhance the filter estimation performance, an appropriate lower boundary is set to the target states' variances according to a carefully designed relative convergence index. The variance-constraint Kalman filter (VCKF) approach is proposed in this paper, and the shipborne experiments validate its effectiveness. The results demonstrate that the VCKF approach significantly reduces the time requirement for fine alignment to achieve the same accuracy on a swaying base, from 90 min in the classic Kalman filter to 30 min. Additionally, the parameter estimation performance in the Kalman filter is also improved, particularly in situations where unpredicted external interference is involved during fine alignment.

Deep Transfer Learning for Cerebral Cortex Using Area-Preserving Geometry Mapping.

Limited sample size hinders the application of deep learning in brain image analysis, and transfer learning is a possible solution. However, most pretrained models are 2D based and cannot be applied directly to 3D brain images. In this study, we propose a novel framework to apply 2D pretrained models to 3D brain images by projecting surface-based cortical morphometry into planar images using computational geometry mapping. Firstly, 3D cortical meshes are reconstructed from magnetic resonance imaging (MRI) using FreeSurfer and projected into 2D planar meshes with topological preservation based on area-preserving geometry mapping. Then, 2D deep models pretrained on ImageNet are adopted and fine-tuned for cortical image classification on morphometric shape metrics. We apply the framework to sex classification on the Human Connectome Project dataset and autism spectrum disorder (ASD) classification on the Autism Brain Imaging Data Exchange dataset. Moreover, a 2-stage transfer learning strategy is suggested to boost the ASD classification performance by using the sex classification as an intermediate task. Our framework brings significant improvement in sex classification and ASD classification with transfer learning. In summary, the proposed framework builds a bridge between 3D cortical data and 2D models, making 2D pretrained models available for brain image analysis in cognitive and psychiatric neuroscience.

Genetic predisposition to female infertility in relation to epithelial ovarian and endometrial cancers.

BACKGROUND: The associations between female infertility and epithelial ovarian cancer (EOC) or endometrial cancer (EC) have been reported in observational studies, but its causal relationship remains unknown. We intended to assess the causal effect of female infertility on EOCs and ECs using a two-sample Mendelian Randomization (MR) approach. METHODS: Large pooled genome-wide association study (GWAS) datasets for female infertility (6481 cases and 68 969 controls), EOC (25 509 cases and 40 941 controls), and EC (12 906 cases and 108 979 controls) were derived from public GWAS databases and published studies. The Inverse Variance Weighted method, Weighted Median method, MR-Egger regression, and MR-Pleiotropy Residual Sum and Outlier test were adopted for MR analyses. RESULTS: Our results suggested that genetically predicted infertility was positively associated with the risk of EOC (OR = 1.117, 95% CI = 1.003-1.245, P = .045), but did not find a causal relationship between infertility and EC (OR = 1.081, 95% CI = 0.954-1.224, P = .223). As to the reverse direction, our study did not obtain evidence from genetics that EOCs (OR = 0.974, 95% CI = 0.825-1.150, P = .755) and ECs (OR = 1.039, 95% CI = 0.917-1.177, P = .548) were associated with an increased risk of infertility. CONCLUSIONS: This large MR analysis supported a causal association between female infertility and increased risk of EOCs, but did not find a causal relationship between infertility and ECs.

tRNA-derived small RNA changes in bone marrow stem cells under hypoxia and osteogenic conduction.

BACKGROUND: Tissue engineering using bone mesenchymal stem cells (BMSCs) transplantation is a promising therapeutic for bone regeneration. However, the effect of bone regeneration remains unsatisfactory due to the BMSCs' functional abnormality influenced by hypoxia. In this study, we attempt to explore the mechanism of osteogenic differentiation of BMSCs under hypoxic conditions from the perspective of non-coding RNA regulation. METHODS: The study employed BMSCs obtained from healthy donors and simulated hypoxia using CoCl2 stimulation. High-throughput sequencing technique was used to identify differential expression profiles of tRNA-derived small RNA (tsRNA) in three experimental groups: BMSCs-0d, BMSCs-7d and BMSCs-0d-CoCl2 . TargetScan and miRanda algorithms were used to determine tsRNA target genes, while Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis were employed for the prediction of biological functions. Real-time reverse transcriptase-polymerase chain reaction (Real-time RT-PCR) was carried out on four selected differentially expressed tsRNAs. RESULTS: After the osteogenic induction and CoCl2 stimulated separately, there were 19 tsRNAs differentially expressed in BMSCs, including 14 upregulated and five downregulated. According to the analysis of biological information, these tsRNAs may regulate 311 potential target genes and mainly enrich the pathways such as metabolic pathways, Wnt signalling pathway, osteoclast differentiation, cellular senescence and mTOR signalling pathway. The results of Real-time RT-PCR for 3'tiRNA-41-GlnTTG-6, 3'tiRNA-42-LysTTT-8, 5'tiRNA-35-CysACA-1 and tRF3a-AsnGTT-9 were consistent with small RNA sequencing data. CONCLUSION: We discovered the tsRNA that changes the process of osteogenesis and hypoxia, which provides new targets for promoting survival and regeneration functions after BMSCs transplantation.

KDM6B Negatively Regulates the Neurogenesis Potential of Apical Papilla Stem Cells via HES1.

Stem cells from the apical papilla (SCAPs) are used to regulate the microenvironment of nerve defects. KDM6B, which functions as an H3K27me3 demethylase, is known to play a crucial role in neurogenesis. However, the mechanism by which KDM6B influences the neurogenesis potential of SCAPs remains unclear. We evaluated the expression of neural markers in SCAPs by using real-time RT-PCR and immunofluorescence staining. To assess the effectiveness of SCAP transplantation in the SCI model, we used the BBB scale to evaluate motor function. Additionally, toluidine blue staining and Immunofluorescence staining of NCAM, NEFM, ß-III-tubulin, and Nestin were used to assess nerve tissue remodeling. Further analysis was conducted through Microarray analysis and ChIP assay to study the molecular mechanisms. Our results show that KDM6B inhibits the expression of NeuroD, TH, ß-III tubulin, and Nestin. In vivo studies indicate that the SCAP-KDM6Bsh group is highly effective in restoring spinal cord structure and motor function in rats suffering from SCI. Our findings suggest that KDM6B directly binds to the HES1 promoter via regulating H3K27me3 and HES1 expression. In conclusion, our study can help understand the regulatory role of KDM6B in neurogenesis and provide more effective treatments for nerve injury.

A Dual-Modality Imaging Method Based on Polarimetry and Second Harmonic Generation for Characterization and Evaluation of Skin Tissue Structures.

The characterization and evaluation of skin tissue structures are crucial for dermatological applications. Recently, Mueller matrix polarimetry and second harmonic generation microscopy have been widely used in skin tissue imaging due to their unique advantages. However, the features of layered skin tissue structures are too complicated to use a single imaging modality for achieving a comprehensive evaluation. In this study, we propose a dual-modality imaging method combining Mueller matrix polarimetry and second harmonic generation microscopy for quantitative characterization of skin tissue structures. It is demonstrated that the dual-modality method can well divide the mouse tail skin tissue specimens' images into three layers of stratum corneum, epidermis, and dermis. Then, to quantitatively analyze the structural features of different skin layers, the gray level co-occurrence matrix is adopted to provide various evaluating parameters after the image segmentations. Finally, to quantitatively measure the structural differences between damaged and normal skin areas, an index named Q-Health is defined based on cosine similarity and the gray-level co-occurrence matrix parameters of imaging results. The experiments confirm the effectiveness of the dual-modality imaging parameters for skin tissue structure discrimination and assessment. It shows the potential of the proposed method for dermatological practices and lays the foundation for further, in-depth evaluation of the health status of human skin.

PRMT6/LMNA/CXCL12 signaling pathway regulated the osteo/odontogenic differentiation ability in dental stem cells isolated from apical papilla.

Tooth loss and maxillofacial bone defect are common diseases, which seriously affect people's health. Effective tooth and maxillofacial bone tissue regeneration is a key problem that need to be solved. In the present study, we investigate the role of PRMT6 in osteo/odontogenic differentiation and migration capacity by using SCAPs. Our results showed that knockdown of PRMT6 promoted the osteo/odontogenic differentiation compared with the control group, as detected by alkaline phosphatase activity, alizarin red staining, and the indicators of osteo/odontogenic differentiation measured by Western blot. In addition, overexpression of PRMT6 inhibited the osteo/odontogenic differentiation potentials of SCAPs. Then, knockdown of PRMT6 promoted the migration ability and overexpression of PRMT6 inhibited the migration ability in SCAPs. Mechanically, we discovered that the depletion of PRMT6 promoted the expression of CXCL12 by decreasing H3R2 methylation in the promoter region of CXCL12. In addition, PRMT6 formed a protein complex with LMNA, a nuclear structural protein. Depletion of LMNA inhibited the osteo/odontogenic differentiation and CXCL12 expression and increased the intranucleus PRMT6 in SCAPs. To sum up, PRMT6 might inhibit the osteo/odontogenic differentiation and migration ability of SCAPs via inhibiting CXCL12. And LMNA might be a negative regulator of PRMT6. It is suggested that PRMT6 may be a key target for SCAP-mediated bone and tooth tissue regeneration.

LncRNA, PLXDC2-OT promoted the osteogenesis potentials of MSCs by inhibiting the deacetylation function of RBM6/SIRT7 complex and OSX specific isoform.

Bone regeneration and remodeling are complex physiological processes that are regulated by key transcription factors. Understanding the regulatory mechanism of key transcription factors on the osteogenic differentiation of mesenchymal stem cells (MSCs) is a key issue for successful bone regeneration and remodeling. In the present study, we investigated the regulatory mechanism of the histone deacetylase Sirtuin 7 (SIRT7) on the key transcription factor OSX and osteogenesis of MSCs. In this study, we found that SIRT7 knockdown increased ALP activity and in vitro mineralization and promoted the expression of the osteogenic differentiation markers DSPP, DMP1, BSP, OCN, and the key transcription factor OSX in MSCs. In addition, SIRT7 could associate with RNA binding motif protein 6 (RBM6) to form a protein complex. Moreover, RBM6 inhibited ALP activity, the expression of DSPP, DMP1, BSP, OCN, and OSX in MSCs, and the osteogenesis of MSCs in vivo. Then, the SIRT7/RBM6 protein complex was shown to downregulate the level of H3K18Ac in the OSX promoter by recruiting SIRT7 to the OSX promoter and inhibiting the expression of OSX isoforms 1 and 2. Furthermore, lncRNA PLXDC2-OT could associate with the SIRT7/RBM6 protein complex to diminish its binding and deacetylation function in the OSX promoter and its inhibitory function on OSX isoforms 1 and 2 and to promote the osteogenic potential of MSCs.

Altered cerebellar-motor loop in benign adult familial myoclonic epilepsy type 1: The structural basis of cortical tremor.

OBJECTIVE: Cortical tremor/myoclonus is the hallmark feature of benign adult familial myoclonic epilepsy (BAFME), the mechanism of which remains elusive. A hypothesis is that a defective control in the preexisting cerebellar-motor loop drives cortical tremor. Meanwhile, the basal ganglia system might also participate in BAFME. This study aimed to discover the structural basis of cortical tremor/myoclonus in BAFME. METHODS: Nineteen patients with BAFME type 1 (BAFME1) and 30 matched healthy controls underwent T1-weighted and diffusion tensor imaging scans. FreeSurfer and spatially unbiased infratentorial template (SUIT) toolboxes were utilized to assess the motor cortex and the cerebellum. Probabilistic tractography was generated for two fibers to test the hypothesis: the dentato-thalamo-(M1) (primary motor cortex) and globus pallidus internus (GPi)-thalamic projections. Average fractional anisotropy (FA), axial diffusivity (AD), mean diffusivity (MD), and radial diffusivity (RD) of each tract were extracted. RESULTS: Cerebellar atrophy and dentate nucleus alteration were observed in the patients. In addition, patients with BAFME1 exhibited reduced AD and FA in the left and right dentato-thalamo-M1 nondecussating fibers, respectively false discovery rate (FDR) correction q < .05. Cerebellar projections showed negative correlations with somatosensory-evoked potential P25-N33 amplitude and were independent of disease duration and medication. BAFME1 patients also had increased FA and decreased MD in the left GPi-thalamic projection. Higher FA and lower RD in the right GPi-thalamic projection were also observed (FDR q < .05). SIGNIFICANCE: The present findings support the hypothesis that the cerebello-thalamo-M1 loop might be the structural basis of cortical tremor in BAFME1. The basal ganglia system also participates in BAFME1 and probably serves a regulatory role.

Identification and profiles of microRNAs in different development stages of miniature pig secondary palate.

Cleft palate is one of the most frequent craniofacial malformation birth defects. Miniature pigs (Sus scrofa) are a valuable alternative large animal model to explore human palate development. Presently, the microRNA (miRNA) expression profiles in miniature pigs during palatogenesis from embryonic day (E) 30 to 50 were identified. A total of 2044 known miRNAs and 192 novel miRNAs were identified. The functional characteristics of their potential target genes were identified using Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway analysis. MiRNAs displayed diverse expression levels among the different stages. Using Short Time-series Expression Miner software to investigate the expression patterns of miRNAs from E30-50, all miRNAs were clustered into 20 profiles. The profiles showing miRNAs expression decreased (profile 0)/increased (profile 19) from E30-50 were the main patterns during palatogenesis. Hub genes of four significant modules were identified by weighted correlation network analysis, including ssc-miR-98, ssc-miR-27a_R + 1, and ssc-miR-150, etc. which might be novel potential targets for regulating palate development. The data are expected to improve the understanding of palate development and the etiology of cleft palate in further studies.

GREM1 inhibits osteogenic differentiation, senescence and BMP transcription of adipose-derived stem cells.

Purpose: Adipose-derived stem cells (ADSCs) are ideal for cell-based therapies to support bone regeneration. It is vital to understand the critical genes and molecular mechanisms involved in the functional regulation of ADSCs for enhancing bone regeneration. In the present study, we investigated the Gremlin 1 (GREM1) effect on ADSCs osteogenic differentiation and senescence.Materials and methods: The in vitro ADSCs osteogenic differentiation potential was evaluated by determining alkaline phosphatase (ALP) activity, mineralization ability, and the expression of osteogenic markers. Cell senescence is determined by SA-ß-gal staining, telomerase assay, and the expression of aging markers.Results: GREM1 overexpression in ADSCs reduced ALP activity and mineralization, inhibited the expression of osteogenic related genes OCN, OPN, DSPP, DMP1, and BSP, and key transcription factors, RUNX2 and OSX. GREM1 knockdown in ADSCs enhanced ALP activity and mineralization, promoted the expression of OCN, OPN, DSPP, DMP1, BSP, RUNX2, and OSX. GREM1 overexpression in ADSCs reduced the percent SA-ß-Gal positive cells, P16 and P53 expressions, and increased telomerase activity. GREM1 knockdown in ADSCs increased the percentage of SA-ß-Gal positive cells, P16 and P53 expressions, and reduced telomerase activity. Furthermore, GREM1 reduced the mRNA expression levels of BMP2, BMP6, and BMP7.Conclusions: In summary, our findings suggested that GREM1 inhibited ADSCs senescence and osteogenic differentiation and antagonized BMP transcription.

Pleiotropin enhances the osteo/dentinogenic differentiation potential of dental pulp stem cells.

Purpose: Pleiotrophin (PTN) is a heparin-binding growth-associated molecule and expressed in ameloblasts and odontoblasts throughout tooth maturation. Our previous study has shown that PTN expressed more than 20-fold higher in dental tissue than dental stem cells. However, the role of PTN on proliferation and osteo/dentinogenesis of dental pulp stem cells (DPSCs) is unclear. The purpose of the present study was to investigate the role of PTN on the DPSCs' function.Methods: DPSCs were isolated from human third molars. Short hairpin RNAs (shRNAs) was used to knock down the PTN expression in DPSCs. Real-time RT-PCR, alizarin red staining, quantitative calcium analysis, in vivo transplantation and cell counting kit-8 (CCK8) assay were used to study the function of DPSCs. Possible mechanism was studied by RNA sequencing.Results: After PTN depletion, ALP activity and mineralization ability of DPSCs decreased. Expression of DMP-1 and BSP weakened. Proliferation of DPSCs at 48 h and 72 h was inhibited. Furthermore, 50 pg/mL PTN recombinant protein rescued the impaired osteo/dentinogenic differentiation potential and proliferation ability caused by PTN depletion. In addition, RNA sequencing showed 221 genes were downregulated and 233 genes upregulated in PTN depleted DPSCs. Several genes including BMP2 and IGFBP5 might be associated with PTN function on the DPSCs. P53 and the AMPK signaling pathways were involved. LncRNA analysis displayed 47 significantly upregulated lncRNA and 31 downregulated lncRNA comparing PTN depleted DPSCs with the control.Conclusion: Our research demonstrated that PTN has a positive role in maintaining DPSCs proliferation and osteo/dentinogenic differentiation potential.

Understanding the Doping Chemistry of High Oxidation States in Scheelite CaWO4 by Hydrothermal Conditions.

Doping chemistry has become one of the most effective means of tuning materials' properties for diverse applications. In particular for scheelite-type CaWO4, high-oxidation-state doping is extremely important, since one may expand the scheelite family and further create prospective candidates for novel applications and/or useful spectral signatures for nuclear forensics. However, the chemistry associated with high-valence doping in scheelite-type CaWO4 is far from understanding. In this work, a series of scheelite-based materials (Ca1-x-y-zEuxKy□z)WO4 (□ represents the cation vacancy of the Ca2+ site) were synthesized by hydrothermal conditions and solid-state methods and comparatively studied. For the bulk prepared by the solid-state method, occupation of high-oxidation-state Eu3+ at the Ca2+ sites of CaWO4 is followed by doping of the low-oxidation-state K+ at a nearly equivalent molar amount. The Eu3+ local symmetry is thus varied from the original S4 point group symmetry to C2v point group symmetry. Surprisingly different from the cases in bulk, for the nanoscale counterparts prepared by hydrothermal conditions, the high-oxidation-state Eu3+ was incorporated in CaWO4 at two distinct sites, and its amount is higher than that of the low-oxidation-state K+ even though KOH was used as a mineralizer, creating a certain amount of cation vacancies. Consequently, an apparent split emission of 5D0 → 7F0 was first demonstrated for (Ca1-x-y-zEuxKy□z)WO4. The doping chemistry of high oxidation states uncovered in this work not only provides an explanation for the commonly observed spectral changes in rare-earth-ion-modified scheelite structures, but also points out an advanced direction that can guide the design and synthesis of novel functional oxides by solution chemistry routes.

SFRP2 enhances dental pulp stem cell-mediated dentin regeneration in rabbit jaw.

OBJECTIVES: Dental tissue-derived mesenchymal stem cell (MSC)-mediated tooth regeneration may be a useful therapeutic tool for repairing tooth loss. However, the low success rate of tooth regeneration restricts its clinical application. Identifying key factors for enhancing dentinogenesis in MSCs is crucial for promoting tooth regeneration. MATERIALS AND METHODS: Human dental pulp stem cells (DPSCs) were transfected with retrovirus to obtain SFRP2-over-expressing DPSCs. Alkaline phosphatase (ALP) activity assay, Alizarin red staining, quantitative analysis of calcium, and dentinogenesis-related genes were detected. Additionally, transplantation in a rabbit tooth extraction model was used to explore the role of SFRP2 in dentin regeneration. RESULTS: We found SFRP2 over-expression greatly enhanced ALP activity, and mineralization in DPSCs. Real-time RT-PCR revealed SFRP2 over-expression promoted the expressions of OSX, RUNX2, DSPP, DMP1, and BSP. Moreover, Micro CT analysis showed high-density calcification occurred to a much higher extent in SFRP2 over-expressing group compared to control group in vivo. Additionally, HE staining, immmunohistochemistry staining, and scanning electron microscopy results showed much more dentin-like tissue formed in SFRP2 over-expressing group compared to control group. CONCLUSIONS: Our findings revealed SFRP2 is an important regulator that enhances the dentinogenesis of DPSCs and dentin regeneration in the jaw, which may have clinical applications.

[The Mechanism of GREM1's Effect on Osteogenic/Odontogenic Differentiation of Stem Cells from Apical Papilla].

OBJECTIVE: To study the effect of bone morphogenetic protein (BMP) antagonist Gremlin 1 (GREM1) on the function of stem cells from apical papilla (SCAPs) and explore its mechanism. METHODS: After isolation and culturing of stem cells from apical papilla in vitro, immunofluorescent staining was done to examine the subcellular localization of GREM1 in SCAPs. Transfection with lentiviral GREM1 shRNA was done to knock-down the GREM1. The SCAPs were subjected to osteogenic induction in both the GREM1 knockdown group and the control group, and the knockdown effect of GREM1 was examined using real time-PCR and Western blot. Two groups of cells were collected and the alkaline phosphatase (ALP) activity was measured 7 d after osteogenic induction. Alizarin red staining was done 3 weeks after osteogenic/odontogenic induction and real time-PCR was done after 0, 1, 2, 3 weeks of osteogenic induction to examine the expression of osteogenic/odontogenic marker genes, including osteocalcin ( OCN), osteopontin ( OPN), bone sialoprotein ( BSP), dentin matrix protein 1 ( DMP1), dentin sialophosphoprotein ( DSPP) and and the critical transcription factor osterix ( OSX), Runt-related transcription factor 2 ( RUNX2), and distal-less homebox 2 ( DLX2). Two groups of cells were collected, and CCK-8 and CFSE assay were used to evaluate changes in cell proliferation. In addition, real time-PCR was used to examine the expression of senescence-related genes p53 and wide-type activated factor 1 ( Waf1), a regulatory factor of the cell cycle, stemness associated gene krupple-like factor 4 ( KLF4), and SRY related HMG box-2 ( SOX2), and the expression of bone morphogenetic protein ( BMP) 2, 4, 5, 6, 7, 9 after GREM1 knockdown. RESULTS: Immunofluorescence staining showed that the expression of GREM1 in the nucleus was higher than that in the cytoplasm. Real time-PCR and Western blot affirmed that GREM1 was knocked down steadily. The ALP activity of the GREM1 knockdown group was higher than that of the control group ( P<0.05), and the alizarin red staining was lighter than that of the control group. The expression of OCN and DMP1 increased in the first, second and third week, OPN was increased in the second week, BSP increased in the third week, DSPP increased in the first week, and the difference was statistically significant ( P<0.05). The key osteogenic transcription factors RUNX2, OSX, and DLX2 all increased at different stages, and the difference was statistically significant ( P<0.05). CCK-8 and CFSE assay showed that the proliferation ability of the GREM1 knockdown group decreased ( P<0.05). In the GREM1 knockdown group, the expression of BMP2, 6, and 7 increased, the expression of SOX2 and KLF4 increased, while the expression of p53 and Waf1 decreased ( P<0.05). CONCLUSIONS: The knockdown of GREM1 enhanced the osteogenic/odontogenic differentiation and stemness of SCAPs and inhibited the proliferation and senescence of SCAPs. Effects of GREM1 on the function of SCAPs maybe achieved through regulating the gene expression of BMP2, BMP6, and BMP7 at the mRNA level.

Depletion of SNRNP200 inhibits the osteo-/dentinogenic differentiation and cell proliferation potential of stem cells from the apical papilla.

BACKGROUND: Tissue regeneration mediated by mesenchymal stem cells (MSCs) is deemed a desirable way to repair teeth and craniomaxillofacial tissue defects. Nevertheless, the molecular mechanisms about cell proliferation and committed differentiation of MSCs remain obscure. Previous researches have proved that lysine demethylase 2A (KDM2A) performed significant function in the regulation of MSC proliferation and differentiation. SNRNP200, as a co-binding factor of KDM2A, its potential effect in regulating MSCs' function is still unclear. Therefore, stem cells from the apical papilla (SCAPs) were used to investigate the function of SNRNP200 in this research. METHODS: The alkaline phosphatase (ALP) activity assay, Alizarin Red staining, and osteogenesis-related gene expressions were used to examine osteo-/dentinogenic differentiation potential. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) and cell cycle analysis were applied to detect the cell proliferation. Western blot analysis was used to evaluate the expressions of cell cycle-related proteins. RESULTS: Depletion of SNRNP200 caused an obvious decrease of ALP activity, mineralization formation and the expressions of osteo-/dentinogenic genes including RUNX2, DSPP, DMP1 and BSP. Meanwhile, CFSE and cell cycle assays revealed that knock-down of SNRNP200 inhibited the cell proliferation and blocked cell cycle at the G2/M and S phase in SCAPs. In addition, it was found that depletion of SNRNP200 up-regulated p21 and p53, and down-regulated the CDK1, CyclinB, CyclinE and CDK2. CONCLUSIONS: Depletion of SNRNP200 repressed osteo-/dentinogenic differentiation potentials and restrained cell proliferation through blocking cell cycle progression at the G2/M and S phase, further revealing that SNRNP200 has crucial effects on preserving the proliferation and differentiation potentials of dental tissue-derived MSCs.

Homeobox C8 inhibited the osteo-/dentinogenic differentiation and migration ability of stem cells of the apical papilla via activating KDM1A.

Enhancing the functions of mesenchymal stem cells (MSCs) is considered a potential approach for promoting tissue regeneration. In the present study, we investigate the role of HOXC8 in regulating differentiation and migration by using stem cells of the apical papilla (SCAPs). Our results showed that overexpression of HOXC8 suppressed the osteo-/dentinogenic differentiation, as detected by measuring alkaline phosphatase activity, in vitro mineralization, and the expressions of dentin sialophosphoprotein, dentin matrix acidic phosphoprotein 1, bone sialoprotein, runt-related transcription factor 2, and osterix in SCAPs, and inhibited in vivo osteo-/dentinogenesis of SCAPs. In addition, knockdown of HOXC8 promoted the osteo-/dentinogenic differentiation potentials of SCAPs. Mechanically, HOXC8 enhanced KDM1A transcription by directly binding to its promoter. HOXC8 and KDM1A also inhibited the migration and chemotaxis abilities of SCAPs. To sum up, HOXC8 negatively regulated the osteo-/dentinogenic differentiation and migration abilities of SCAPs by directly enhancing KDM1A transcription and indicated that HOXC8 and KDM1A could serve as potential targets for enhancing dental MSC mediated tissue regeneration.