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Objective @#To study the effect of light-emitting diode (LED) red light on the osteogenic/odontogenic differentiation of human dental pulp stem cells (hDPSCs) and its mechanism were discussed. @*Methods@#This study has been reviewed and approved by the Ethics Committee. hDPSCs were cultured by tissue block enzyme digestion. The proliferative capacity of hDPSCs was detected by the CCK-8 at days 1, 3, 5 and 7 under stimulation with 0, 1, 5 and 10 μg/mL lipopolysaccharide (LPS), and the LPS stimulatory concentration was screened. The CG group (mineralization induction), LPS+CG group, and LPS+CG+ (2, 4, 6, 8, and 10 J/cm2) LED red light groups were set. On day 7, alkaline phosphatase (ALP) staining and ALP activity were determined. Relative expression levels of the ALP, osterix (OSX), dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP) genes were measured by qRT-PCR. On day 21, alizarin red staining and calcium nodule quantitative determination were performed to screen the best light energy. The LPS+CG group and LPS+CG+LED group (optimal energy) were set up, and the secretion and expression levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected by ELISAs on days 1, 3, 5 and 7. The relative expression levels of the extracellular regulated protein kinases 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and extracellular regulated protein kinases 5 (ERK5) proteins and their phosphorylated proteins in the MAPK signaling pathway were detected by Western blots. After the pathway was blocked, the relative expression levels of the ALP, OSX, DMP-1, and DSPP proteins after LED red light irradiation on day 7 were detected by Western blots.@*Results@# CCK-8 assays showed that the proliferation of hDPSCs induced by 10 μg/mL LPS was lower than that of the 0, 1, and 5 μg/mL groups on the 5th and 7th days (P<0.05), and 10 μg/mL was selected as the LPS stimulatory concentration in the follow-up experiment. ALP staining, ALP activity, gene expression levels of ALP, OSX, DMP-1 and DSPP and calcium nodule quantification in the LPS+CG+4 J/cm2 group were higher than those in the other treatment groups (P<0.05). 4 J/cm2 LED red light had the strongest ability to promote osteogenic/odontogenic differentiation and was used as the LED light energy density in subsequent experiments. ELISA showed that the secretion and expression levels of TNF-α and IL-1β in the LPS+CG+LED group were lower than those in the LPS+CG group on the 5th and 7th days (P<0.05). Western blot analysis showed that 4 J/cm2 LED red light promoted the expression levels of the p-ERK1/2, p-p38, p-JNK and p-ERK5 proteins. After the MAPK pathway was blocked, the expression levels of the ALP, OSX, DMP-1, and DSPP proteins in the LPS+CG+LED+U0126 (ERK1/2 inhibitor), SP600125 (JNK inhibitor), and BIX02189 (ERK5 inhibitor) groups were lower than those in the LPS+CG+LED group (P<0.001). The protein expression levels of ALP, OSX and DMP-1 in the LPS+CG+LED+SB203580 (p38 inhibitor) group were not significantly different from those in the LPS+CG+LED group (P>0.05).@*Conclusion@#In inflammatory conditions, LED red light promotes osteogenic/odontogenic differentiation of hDPSCs. This effect may be attributed to enhancement of the ERK1/2, JNK, and ERK5 signaling pathways, which reduces the production of the inflammatory cytokines TNF-α and IL-1β.
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BACKGROUND: Dental pulp stem cells can differentiate into dentin under appropriate induction conditions, which are important seed cells i n dental tissue engineering. However, the commonly used inducers are chemical agents, which are not available for in vivo application. Mesenchymal stem cells can differentiate with the material hardness, and the physical property-induced cell differentiation is little reported. OBJECTIVE: To observe the extension characteristics and dentin differentiation potential of dental pulp stem cells from human deciduous teeth on the stiff matrix surface. METHODS: Dental pulp stem cells from naturally shed deciduous teeth were isolated, cultured and identified. Four solid gel matrixes with elasticity modulus of (9.12±0.94), (27.18±3.55), (59.37±4.05) and (86.45±5.33) kPa were made using low melting point agarose. The extension ability of passage 4 dental pulp stem cells on the surface of the above solid matrixes was detected by two-dimensional clone formation and cell scratch tests. The protein expression levels of dentin matrix protein-1, dentin phosphoprotein and dentin sialoprotein were detected by western blot assay. RESULTS AND CONCLUSION: Dental pulp stem cells from human deciduous teeth seeded on the gel matrix with extremely low and low hardness almost existed as cell clones with neat edges, and cell spreading and extension were rare. When seeded on the gel matrix with moderate and high hardness, the cloned edge of deciduous dental pulp stem cells spread and extended obviously. The cell body became large and the cell edge extended significantly. The cell scratch test revealed the similar phenomenon. When seeded on the gel matrix with moderate and high hardness, dental pulp stem cells from human deciduous teeth exhibited high expression levels of of dentin matrix protein-1, dentin phosphoprotein and dentin sialoprotein. In summary, with the increase of matrix hardness, the abilities of extension and differentiation into dentin of dental pulp stem cells from human deciduous teeth are increased gradually, which provides a method for dental tissue engineering.
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BACKGROUND: Bisphosphonates are a novel inhibitor of bone resorption that can inhibit the activity and function of osteoclasts. OBJECTIVE: To observe the effects of sodium ibandronate on the expression of dentin matrix protein 1, Caspace3, Bcl-2 and Bax in condylar cartilage in osteoporosis rats. METHODS: Thirty-six female rats were randomly divided into sham group, osteoporosis group and sodium ibandronate group, twelve in each group. The sham group did not excise ovaries during surgery. Bilateral ovaries of rats were removed in the osteoporosis and sodium ibandronate groups. On the 7th day after operation, rats in the sodium ibandronate group were intraperitoneally given sodium ibandronate 10 µg/kg, once every 7 days. After 90 days, the rat ovaries were taken. Bone mineral density was measured in each group. The changes of condylar cartilage were observed by toluidine blue staining and TUNEL staining. The expression of dentin matrix protein 1 protein was detected by immunohistochemistry. The levels of Caspase3, Bcl-2 and Bax were detected by western blot assay. The study protocol was approved by the Animal Ethics Committee of Nanhua Hospital in China with the approval No. SLXD_201804010. RESULTS AND CONCLUSION: Compared with the sham group or sodium ibandronate group, the bone mineral density in the osteoporosis group was significantly decreased (P < 0.05). The results of toluidine blue staining showed that the hypertrophic layer of condylar cartilage in the sodium ibandronate group was significantly thicker than that in the osteoporosis group. Compared with the sham group or sodium ibandronate group, the number of apoptotic cells in condylar cartilage and subchondral bone increased significantly in the osteoporosis group (P < 0.05). The expression of dentin matrix protein 1 protein was significantly lower in the osteoporosis group than the sham group, but it increased after treatment with sodium ibandronate (P < 0.05). Compared with the sham group, the expression of Caspase 3 and Bax in the osteoporosis group increased significantly, and the expression of Bcl-2 decreased. However, treatment with sodium ibandronate decreased the expression of Caspase 3 and Bax and increased the expression of Bcl-2 significantly. Overall, our findings reveal that sodium ibandronate can inhibit the apoptosis of condylar chondrocytes and the number of osteoclasts in osteoporotic state, which may be related to the regulation of Caspase 3, Bcl-2, Bax and dentin matrix protein 1 expression.
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Objective@#To investigate the effects of leptin on the proliferation of stem cells from human stem cells from the apical papilla (hSCAPs) and the expression of osteogenic/dentinogenic genes in vitro to provide an experimental basis for the sustainable development of young permanent teeth. @* Methods @#The tissue block method was used to isolate and culture hSCAPs from the apical papilla of the immature third permanent molar. The expression of leptin and OBRb in hSCAPs was detected using immunocytofluorescence staining, western blotting and reverse transcription polymerase chain reaction (RT-PCR) analysis. The hSCAPs was treated with 0.1 μg/mL of leptin (0.1 μg/mL group) or 1.5 μg/mL of leptin (1.5 μg/mL group) at different time points. The control group was treated with alpha-MEM medium. Cell proliferation was measured using the CCK8 assay and cell cycle analysis. QRT-PCR was used to detect the expression of related osteoblast/odontogenic genes for alkaline phosphatase (ALP), dentin matrix protein -1 (DMP-1), dentin sialophosphoprotein (DSPP), and osteocalcin (OCN) mRNA. The differences between the treatment groups and the control group were analyzed statistically using one-way ANOVA followed by Bonferroni analysis.@*Results@#The expression of both leptin and OBRb were found in hSCAPs. Compared with the control group, the cell proliferation capacity and S phase cells in the treatment groups were higher than those in the control group, with the 1.5 μg /mL group displaying higher levels than 0.1 μg /mL group, and the treated hSCAPs demonstrated a higher proliferation rate and a higher expression of ALP, DSPP, and DMP-1 from day 3 to day 7, with the 1.5 μg /mL group displaying higher levels than 0.1 μg /mL group , and the difference was statistically significant (P < 0.05), at day 7. The treated hSCAPs demonstrated a lower expression of ALP, DSPP, and DMP-1. Compared with the control group, the treated hSCAPs demonstrated a higher expression of OCN from day 7 to day 14, with significantly higher expression in the 1.5 μg /mL group compared to the 0.1 μg /mL group.@*Conclusion@#Leptin may promote cell proliferation and upregulate the expression of relative osteogenic/dentinogenic genes.
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Fractures are frequently occurring diseases that endanger human health. Crucial to fracture healing is cartilage formation, which provides a bone-regeneration environment. Cartilage consists of both chondrocytes and extracellular matrix (ECM). The ECM of cartilage includes collagens and various types of proteoglycans (PGs), which play important roles in maintaining primary stability in fracture healing. The PG form of dentin matrix protein 1 (DMP1-PG) is involved in maintaining the health of articular cartilage and bone. Our previous data have shown that DMP1-PG is richly expressed in the cartilaginous calluses of fracture sites. However, the possible significant role of DMP1-PG in chondrogenesis and fracture healing is unknown. To further detect the potential role of DMP1-PG in fracture repair, we established a mouse fracture model by using a glycosylation site mutant DMP1 mouse (S89G-DMP1 mouse). Upon inspection, fewer cartilaginous calluses and down-regulated expression levels of chondrogenesis genes were observed in the fracture sites of S89G-DMP1 mice. Given the deficiency of DMP1-PG, the impaired IL-6/JAK/STAT signaling pathway was observed to affect the chondrogenesis of fracture healing. Overall, these results suggest that DMP1-PG is an indispensable proteoglycan in chondrogenesis during fracture healing.
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The blood-brain barrier (BBB) is a tight boundary formed between endothelial cells and astrocytes, which separates and protects brain from most pathogens as well as neural toxins in circulation. However, detailed molecular players involved in formation of BBB are not completely known. Dentin matrix protein 1 (DMP1)-proteoglycan (PG), which is known to be involved in mineralization of bones and dentin, is also expressed in soft tissues including brain with unknown functions. In the present study, we reported that DMP1-PG was expressed in brain astrocytes and enriched in BBB units. The only glycosylation site of DMP1 is serine89 (S89) in the N-terminal domain of the protein in mouse. Mutant mice with DMP1 point mutations changing S89 to glycine (S89G), which completely eradicated glycosylation of the protein, demonstrated severe BBB disruption. Another breed of DMP1 mutant mice, which lacked the C-terminal domain of DMP1, manifested normal BBB function. The polarity of S89G-DMP1 astrocytes was disrupted and cell-cell adhesion was loosened. Through a battery of analyses, we found that DMP1 glycosylation was critically required for astrocyte maturation both in vitro and in vivo. S89G-DMP1 mutant astrocytes failed to express aquaporin 4 and had reduced laminin and ZO1 expression, which resulted in disruption of BBB. Interestingly, overexpression of wild-type DMP1-PG in mouse brain driven by the nestin promoter elevated laminin and ZO1 expression beyond wild type levels and could effectively resisted intravenous mannitol-induced BBB reversible opening. Taken together, our study not only revealed a novel element, i.e., DMP1-PG, that regulated BBB formation, but also assigned a new function to DMP1-PG.
Subject(s)
Animals , Female , Male , Mice , Astrocytes , Cell Biology , Metabolism , Blood-Brain Barrier , Cell Biology , Metabolism , Cells, Cultured , Extracellular Matrix Proteins , Genetics , Metabolism , Glycosylation , Proteoglycans , Metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Objective To examine the distribution and expression of dentin matrix protein1 ( DMP1 ) in the condylar cartilage and subchondral bone of osteoporosis rats. Methods Female Sprague-Dawley rats aged 6 months (n=30)wererandomlydividedinto3groups. TheShamgroupunderwentshamoperationonly(n=10),theOVX group ( n = 10 ) received a bilateral ovariectomy first and then saline solution treatment subcutaneously for 3 months. The RIS group ( n=10 ) also received a bilateral ovariectomy and then with risedronate treatment ( 2. 4μg/kg) subcutaneously for 3 months. Three months after the operation, the animals were sacrificed. Toluidine blue staining showed the structure changes of rat condylar cartilage region. The changes of osteoclasts in the bony subcondylar region were evaluated by tartrate-resistant acid phosphatase ( TRAP) staining. The expression of DMP1 was analyzed immunohistochemically and then performed by semi-quantitative imaging analyses. Results Toluidine blue staining showed a thickened hypertrophic layers of condylar cartilage in RIS group. The results of TRAP staining indicated that the number of osteoclasts was significantly greater in OVX group than RIS group (P<0. 05). Immunohistochemistry showed that DMP1 localized mainly in the chondrogenic layers and osteocytes, bony subcondylar region in three groups. The expression levels of DMP1 proteins statistically decreased in OVX group than the other two groups(both P<0. 05). Conclusion Bisphophonates may reduce the the number of osteoclasts in the condyle from osteoporosis rats, with increasing of the expression of DMP1, which may influences condylar cartilage biomineralization.
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Objective:To examine the expression of odontoblast related proteins in dental pulp stem cells(DPSCs)induced by BMP-7.Methods:DPSCs were cultured in the common culture medium or medium supplemented with 1 00 ng/ml BMP-7.Electron microscope,CCK8 and immunohistochemical staining were carried out to estimate the cell morphology and differentiation.Results:In-duced by BMP-7,the morphology of DPSCs was not changed,the proliferation of DPSCs was slower than that of the cells without BMP-7 treatment.DPSCs were negative for the expression of DSPP,DMP-7 and ALP.However,DPSCs were found strongly positive for DSPP,DMP-7 and ALP after the induction of BMP-7.Conclusion:BMP-7 induction may promote the differentiation of DPSCs.
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Dentin matrix protein 1 (DMP1) is an acidic phosphoprotein that plays an important role in mineralized tissue formation by initiation of nucleation and modulation of mineral phase morphology. The purpose of the present study was to examine the immunoexpression of DMP1 in tooth germs of 7 human fetuses at different gestational ages (14, 16, 19, 20, 21, 23 and 24 weeks) comparing with completed tooth formation erupted teeth. The results showed the presence of DMP1 in the dental lamina, as well as in the cells of the external epithelium, stellate reticulum and stratum intermedium of the enamel organ. However, in the internal dental epithelium, cervical loop region and dental papilla some cells have not labeled for DMP1. In the crown stage, DMP1 was expressed in the ameloblast and odontoblast layer, as well as in the dentinal tubules of coronal dentin near the odontoblast area. Erupted teeth with complete tooth formation exhibited immunolabeling for DMP1 only in the dentinal tubules mainly close to the dental pulp. No staining was observed in the enamel, predentin or dental pulp matrix. DMP1 is present in all developing dental structures (dental lamina, enamel organ, dental papilla) presenting few immunoexpression variations, with no staining in mineralized enamel and dentin.
A proteína da matriz dentinária 1 (DMP1) é uma fosfoproteína ácida que tem sido relacionada diretamente ao processo de mineralização dos tecidos em formação sendo iniciadora do processo de nucleação e modulação da fase mineral. O objetivo desse trabalho foi avaliar a imunoexpressão da DMP1 em germes dentários em diferentes fases da odontogênese, obtidos de 7 fetos humanos em diversos estágios gestacionais (14, 16, 19, 20, 21, 23 e 24 semanas), comparando-se com dentes com rizogênese completa. Os resultados mostraram que a DMP1 esteve expressa na lâmina dentária, bem como, nas células do epitélio externo, retículo estrelado e estrato intermediário do órgão do esmalte. Diferentemente, no epitélio interno do órgão do esmalte, alça cervical e papila dentária algumas células não apresentaram a DMP1. Nas fases de coroa, os ameloblastos e odontoblastos apresentaram marcação positiva para a DMP1, bem como os túbulos dentinários da dentina coronária próximos à região odontoblástica. Os dentes com rizogênese completa exibiram marcação para a DMP1 apenas nos túbulos dentinários principalmente próximos à polpa dentária. Nenhuma marcação foi observada na matriz de esmalte ou pré-dentina, nem na polpa dentária. Concluímos que a DMP1 está presente em todas as fases da odontogênese, tanto na lâmina dentária, órgão do esmalte, bem como na papila dentária, com pequenas variações de nuances de expressão, estando ausente na dentina e esmalte mineralizados.