Expression Of Dentin Matrix Protein Research Articles

A novel miR-466l-3p/FGF23 axis promotes osteogenic differentiation of human bone marrow mesenchymal stem cells

BackgroundMicroRNAs (miRNAs) regulate osteogenic differentiation processes and influence the development of osteoporosis (OP). This study aimed to investigate the potential role of miR-466 l-3p in OP. MethodsThe expression levels of miR-466 l-3p and fibroblast growth factor 23 (FGF23) were quantified in the trabeculae of the femoral neck of 40 individuals with or without OP using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The impact of miR-466 l-3p or FGF23 expression on cell proliferation and the expression levels of runt-related transcription factor 2 (RUNX2), type I collagen (Col1), osteocalcin (OCN), osterix (OSX) and dentin matrix protein 1 (DMP1) was quantified in human bone marrow mesenchymal stem cells (hBMSCs) overexpressing miR-466 l-3p. Furthermore, alkaline phosphatase (ALP) staining and alizarin red staining were performed to measure ALP activity and the levels of calcium deposition, respectively. In addition, bioinformatics analysis, luciferase reporter assays, and RNA pull-down assays were conducted to explore the molecular mechanisms underlying the effects of miR-466 l-3p and FGF23 in osteogenic differentiation of hBMSCs. ResultsThe expression levels of miR-466 l-3p were significantly lower in femoral neck trabeculae of patients with OP than in the control cohort, whereas FGF23 levels exhibited the opposite trend. Furthermore, miR-466 l-3p levels were upregulated and FGF23 levels were downregulated in hBMSCs during osteogenic differentiation. Moreover, the high miR-466 l-3p expression enhanced the mRNA expression of RUNX2, Col1, OCN, OSX and DMP1, as well as cell proliferation, ALP activity, and calcium deposition in hBMSCs. FGF23 was found to be a direct target of miR-466 l-3p. FGF23 overexpression downregulated the expression of osteoblast markers and inhibited the osteogenic differentiation induced by miR-466 l-3p overexpression. qRT-PCR and Western blot assays showed that miR-466 l-3p overexpression decreased the expression levels of mRNAs and proteins associated with the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, whereas FGF23 upregulation exhibited the opposite trend. ConclusionIn conclusion, these findings suggest that miR-466 l-3p enhances the osteogenic differentiation of hBMSCs by suppressing FGF23 expression, ultimately preventing OP.

BDNF/TrkB Is a Crucial Regulator in the Inflammation-Mediated Odontoblastic Differentiation of Dental Pulp Stem Cells.

The odontoblastic differentiation of dental pulp stem cells (DPSCs) associated with caries injury happens in an inflammatory context. We recently demonstrated that there is a link between inflammation and dental tissue regeneration, identified via enhanced DPSC-mediated dentinogenesis in vitro. Brain-derived neurotrophic factor (BDNF) is a nerve growth factor-related gene family molecule which functions through tropomyosin receptor kinase B (TrkB). While the roles of BDNF in neural tissue repair and other regeneration processes are well identified, its role in dentinogenesis has not been explored. Furthermore, the role of BDNF receptor-TrkB in inflammation-induced dentinogenesis remains unknown. The role of BDNF/TrkB was examined during a 17-day odontogenic differentiation of DPSCs. Human DPSCs were subjected to odontogenic differentiation in dentinogenic media treated with inflammation inducers (LTA or TNFα), BDNF, and a TrkB agonist (LM22A-4) and/or antagonist (CTX-B). Our data show that BDNF and TrkB receptors affect the early and late stages of the odontogenic differentiation of DPSCs. Immunofluorescent data confirmed the expression of BDNF and TrkB in DPSCs. Our ELISA and qPCR data demonstrate that TrkB agonist treatment increased the expression of dentin matrix protein-1 (DMP-1) during early DPSC odontoblastic differentiation. Coherently, the expression levels of runt-related transcription factor 2 (RUNX-2) and osteocalcin (OCN) were increased. TNFα, which is responsible for a diverse range of inflammation signaling, increased the levels of expression of dentin sialophosphoprotein (DSPP) and DMP1. Furthermore, BDNF significantly potentiated its effect. The application of CTX-B reversed this effect, suggesting TrkB`s critical role in TNFα-mediated dentinogenesis. Our studies provide novel findings on the role of BDNF-TrkB in the inflammation-induced odontoblastic differentiation of DPSCs. This finding will address a novel regulatory pathway and a therapeutic approach in dentin tissue engineering using DPSCs.

Dentin Matrix Protein 1 Regulates Mineralization of MC3T3-E1 Cells via the TNAP-ANK-ENPP1 Axis.

Dentin matrix protein 1 (DMP1) is central to matrix mineralization. Clarification of the function of DMP1 is crucial to understanding normal bone formation and pathological calcification. The tissue-nonspecific alkaline phosphatase (TNAP) -progressive ankylosing enzyme (ANK) -extracellular nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) axis induces deposition of hydroxyapatite (HA) and pyrophosphate dehydrate (CPPD) by regulating pyrophosphate (PPi). Here, we investigated the mechanism by which DMP1 and the TNAP-ANK-ENPP1 axis participate in mineralization. Expression of DMP1, TNAP, NPP1, and ANK genes in MC3T3-E1 cells was detected by RT-qPCR before and after treatment with DMP1 siRNA. An enzyme-linked immunosorbent assay was used to determine expression of DMP1 protein, TNAP activity was detected by SIGMAFAST p-nitrophenyl phosphate tablets, and mineralization of osteoblasts was determined by alizarin red staining. PPi levels were determined radiometrically and equalized for cell DNA. Levels of calcium, inorganic phosphate, zinc, and magnesium were assessed by standard laboratory techniques. After DMP1 gene silencing, expressions of TNAP, ENPP1, and ANK were correspondingly reduced. DMP1 altered extravesicular and intravesicular ion levels through the TNAP-ENPP1-ANK axis in MC3T3-E1 cells. DMP1 regulated mineralization of MC3T3-E1 cells via the TNAP-ANK-ENPP1 axis and affected TNAP activity by two processes-rapid regulation of the Zn2+ transporter (ZnT) and transcriptional regulation of hysteresis. However, DMP1 may affect expression of ENPP1 and ANK only via hysteresis transcriptional regulation. DMP1, as a calcium trap or catalytic enzyme, appears to have a role in collagen mineralization.

Mesenchymal stromal cell exosomes enhance dental pulp cell functions and promote pulp-dentin regeneration

Mesenchymal stromal/stem cell (MSC) therapies are currently being explored for dental pulp regeneration. As the therapeutic effects of MSCs in tissue repair are mediated mainly through the release of extracellular vesicles (EVs) including exosomes, we investigated here the cellular processes and molecular mechanisms modulated by MSC exosomes in dental pulp regeneration. Using dental pulp cell (DPC) cultures, we demonstrated that MSC exosomes could increase DPC migration, proliferation, and odontogenic differentiation. The enhancement of these cellular processes was mediated through exosomal CD73-mediated adenosine receptor activation of AKT and ERK signaling. Consistent with these observations, MSC exosomes increased the expression of dentin matrix proteins and promoted the formation of dentin-like tissue and bridge-like structures in a rat pulp defect model. These effects were comparable to that of mineral trioxide aggregate (MTA) treatment. MSC exosomes also yielded recellularized pulp-dentin tissues in the root canal of endodontically-treated human premolars, following subcutaneous implantation in the mouse dorsum. Together, our findings suggest that MSC exosomes could exert a multi-faceted effect on DPC functions including migration, proliferation and odontogenic differentiation to promote dental pulp regeneration. This study provides the basis for development of MSC exosomes as a cell-free MSC therapeutic alternative for pulp-dentin regeneration.

DMP1 and IFITM5 Regulate Osteogenic Differentiation of MC3T3-E1 on PEO-Treated Ti-6Al-4V-Ca2+/Pi surface.

Despite numerous studies on various surface modifications on titanium and its alloys, it remains unclear what kind of titanium-based surface modifications are capable of controlling cell activity. This study aimed to understand the mechanism at the cellular and molecular levels and investigate the in vitro response of osteoblastic MC3T3-E1 cultured on the Ti-6Al-4V surface modified by plasma electrolytic oxidation (PEO) treatment. A Ti-6Al-4V surface was prepared by PEO at 180, 280, and 380 V for 3 or 10 min in an electrolyte containing Ca2+/Pi ions. Our results showed that PEO-treated Ti-6Al-4V-Ca2+/Pi surfaces enhanced the cell attachment and differentiation of MC3T3-E1 compared to the untreated Ti-6Al-4V control but did not affect cytotoxicity as shown by cell proliferation and cell death. Interestingly, on the Ti-6Al-4V-Ca2+/Pi surface treated by PEO at 280 V for 3 or 10 min, MC3T3-E1 showed a higher initial adhesion and mineralization. In addition, the alkaline phosphatase (ALP) activity significantly increased in MC3T3-E1 on the PEO-treated Ti-6Al-4V-Ca2+/Pi (280 V for 3 or 10 min). In RNA-seq analysis, the expression of dentin matrix protein 1 (DMP1), sortilin 1 (Sort1), signal-induced proliferation-associated 1 like 2 (SIPA1L2), and interferon-induced transmembrane protein 5 (IFITM5) was induced during the osteogenic differentiation of MC3T3-E1 on the PEO-treated Ti-6Al-4V-Ca2+/Pi. DMP1 and IFITM5 silencing decreased the expression of bone differentiation-related mRNAs and proteins and ALP activity in MC3T3-E1. These results suggest that the PEO-treated Ti-6Al-4V-Ca2+/Pi surface induces osteoblast differentiation by regulating the expression of DMP1 and IFITM5. Therefore, surface microstructure modification through PEO coatings with Ca2+/Pi ions could be used as a valuable method to improve biocompatibility properties of titanium alloys.

Response of stem cells derived from human exfoliated deciduous teeth to Bio-C Repair and Mineral Trioxide Aggregate Repair HP: Cytotoxicity and gene expression assessment

Background: The aim of this study was to investigate and compare the cytotoxicity and gene expression of Bio-C Repair, Mineral Trioxide Aggregate (MTA) HP Repair, and Biodentine on stem cells derived from exfoliated deciduous teeth.Materials and Methods: In this in vitro study MTT assay was used to assess the cellular viability at three different dilutions. The gene expression of Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteocalcin [OCN], and dentin matrix protein-1 (DMP-1) was measured with real-time polymerase chain reaction after 7 days, 14 days, and 21 days of incubation. One-way analysis of variance and Bonferroni posttest were used for statistical analysis (p=o.o5).Results: After 72 h of incubation at dilution 1:4, stem cells derived from human exfoliated deciduous teeth (SHEDs) cultivated in Biodentine, followed by Bio-C Repair and MTA Repair HP reported with highest cellular viability. The highest mRNA expression of Runx2, ALP, OCN, and DMP-1 was reported in SHEDs cultured in Biodentine (after 21 days of incubation). Conclusion: Bio-C Repair and MTA HP Repair are biocompatible and capable of odontogenic differentiation similar to Biodentine when cultured in stem cells derived from exfoliated primary teeth.

Characterization of SIBLING Proteins in the Mineralized Tissues.

The SIBLING proteins are a family of non-collagenous proteins (NCPs) previously thought to be expressed only in dentin but have been demonstrated in other mineralized and non-mineralized tissues. They are believed to play vital roles in both osteogenesis and dentinogenesis. Since they are tightly regulated lifelong processes and involve a peak of mineralization, three different age groups were investigated. Fifteen wild-type (WT) mice were euthanized at ages 1, 3, and 6 months. Hematoxylin and eosin staining (H&E) was performed to localize various microscopic structures in the mice mandibles and tibias. The immunostaining pattern was compared using antibodies for dentin sialoprotein (DSP), dentin matrix protein 1 (DMP1), bone sialoprotein (BSP), and osteopontin (OPN). Immunostaining of DSP in tibia showed its most noticeable staining in the 3-month age group. DSP was expressed in alveolar bone, cellular cementum, and PDL. A similar expression of DMP1 was seen in the tibia and dentin. BSP was most noticeably detected in the tibia and acellular cementum. OPN was mainly expressed in the bone. A lower level of OPN was observed at all age groups in the teeth. The immunostaining intensity was the least detected for all proteins in the 6-month tibia sample. The expression patterns of the four SIBLING proteins showed variations in their staining intensity and temporospatial patterning concordant with skeletal and dental maturity. These findings suggest some role in this tightly regulated mineralization process.

Intermittent compressive force regulates dentin matrix protein 1 expression in human periodontal ligament stem cells

Mechanical force differentially regulates periodontal ligament functions depending on types, magnitudes, and duration of stimulation. Intermittent compressive force (ICF) promotes an in vitro mineralization in human periodontal ligament cells. The present study investigated the effect of ICF on dentin matrix protein-1 (DMP1) expression in human periodontal ligament stem cells (hPDLSCs). Cells were treated with ICF in a serum-free culture medium for 24h The mRNA and protein expression were examined using real-time polymerase chain reaction, immunofluorescence staining and Western blot analysis, respectively. The exposure to ICF in a serum-free condition significantly induced DMP1 expression in both mRNA and protein levels. The effect of ICF-induced DMP1 expression was inhibited by pretreatment with cycloheximide, indicating the requirement of the intermediated molecule(s). Pretreatment with transforming growth factor β (TGF-β) receptor inhibitor (SB431542) or neutralized antibody against TGF-β1 prior to ICF application abolished the effect of ICF-induced DMP1 expression. Further, recombinant TGF-β1 treatment stimulated DMP1 expression. The present study illustrated that ICF induces DMP1 expression in hPDLSCs via the regulation of TGF-β signaling pathway.

Dentin Matrix Protein 1 Silencing Inhibits Phosphorus Utilization in Primary Cultured Tibial Osteoblasts of Broiler Chicks.

Three experiments were carried out in the present study to investigate whether dentin matrix protein 1 (DMP1) was involved in regulating phosphorus (P) metabolic utilization in primary cultured tibial osteoblasts of broiler chicks. Experiment 1 was conducted to select the optimal osteogenic inductive culture medium and the optimal induction time in primary cultured tibial osteoblasts of broiler chicks. In experiment 2, the siRNAs against DMP1 were designed, synthesized and transfected into primary cultured tibial osteoblasts of broiler chicks, and then the inhibitory efficiencies of siRNAs against DMP1 were determined, and the most efficacious siRNA was selected to be used for the DMP1 silencing. In experiment 3, with or without siRNA against DMP1, primary cultured tibial osteoblasts of broiler chicks were treated with the medium supplemented with 0.0, 1.0 or 2.0 mmol/L of P as NaH2PO4 for 12 days. The P metabolic utilization-related parameters were measured. The results showed that the osteogenic induced medium 2 and 12 days of the optimal induction time were selected; Among the designed siRNAs, the si340 was the most effective (P < 0.05) in inhibiting the DMP1 expression; DMP1 silencing decreased (P < 0.05) the expressions of DMP1 mRNA and protein, P retention rate, mineralization formation, alkaline phosphatase activity and bone gla-protein content in tibial osteoblasts at all of added P levels. It is concluded that DMP1 silencing inhibited P utilization, and thus DMP1 was involved in regulating P metabolic utilization in primary cultured tibial osteoblasts of broiler chicks, which provides a novel insight into the regulation of the P utilization in the bone of broilers, and will contribute to develop feasible strategies to improve the bone P utilization efficiency of broilers so as to decrease its excretion.

Effect of mesoporous bioactive glass on odontogenic differentiation of human dental pulp stem cells.

Healthy pulp tissue plays an important role in normal function and long-term retention of teeth. Mesoporous bioactive glass (MBG) as a kind of regenerative biomaterials shows the potential in preserving the vital pulp. In this study, MBG prepared by organic template method combined with sol-gel method were used in human dental pulp cell culture and ectopic mineralization experiment. Real-Time PCR was used to explore its ability to induce odontogenic differentiation of dental pulp cells. MBG and rat crowns were implanted under the skin of nude mice for 4 weeks to observe the formation of pulp dentin complex. We found that MBG can release Si and Ca ions and has a strong mineralization activity in vitro. The co-culture of MBG with human dental pulp cells promoted the expression of DMP-1 (dentin matrix protein-1) and ALP (alkalinephosphatase) without affecting cell proliferation. After 4 weeks of subcutaneous implantation in nude mice, the formation of hard tissue with regular structure under the material could be seen, and the structure was similar to dentin tubules. These results indicate that MBG can induce the differentiation of dental pulp cells and the formation of dental pulp-dentin complex and has the potential to promote the repair and regeneration of dental pulp injuries.

The Role of DMP1 in CKD-MBD.

Chronic kidney disease-mineral and bone disorder (CKD-MBD) has become a global health crisis with very limited therapeutic options. Dentin matrix protein 1 (DMP1) is a matrix extracellular protein secreted by osteocytes that has generated recent interest for its possible involvement in CKD-MBD pathogenesis. This is a review of DMP1 established regulation and function, and early studies implicating DMP1 in CKD-MBD. Patients and mice with CKD show perturbations of DMP1 expression in bone, associated with impaired osteocyte maturation, mineralization, and increased fibroblast growth factor 23 (FGF23) production. In humans with CKD, low circulating DMP1 levels are independently associated with increased cardiovascular events. We recently showed that DMP1 supplementation lowers circulating FGF23 levels and improves bone mineralization and cardiac outcomes in mice with CKD. Mortality rates are extremely high among patients with CKD and have only marginally improved over decades. Bone disease and FGF23 excess contribute to mortality in CKD by increasing the risk of bone fractures and cardiovascular disease, respectively. Previous studies focused on DMP1 loss-of-function mutations have established its role in the regulation of FGF23 and bone mineralization. Recent studies show that DMP1 supplementation may fill a crucial therapeutic gap by improving bone and cardiac health in CKD.

Inactivation of PI3K/Akt promotes the odontoblastic differentiation and suppresses the stemness with autophagic flux in dental pulp cells

Background/purposeAutophagy is involved in controlling differentiation of various cell types. The present study aimed to investigate the mechanism related to autophagy in regulating odontogenic differentiation of dental pulp cells. Materials and methodsHuman dental pulp cells (HDPCs) were cultured in differentiation inductive medium (DM) and odontoblastic differentiation and mineralization were evaluated by alkaline phosphatase (ALP) staining and Alizarin red S staining, respectively. Tooth cavity preparation was made on the mesial surface of lower first molars in rat. The expression of autophagy-related signal molecules was detected using Western blot analysis and Immunohistochemistry. ResultsHDPCs cultured in DM showed increased autophagic flux and declined phosphorylation of phosphoinositide 3-kinases (PI3K), protein kinase B (Akt), and mTOR. Dentin matrix protein-1 (DMP-1) and dentin sialoprotein (DSP), markers of odontoblastic differentiation, were upregulated and autophagic activation showing increased LC3-II and decreased p62 levels was observed during odontogenic differentiation of HDPCs. However, PI3K blocker 3-methyladenine (3MA), lentiviral shLC3 and Akt activator SC79 attenuated the expression of LC3II as well as DMP-1, ALP activity and mineralization enhanced in HDPCs under DM condition. In addition, 3MA, shLC3 and SC79 recovered the expression of pluripotency factor CD146, Oct4 and Nanog downregulated in DM condition. In rat tooth cavity preparation model, the expression of LC3B and DMP-1 was elevated near odontoblast-dentin layer during reparative dentin formation, whereas 3MA significantly reduced the expression of LC3B and DMP-1. ConclusionThese findings indicated autophagy promotes the odontogenic differentiation of dental pulp cells modulating stemness via PI3K/Akt inactivation and the repair of pulp.

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

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. 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. 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). 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.

Dentin Matrix Protein-1 (DMP-1) Expression after Application of Haruan Fish Extract (Channa striata) on Inflamed Wistar Rat Dental Pulp

Haruan fish (Channa striata) contains essential compounds such as albumin, amino acids, zinc (Zn), iron (Fe), and hydroxyapatite minerals that can play a role in the process of inflammation, proliferation and cell differentiation. Odontoblast cell activity can be analyzed through DMP-1 expression. The purpose of this study is to determine the effect of administration of extract of Haruan fish (Channa striata) on the interpretation of DMP-1 on inflamed rat dental pulp. This study was an experimental laboratory with a posttest only study design with a control group design. The research sample was nine groups of rats with 27 animals divided into 3, 7, and 14 days of observation, and occlusal preparation was performed on the first molar. Calcium hydroxide was given to the positive control group, extract of the Haruan fish in the treatment group, and the group negative control no application. Then the expression of DMP-1 was examined by immunohistochemical examination. Data were analyzed using Anova statistical test, Kruskal-Wallis, and chi-square test. The extract of the Haruan fish (Channa striata) can increase the expression of DMP-1 because the calcium and phosphate ion content in the Haruan fish extract can be a carrier of bioactive molecules and can increase the differentiation of odontoblast cells. The expression of DMP1 has increased after applying the extract of the Haruan fish (Channa striata) so that it can be considered a dent in biomineral material that can increase the proliferation and differentiation of odontoblast cells. How to Cite this Article Pubmed Style Andi Sumidarti, Christine Anastasia Rovani, Juni Jekti Nugroho, Bulkis Thahir. Matrix Protein-1 (DMP-1) Expression after Application of Haruan Fish Extract (Channa striata) on Inflamed Wistar Rat Dental Pulp. SRP. 2020; 11(10): 31-35. doi:10.31838/srp.2020.10.6 Web Style Andi Sumidarti, Christine Anastasia Rovani, Juni Jekti Nugroho, Bulkis Thahir. Matrix Protein-1 (DMP-1) Expression after Application of Haruan Fish Extract (Channa striata) on Inflamed Wistar Rat Dental Pulp. http://www.sysrevpharm.org/?mno=127064 [Access: March 28, 2021]. doi:10.31838/srp.2020.10.6 AMA (American Medical Association) Style Andi Sumidarti, Christine Anastasia Rovani, Juni Jekti Nugroho, Bulkis Thahir. Matrix Protein-1 (DMP-1) Expression after Application of Haruan Fish Extract (Channa striata) on Inflamed Wistar Rat Dental Pulp. SRP. 2020; 11(10): 31-35. doi:10.31838/srp.2020.10.6 Vancouver/ICMJE Style Andi Sumidarti, Christine Anastasia Rovani, Juni Jekti Nugroho, Bulkis Thahir. Matrix Protein-1 (DMP-1) Expression after Application of Haruan Fish Extract (Channa striata) on Inflamed Wistar Rat Dental Pulp. SRP. (2020), [cited March 28, 2021]; 11(10): 31-35. doi:10.31838/srp.2020.10.6 Harvard Style Andi Sumidarti, Christine Anastasia Rovani, Juni Jekti Nugroho, Bulkis Thahir (2020) Matrix Protein-1 (DMP-1) Expression after Application of Haruan Fish Extract (Channa striata) on Inflamed Wistar Rat Dental Pulp. SRP, 11 (10), 31-35. doi:10.31838/srp.2020.10.6 Turabian Style Andi Sumidarti, Christine Anastasia Rovani, Juni Jekti Nugroho, Bulkis Thahir. 2020. Matrix Protein-1 (DMP-1) Expression after Application of Haruan Fish Extract (Channa striata) on Inflamed Wistar Rat Dental Reviews in Pharmacy, 11 (10), 31-35. doi:10.31838/srp.2020.10.6 Chicago Style Andi Sumidarti, Christine Anastasia Rovani, Juni Jekti Nugroho, Bulkis Thahir. Dentin Matrix Protein-1 (DMP-1) Expression after Application of Haruan Fish Extract (Channa striata) on Inflamed Wistar Rat Dental Pulp. Systematic Reviews in Pharmacy 11 (2020), 31-35. doi:10.31838/srp.2020.10.6 MLA (The Modern Language Association) Style Andi Sumidarti, Christine Anastasia Rovani, Juni Jekti Nugroho, Bulkis Thahir. Dentin Matrix Protein-1 (DMP-1) Expression after Application of Haruan Fish Extract (Channa striata) on Inflamed Wistar Rat Dental Pulp. Systematic Reviews in Pharmacy 11.10 (2020), 31-35. Print. doi:10.31838/srp.2020.10.6 APA (American Psychological Association) Style Andi Sumidarti, Christine Anastasia Rovani, Juni Jekti Nugroho, Bulkis Thahir (2020) Matrix Protein-1 (DMP-1) Expression after Application of Haruan Fish Extract (Channa striata) on Inflamed Wistar Rat Dental Reviews in Pharmacy, 11 (10), 31-35. doi:10.31838/srp.2020.10.6

Distinguished properties of cells isolated from the dentin-pulp interface

BackgroundDental odontoblasts produce dentin mineralized matrix, trigger immune responses and act as sensory cells. The understanding of the mechanisms of these functions has been particularly restricted due to the lack of odontoblasts being cultivable in vitro. Because of the lack of specific markers to identify cells of the odontoblastic lineage, properties of the cells isolated from the dentin-pulp interface were compared to dental pulp cells, periodontal ligament cells, osteoblasts, skin fibroblasts, epithelial cells (A549) and HeLa in the present study. MethodsAfter surgical procedures, the pulp tissue was removed from the tooth crown, and cells were scrapped off the dentin-pulp interface. Explants from teeth of three patients were routinely cultivated, and cells were harvested after several weeks. Cell morphology and ultrastructure was studied by light microscopy (LM), scanning (SEM) or transmission electron microscopy (TEM). Expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), TRPV4, and S100 calcium binding protein A4 (S100A4) were analyzed at the protein level by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using specific antibodies. The differential expression of S100A4 in the various cell lines was further investigated at the gene level by semiquantitative real-time PCR. Mineralization in the various cell types was observed after alizarin red staining after a 28 days incubation period. The immunophenotype of the cells was examined by flow cytometry using monoclonal anti-human antibodies CD90-FITC, CD73-PE, CD105-PE, CD29-PE, CD140a-FITC, CD144-PE, CD45-FITC or CD34-FITC. Differences between median values were statistically analyzed (Mann–Whitney U-test). ResultsCells from the dentin-pulp interface retain the polarity of odontoblast morphology in culture with an elongated, rounded cell body, and an extended cellular process. Ultrastructural analysis of the cells indicates high secretory activity including the extracellular deposition of fibrillar collagen. An extended rough endoplasmic reticulum is lined by a large number of ribosomes, and a vast number of secretory granules merges with the cell membrane. Protein expression of DSPP, DMP1, and TRPV4 as a transient receptor potential cation was detected in all cell lines. S100A4 was found differentially expressed in cultures of cells from tooth tissues. High expression of S100A4 was observed at the protein and gene level in two fractions of cells isolated from the dentin-pulp interface, but was absent or only weakly expressed in pulp cells. S100A4 expression in cells from the dentin-pulp interface and pulp cells is consistent with the intensity of the formation of mineralized nodules detected by alizarin red staining. Immunophenotyping revealed that a high percentage of CD73 (ecto-5-nucleotidase), an enzyme active on the surface of immune-competent cells, was expressed in cells of the dentin-pulp interface. While 72%–78% of positive cells were detected in dentin-pulp interface fractions, only 28–64% of the cells in pulp cell cultures were stained. ConclusionsThe present findings obtained with a variety of cells of different origin provide experimental evidence that cells isolated from the dentin-pulp interface express unique properties different from dental pulp cells in particular. The differential expression of S100A4 is a relevant marker candidate for differentiating between dental pulp cells and cells of the odontoblast lineage.

CRISPR/Cas9 mediated GFP-human dentin matrix protein 1 (DMP1) promoter knock-in at the ROSA26 locus in mesenchymal stem cell for monitoring osteoblast differentiation.

Dentin matrix protein 1 (DMP1) is highly expressed in mineralized tooth and bone, playing a critical role in mineralization and phosphate metabolism. One important role for the expression of DMP1 in the nucleus of preosteoblasts is the up-regulation of osteoblast-specific genes such as osteocalcin and alkaline phosphatase1 . The present study aimed to investigate the potential application of human DMP1 promoter as an indicator marker of osteoblastic differentiation. In the present study, we developed DMP1 promoter-DsRed-GFP knock-in mesenchymal stem cell (MSCs) via the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system that enabled automatic detection of osteoblast differentiation. With the application of a homology-directed knock-in strategy, a 2-kb fragment of DMP1 promoter, which was inserted upstream of the GFP and DsRed reporter cassette, was integrated into the human ROSA locus to generate double fluorescent cells. We further differentiated MSCs under osteogenic media to monitor the fate of MSCs. First, cells were transfected using CRISPR/Cas9 plasmids, which culminated in MSCs with a green fluorescence intensity, then GFP-positive cells were selected using puromycin. Second, the GFP-positive MSCs were differentiated toward osteoblasts, which demonstrated an increased red fluorescence intensity. The osteoblast differentiation of MSCs was also verified by performing alkaline phosphatase and Alizarin Red assays. We have exploited the DMP1 promoter as a predictive marker of MSC differentiation toward osteoblasts. Using the CRISPR/Cas9 technology, we have identified a distinctive change in the fluorescence intensities of GFP knock-in (green) and osteoblast differentiated MSCs 2 . The data show that DMP1-DsRed-GFP knock-in MSCs through CRISPR/Cas9 technology provide a valuable indicator for osteoblast differentiation. Moreover, The DMP1 promoter might be used as a predictive marker of MSCs differentiated toward osteoblasts.

The influence of experimental bioactive glasses on pulp cells behavior in vitro

ObjectivesTo assess in vitro the effect of experimental mesoporous BAG, on human dental pulp cells (hDPCs) behavior in terms of cytocompatiblity and bioactivity via mineralization potential. MethodsFine (FP) and large particles (LP) of a fixed BAG composition named 0NaMBG have been elaborated by a sol-gel process. In vitro assessment was achieved on cultured primary hDPCs using indirect contact. The effect of the concentration of 800μg/mL on cell metabolic activity and cytotoxicity were examined by performing Alamar blue and crystal violet assays. Alizarin Red staining was used to detect and quantify the formation of mineralized nodules and ALP activity was colourimetrically quantified. The expression of Odontogenic markers: DMP-1 and osteopontin (OPN) expression and cell morphology was evaluated by confocal microscopy. ResultsAccording to the Alamar blue and crystal violet assay, 0NaMBG samples were non-cytotoxic. Cells treated with 0NaMBG particles expressed higher metabolic activity than control cells, especially for LP. Both FP and LP significantly increased both extra and intra cellular ALP activity. hDPCs exhibited good cell spreading and adhesion in the presence of FP and LP extracts by confocal imaging. Further, Alizarin red S assay demonstrated more mineralization nodules and significant enhancement of the extracellular calcium deposition when cells were interfaced with both FP and LP compared to the control cells. Moreover, LP extracts enhanced the production and secretion of odontogenic markers: dentin matrix protein 1 (DMP-1) and osteopontin. SignificanceLP have a higher surface area and pore volume, which could explain their greater bioactivity in contact with pulp cells. The clinical relevance of these findings implicate that 0NaMBG could be used as fillers in dental therapeutic materials suitable for dentin and/or pulp tissues preservation.

Comparison of odontoblast differentiation capacity between stem cells from human exfoliated deciduous teeth and dental pulp stem cells.

To explore the difference in odontoblast differentiation capacity between stem cells from human exfoliated deciduous teeth (SHED) and dental pulp stem cells (DPSCs), and to examine the expression level of ephrinB1 in odontoblast differentiation of these stem cells. The stems cells were divided into a SHED group and a DPSCs group. After odontoblast differentiation induction, the above 2 groups were also randomly divided into a 3 d group and a 7 d group, respectively.The calcium deposition was detected by alkaline phosphatase (ALP) staining and alizarin red staining.The mRNA and protein expressions of ephrinB1, dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP) were detected by real-time PCR and Western blotting. ALP staining and alizarin red staining showed that there was stronger mineralization capacity in the SHED group than that in the DPSCs group. The relative mRNA and protein expressions of DMP-1, DSPP, and ephrinB1 in the SHED group were higher than those in the DPSCs group except for the protein expression of DMP-1 in the SHED 3 d group (all P<0.05). SHED has stronger odontoblast differentiation capacity than DPSCs. In addition, ephrinB1 may be involved in the processes of odontoblast differentiation in the SHED and DPSCs.

Comparative Secretome Analysis of Mesenchymal Stem Cells From Dental Apical Papilla and Bone Marrow During Early Odonto/Osteogenic Differentiation: Potential Role of Transforming Growth Factor-β2.

To understand the functions of secretory proteins in odontogenesis and to further the understanding of the different molecular events during odontogenesis and osteogenesis, we induced the odonto/osteogenic differentiation of stem cells from dental apical papilla (SCAPs) and bone marrow–derived stem cells (BMSCs) in vitro and compared the expression of secretory proteins during early odonto/osteogenic differentiation using high-performance liquid chromatography with tandem mass spectrometry. The results revealed significant changes by at least 50% in 139 SCAP proteins and 203 BMSC proteins during differentiation. Of these, 92 were significantly upregulated and 47 were significantly downregulated during the differentiation of SCAPs. Most of these proteins showed the same trend during the differentiation of BMSCs. Among the proteins that showed significantly changes during the differentiation of SCAPs and BMSCs, we found that transforming growth factor-β2 (TGFβ2) is a key protein in the network with powerful mediation ability. TGFβ2 was secreted more by SCAPs than BMSCs, was significantly upregulated during the differentiation of SCAPs and was significantly downregulated during the differentiation of BMSCs. Furthermore, the effects of recombinant human TGFβ2 and TGFβ1 on the odonto/osteogenic differentiation of SCAPs and BMSCs were investigated. Real-time reverse transcription polymerase chain reaction (RT-PCR) and western blotting data revealed that TGFβ2 enhanced the odontogenic-related markers [dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1)] and inhibited the osteogenic-related marker bone sialoprotein (BSP) in SCAPs, whereas TGFβ1 enhanced the BSP expression and inhibited the DSPP and DMP1 expression at early odonto/osteogenic differentiation of SCAPs. However, in BMSCs, TGFβ2 enhanced the expression of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), DSPP, and DMP1, whereas TGFβ1 enhanced the expression of ALP and RUNX2, with no significant intergroup difference of DSPP at the early odonto/osteogenic differentiation of BMSCs. TGFβ2 is a potentially important molecule with a distinct function in the regulation of odontogenesis and osteogenesis.

Lipopolysaccharide Downregulates the Osteo-/Odontogenic Differentiation of Stem Cells From Apical Papilla by Inducing Autophagy

IntroductionThe dentinogenesis potential of stem cells during dentin-pulp tissue regeneration may be compromised by microorganism components. Here we aimed to investigate the cell viability and osteo-/odontogenic differentiation of stem cells from apical papilla (SCAP) exposed to bacterial lipopolysaccharide (LPS) and to evaluate the molecular mechanism in vitro. MethodsCCK8 assay was used to assess the SCAP proliferation rate on exposure to different concentrations of LPS in medium. Dentin matrix protein-1 (DMP-1), runt-related transcription factor-2 (Runx-2), and alkaline phosphatase (ALP) expression and mineralized nodule formation were detected by Western blotting and alizarin red S staining to evaluate SCAP osteo-/odontogenic differentiation. Autophagosomes in SCAP and the autophagy-related markers Beclin 1, autophagy-related gene 5 (Atg5), and microtubule-associated proteins light chain 3 (LC3) were detected by transmission electron microscopy and Western blotting, respectively. Effects of the autophagy inhibitor 3-methyladenine on LPS-treated SCAP osteo-/odontogenic differentiation were also examined. ResultsSCAP cell viability was decreased by 5 μg/mL LPS treatment on day 3. LPS (5 μg/mL) inhibited SCAP osteo-/odontogenic differentiation and decreased DMP-1, Runx-2, and ALP expression. Moreover, LC3, Atg5, and Beclin 1 expression and autophagosome number were elevated. Conversely, autophagy inhibition rescued the number of mineralized nodules and DMP-1, Runx-2, and ALP expression in the LPS-treated SCAP. ConclusionsOur findings indicated that autophagy was involved in the suppression of SCAP osteo-/odontogenic differentiation in an LPS-induced inflammatory environment. This study discloses autophagy modulation as a potential new strategy to improve SCAP osteo-/odontogenic differentiation in an inflammatory environment.