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International Endodontic Journal Mar 2018To investigate the effects of recombinant human vascular endothelial growth factor (rhVEGF) on odontoblastic differentiation, in vitro angiogenesis, and expression and...
AIM
To investigate the effects of recombinant human vascular endothelial growth factor (rhVEGF) on odontoblastic differentiation, in vitro angiogenesis, and expression and activity of lysyl oxidase (LOX) in human dental pulp cells (HDPCs), compared with rhFGF-2. To identify the underlying molecular mechanisms, the study focused on whether LOX was responsible for the actions of rhVEGF.
METHODOLOGY
Recombinant human vascular endothelial growth factor (rhVEGF) was constructed using the pBAD-HisA plasmid in Escherichia coli. HDPCs were treated with 1-50 μg mL rhVEGF for 14 days. Alkaline phosphatase (ALP) activity was measured, and the formation of calcified nodules was assessed using alizarin red staining after the induction of odontogenic differentiation of HDPCs. The expression level of the odontogenic differentiation markers was detected by reverse transcription polymerase chain reaction. Signal pathways were assessed by Western blot and immunocytochemistry. The data were analysed by anova with Bonferroni's test (α = 0.05).
RESULTS
Recombinant human vascular endothelial growth factor significantly increased cell growth (P < 0.05), ALP activity (P < 0.05) and mineralization nodule formation and upregulated the mRNA expression levels of the osteogenic/odontogenic markers that were lower with rhFGF-2. rhVEGF significantly increased amine oxidase activity (P < 0.05) and upregulated LOX and LOXL mRNA expression in HDPCs. Additionally, rhVEGF dose-dependently upregulated angiogenic gene mRNAs and capillary tube formation to a greater degree than rhFGF-2. Inhibition of LOX using β-aminopropionitrile (BAPN) and LOX or LOXL gene silencing by RNA interference attenuated rhVEGF-induced growth, ALP activity, mineralization, the expression of marker mRNAs and in vitro angiogenesis. Furthermore, treatment with rhVEGF resulted in phosphorylation of Akt, ERK, JNK and p38, and activation of NF-κB, which was inhibited by LOX or LOXL silencing and BAPN.
CONCLUSION
Recombinant human vascular endothelial growth factor promoted cell growth, odontogenic potential and in vitro angiogenesis via modulation of LOX expression. These results support the concept that rhVEGF may offer therapeutic benefits in regenerative endodontics.
Topics: Blotting, Western; Cell Differentiation; Cell Line; Dental Pulp; Humans; Neovascularization, Physiologic; Protein-Lysine 6-Oxidase; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Vascular Endothelial Growth Factor A
PubMed: 28568134
DOI: 10.1111/iej.12796 -
Cellular Reprogramming Oct 2021The osteogenic differentiation of mesenchymal stem cells (MSCs) is strongly related with the inflammatory microenvironment. The ability of osteogenic differentiation of...
The osteogenic differentiation of mesenchymal stem cells (MSCs) is strongly related with the inflammatory microenvironment. The ability of osteogenic differentiation of MSCs is vital for the bone tissue engineering. Interleukin (IL)-10, a well-known anti-inflammatory factor, plays a key role in tissue repair. Dental pulp stem cells (DPSCs), with the advantage of convenience of extraction, are suitable for the bone tissue engineering. Therefore, it is meaning to explore the effects of IL-10 on the osteogenic differentiation of DPSCs. The proliferation activity of DPSCs were evaluated by MTS assay (CellTiter 96 Aqueous One Solution Cell Proliferation Assay [Promega]) and real-time polymerase chain reaction (RT-PCR). The osteogenic differentiation of DPSCs were determined by Alizarin Red staining, RT-PCR, and alkaline phosphatase activity test. The glucose metabolism was detected by Mito Stress test and glycolysis assay. IL-10 (10 or 20 nM) could enhance the osteogenic differentiation of DPSCs and promoted the metabolic switch from glycolysis to oxidative phosphorylation (OXPHOS), whereas IL-10 (5 and 50 nM) has no obvious effects on the osteogenic differentiation of DPSCs. The OXPHOS inhibitor restrained the promotion of osteogenic differentiation induced by IL-10. These findings show that IL-10 can promote the osteogenesis of DPSCs through the activation of OXPHOS, which provides a potential way for enhancing the osteogenic differentiation of DPSCs in bone tissue engineering.
Topics: Adolescent; Adult; Cell Differentiation; Cell Proliferation; Cells, Cultured; Dental Pulp; Glycolysis; Humans; Interleukin-10; Osteogenesis; Oxidative Phosphorylation; Stem Cells; Tissue Engineering; Young Adult
PubMed: 34491831
DOI: 10.1089/cell.2021.0044 -
Cell Proliferation Oct 2017The function of the dental pulp is closely connected to the extracellular matrix (ECM) structure, and ECM has received significant attention due to its biological...
OBJECTIVES
The function of the dental pulp is closely connected to the extracellular matrix (ECM) structure, and ECM has received significant attention due to its biological functions for regulating cells. As such, the interaction between the ECM niche and cells is worth exploring for potential clinical uses.
MATERIALS AND METHODS
In this study, dental pulp stem cell (DPSC)-derived ECM (DPM) was prepared through cell culture and decellularization to function as the cell niche, and changes in DPSC behaviour and histological analysis of dental pulp tissue regeneration were evaluated following the DPM culture. DPM promoted the replication of DPSCs and exhibited retention of their mineralization. Then, the DPM-based culture strategy under odontogenic culture medium was further investigated, and the mineralization-related markers showed that DPSCs were regulated towards odontogenic differentiation. Dental pulp-like tissue with well-arranged ECM was harvested after a 2-month subcutaneous implantation in nude mice with DPM application. Additionally, DPSCs cultured on the plastic culture surface showed the up-regulation of mineralization makers in vitro, but there was a disorder in matrix formation and mineralization when the cells were cultured in vivo.
RESULTS AND CONCLUSIONS
DPM-based cultivation could serve as a cell niche and modulate DPSC behaviour, and this method also provided an alternative to harvest tissue-specific ECM and provided a strategy for ECM-cell interaction.
Topics: Adolescent; Animals; Cell Culture Techniques; Cell Cycle; Cell Differentiation; Cell Fractionation; Cell Proliferation; Cells, Cultured; Child; Dental Pulp; Extracellular Matrix; Humans; Mice, Nude; Odontogenesis; Regeneration; Stem Cell Transplantation; Stem Cells
PubMed: 28741725
DOI: 10.1111/cpr.12361 -
Biochemical and Biophysical Research... Oct 2021Small extracellular vesicles (sEV) facilitate signaling molecule transfer among cells. We examined the therapeutic efficacy of human dental pulp stem cell-derived sEV...
Small extracellular vesicles (sEV) facilitate signaling molecule transfer among cells. We examined the therapeutic efficacy of human dental pulp stem cell-derived sEV (hDPSC-sEV) against cellular senescence in an irradiated-submandibular gland mouse model. Seven-week-old mice were exposed to 25 Gy radiation and randomly assigned to control, phosphate-buffered saline (PBS), or hDPSC-sEV groups. At 18 days post-irradiation, saliva production was measured; histological and reverse transcription-quantitative PCR analyses of the submandibular glands were performed. The salivary flow rate did not differ significantly between the PBS and hDPSC-sEV groups. AQP5-expressing acinar cell numbers and AQP5 expression levels in the submandibular glands were higher in the hDPSC-sEV group than in the other groups. Furthermore, compared with non-irradiated mice, mice in the 25 Gy + PBS group showed a high senescence-associated-β-galactosidase-positive cell number and upregulated senescence-related gene (p16, p19, p21) and senescence-associated secretory phenotypic factor (MMP3, IL-6, PAI-1, NF-κB, and TGF-β) expression, all of which were downregulated in the hDPSC-sEV group. Superoxide dismutase levels were lower in the PBS group than in the hDPSC-sEV group. In summary, hDPSC-sEV reduced inflammatory cytokine and senescence-related gene expression and reversed oxidative stress in submandibular cells, thereby preventing irradiation-induced cellular senescence. Based on these results, we hope to contribute to the development of innovative treatment methods for salivary gland dysfunction that develops after radiotherapy for head and neck cancer.
Topics: Animals; Cellular Senescence; Dental Pulp; Disease Models, Animal; Extracellular Vesicles; Female; Gamma Rays; Humans; Inflammation; Mice; Mice, Inbred ICR; Oxidative Stress; Signal Transduction; Stem Cells; Submandibular Gland
PubMed: 34454177
DOI: 10.1016/j.bbrc.2021.08.046 -
International Journal of Environmental... Jan 2023This study examines 0.6 N-4.8 N as the maximum orthodontic force to be applied to dental pulp and apical NVB on intact and 1-8 mm reduced periodontal-ligament (PDL), in...
This study examines 0.6 N-4.8 N as the maximum orthodontic force to be applied to dental pulp and apical NVB on intact and 1-8 mm reduced periodontal-ligament (PDL), in connection with movement and ischemic, necrotic and resorptive risk. In addition, it examines whether the Tresca finite-element-analysis (FEA) criterion is more adequate for the examination of dental pulp and its apical NVB. Eighty-one (nine patients, with nine models for each patient) anatomically correct models of the periodontium, with the second lower-premolar reconstructed with its apical NVB and dental pulp were assembled, based on X-ray CBCT (cone-beam-computed-tomography) examinations and subjected to 0.6 N, 1.2 N, 2.4 N and 4.8 N of intrusion, extrusion, translation, rotation, and tipping. The Tresca failure criterion was applied, and the shear stress was assessed. Forces of 0.6 N, 1.2 N, and 2.4 N had negligible effects on apical NVB and dental pulp up to 8 mm of periodontal breakdown. A force of 4.8 N was safely applied to apical NVB on the intact periodontium only. Rotation and tipping seemed to be the most invasive movements for the apical NVB. For the dental pulp, only the translation and rotation movements seemed to display a particular risk of ischemia, necrosis, and internal orthodontic-resorption for both coronal (0-8 mm of loss) and radicular pulp (4-8 mm of loss), despite the amount of stress being lower than the MHP. The Tresca failure criterion seems more suitable than other criteria for apical NVB and dental pulp.
Topics: Humans; Bicuspid; Dental Pulp; Computer Simulation; Tooth Movement Techniques; Periodontium; Finite Element Analysis
PubMed: 36673936
DOI: 10.3390/ijerph20021179 -
Dental Materials : Official Publication... Jan 2017The study assessed the cytotoxicity and immunomodulatory/anti-inflammatory effect of extract from zinc oxide-eugenol (ZOE)-based dental materials during setting using...
OBJECTIVE
The study assessed the cytotoxicity and immunomodulatory/anti-inflammatory effect of extract from zinc oxide-eugenol (ZOE)-based dental materials during setting using immortalized human dental pulp stem cells (IHDPSCs) and mouse bone marrow monocytes (IMBMMs), and identified the responsible extract component.
METHODS
In accord with the ISO 10993-12, we extracted a mixture of ZOE cement and sealer after a specified time. The extract was analyzed by two types of mass spectrometry (ICP-MS and GC-MS). Cell viability was evaluated with extract and serial concentrations of ZnCl, ZnSO, and eugenol liquid by WST assay. The immunomodulatory/anti-inflammatory effect of a ZOE component was determined by RT-PCR to detect the downregulatory effect of inflammatory mRNA expression after lipopolysaccharide (LPS)-induced inflammation.
RESULTS
Zn and eugenol (2-20ppm) were detected in the ZOE cement and sealer extracts. During the early stage of setting, significant cytotoxicity was observed in IHDPSCs and IMBMMs (p<0.05). The half maximal effective concentration of Zn was 5-8ppm, whereas that of eugenol could not be detected within 80ppm. After extract treatment, the expression of inflammatory mRNA was significantly lower in inflamed IHDPSCs, but not inflamed IMBMMs, than in the LPS control (p<0.05). However, eugenol, not Zn, at 5-20ppm downregulated inflammatory mRNA expression in the inflamed IMBMMs with and without the exchange of LPS-pretreated medium.
SIGNIFICANCE
ZOE was highly cytotoxic, especially during setting, to both cells due to Zn while the immunomodulatory/anti-inflammatory effect of ZOE was induced by eugenol.
Topics: Animals; Dental Pulp; Eugenol; Humans; Inflammation; Mice; Root Canal Filling Materials; Zinc Oxide-Eugenol Cement
PubMed: 27726970
DOI: 10.1016/j.dental.2016.09.012 -
Stem Cell Research & Therapy Feb 2018Human dental pulp stem cells (DPSCs), which have the ability to differentiate into multiple lineages, were recently identified. DPSCs can be collected readily from...
BACKGROUND
Human dental pulp stem cells (DPSCs), which have the ability to differentiate into multiple lineages, were recently identified. DPSCs can be collected readily from extracted teeth and are now considered to be a type of mesenchymal stem cell with higher clonogenic and proliferative potential than bone marrow stem cells (BMSCs). Meanwhile, the treatment of severe bone defects, such as fractures, cancers, and congenital abnormalities, remains a great challenge, and novel bone regenerative techniques are highly anticipated. Several studies have previously shown that 4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH), a helioxanthin derivative, induces osteogenic differentiation of preosteoblastic and mesenchymal cells. However, the osteogenic differentiation activities of TH have only been confirmed in some mouse cell lines. Therefore, in this study, toward the clinical use of TH in humans, we analyzed the effect of TH on the osteogenic differentiation of DPSCs, and the in-vivo osteogenesis ability of TH-induced DPSCs, taking advantage of the simple transplantation system using cell-sheet technology.
METHODS
DPSCs were obtained from dental pulp of the wisdom teeth of five healthy patients (18-22 years old) and cultured in regular medium and osteogenic medium with or without TH. To evaluate osteogenesis of TH-induced DPSCs in vivo, we transplanted DPSC sheets into mouse calvaria defects.
RESULTS
We demonstrated that osteogenic conditions with TH induce the osteogenic differentiation of DPSCs more efficiently than those without TH and those with bone morphogenetic protein-2. However, regular medium with TH did not induce the osteogenic differentiation of DPSCs. TH induced osteogenesis in both DPSCs and BMSCs, although the gene expression pattern in DPSCs differed from that in BMSCs up to 14 days after induction with TH. Furthermore, we succeeded in bone regeneration in vivo using DPSC sheets with TH treatment, without using any scaffolds or growth factors.
CONCLUSIONS
Our results demonstrate that TH-induced DPSCs are a useful cell source for bone regenerative medicine, and the transplantation of DPSC sheets treated with TH is a convenient scaffold-free method of bone healing.
Topics: Animals; Bone Regeneration; Cell Differentiation; Cells, Immobilized; Dental Pulp; Heterografts; Humans; Lignans; Mice; Mice, Inbred NOD; Mice, SCID; Osteogenesis; Stem Cell Transplantation; Stem Cells
PubMed: 29391049
DOI: 10.1186/s13287-018-0783-7 -
Metallomics : Integrated Biometal... Mar 2019Zinc (Zn) is distributed throughout the body and within cells by saturable processes mediated by the transport proteins of the ZnT (SLC30) and ZIP (SLC39) families. The...
Zinc (Zn) is distributed throughout the body and within cells by saturable processes mediated by the transport proteins of the ZnT (SLC30) and ZIP (SLC39) families. The two families function in opposite directions. ZnT transporters mediate cellular zinc efflux or intracellular sequestration. Zn is found in human tooth enamel and dentine at levels that have been related to environmental exposures, such as pollution, disease, and dietary intake. The mechanism by which Zn in the odontoblast is deposited in the hard tissue of the tooth, however, is unknown but is important in determining the physical properties, and hence resilience, of enamel and in the context of the use of tooth zinc level as a biomarker of exposure. We hypothesised that zinc efflux mediated by members of the ZnT family of 10 transporters is a key step in this process and is regulated by zinc availability through effects on mRNA levels. Thus, we determined the profile of ZnT transporter mRNA in a human active-secretory odontoblast-like cell model under conditions of high- and low-extracellular Zn concentration and determined if the same transporter mRNAs were present in human dental pulp. ZnT1, ZnT5 and ZnT9 mRNAs were detected by RT-PCR in both the secretory odontoblast cells and human dental pulp. ZnT2, ZnT3 and ZnT10 mRNAs were not detected, and ZnT4 mRNA was detected in secretory odontoblasts only, which may be indicative of a specialised zinc efflux function during the active secretory phase of tooth development. ZnT1 mRNA was significantly increased in response to extracellular Zn exposure (60 μM) after 24 h. The presence of Zn transporter mRNAs in secretory odontoblasts and dental pulp indicates that the corresponding transport proteins function to deposit zinc in the dental hard tissues. The responsiveness of ZnT1 in odontoblasts to zinc availability is concordant with this being a process that is regulated to maintain cellular Zn homeostasis and that is a mediator of the relationship between environmental Zn exposure and dental Zn deposition. These findings have likely relevance to human dental health through effects of Zn transporter expression level on the hard tissue properties.
Topics: Cation Transport Proteins; Cells, Cultured; Dental Pulp; Gene Expression; Humans; Odontoblasts; RNA, Messenger; Zinc
PubMed: 30675888
DOI: 10.1039/c8mt00343b -
International Journal of Molecular... Sep 2017There are anatomical and functional differences between human dental pulp (DP) and periodontal ligament (PDL). However, the molecular biological differences and... (Comparative Study)
Comparative Study
There are anatomical and functional differences between human dental pulp (DP) and periodontal ligament (PDL). However, the molecular biological differences and function of these tissues are poorly understood. In the present study, we employed a cDNA microarray array to screen for differentially expressed genes (DEGs) between human DP and PDL tissues, and used the online software WebGestalt to perform the functional analysis of the DEGs. In addition, the STRING database and KEGG pathway analysis were applied for interaction network and pathway analysis of the DEGs. DP and PDL samples were obtained from permanent premolars (n=16) extracted for orthodontic purposes. The results of the microarray assay were confirmed by RT-qPCR. The DEGs were found to be significantly associated with the extracellular matrix and focal adhesion. A total of 10 genes were selected to confirm the results. The mRNA levels of integrin alpha 4 (ITGA4), integrin alpha 8 (ITGA8), neurexin 1 (NRXN1) and contactin 1 (CNTN1) were significantly higher in the DP than in the PDL tissues. However, the levels of collagen type XI alpha 1 (COL11A1), aggrecan (ACAN), collagen type VI alpha 1 (COL6A1), chondroadherin (CHAD), laminin gamma 2 (LAMC2) and laminin alpha 3 (LAMA3) were higher in the PDL than in the DP samples. The gene expression profiles provide novel insight into the characterization of DP and PDL tissues, and contribute to our understanding of the potential molecular mechanisms of dental tissue mineralization and regeneration.
Topics: Adolescent; Child; Databases, Nucleic Acid; Dental Pulp; Female; Gene Expression Profiling; Gene Expression Regulation; Humans; Male; Oligonucleotide Array Sequence Analysis; Periodontal Ligament; Transcriptome
PubMed: 28713908
DOI: 10.3892/ijmm.2017.3065 -
Mediators of Inflammation 2015Pathogen recognition receptors (PRRs) are a class of germ line-encoded receptors that recognize pathogen-associated molecular patterns (PAMPs). The activation of PRRs is... (Review)
Review
Pathogen recognition receptors (PRRs) are a class of germ line-encoded receptors that recognize pathogen-associated molecular patterns (PAMPs). The activation of PRRs is crucial for the initiation of innate immunity, which plays a key role in first-line defense until more specific adaptive immunity is developed. PRRs differ in the signaling cascades and host responses activated by their engagement and in their tissue distribution. Currently identified PRR families are the Toll-like receptors (TLRs), the C-type lectin receptors (CLRs), the nucleotide-binding oligomerization domain-like receptors (NLRs), the retinoic acid-inducible gene-I-like receptors (RLRs), and the AIM2-like receptor (ALR). The environment of the dental pulp is substantially different from that of other tissues of the body. Dental pulp resides in a low compliance root canal system that limits the expansion of pulpal tissues during inflammatory processes. An understanding of the PRRs in dental pulp is important for immunomodulation and hence for developing therapeutic targets in the field of endodontics. Here we comprehensively review recent finding on the PRRs and the mechanisms by which innate immunity is activated. We focus on the PRRs expressed on dental pulp and periapical tissues and their role in dental pulp inflammation.
Topics: Animals; Carrier Proteins; Dental Pulp; Humans; Immunity, Innate; Lectins, C-Type; NLR Family, Pyrin Domain-Containing 3 Protein; Nod1 Signaling Adaptor Protein; Nod2 Signaling Adaptor Protein; Receptors, Pattern Recognition; Toll-Like Receptors
PubMed: 26576076
DOI: 10.1155/2015/794143