RESUMO
Periodontal ligament (PDL) cells are a promising tool for periodontal regeneration therapy. Achieving a sufficient number of PDL cells is essential to PDL regeneration. In our study, appropriate flow shear stress (FSS, 1-6 dyn/cm2) promotes the proliferation of PDL cells. FSS remodels cytoskeleton and focal adhesion in a duration-dependent manner. FSS induces PDL cells to form the actin cap within 10 min, flattens the nuclei, and increases the nuclear pore size, which promotes nuclear translocation of Yes-associated protein (YAP). FSS activates p38, which plays a dual function in YAP regulation. p38 regulates the phosphorylation of Akt and cofilin, as well as induced F-actin polymerization to induce YAP activity. In addition, p38 inhibits pLATS and consecutively regulates angiomotin (AMOT) and YAP phosphorylation. AMOT competitively binds to F-actin and YAP to participate in FSS-mediated YAP nuclear translocation and cell proliferation. Taken collectively, our results provide mechanistic insights into the role of p38-AMOT-YAP in FSS-mediated PDL cells proliferation and indicate potential applications in dental regenerative medicine.
Assuntos
Actinas , Ligamento Periodontal , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Angiomotinas , Proliferação de Células , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Sinalização YAPRESUMO
AIM: Calcium hydroxide is the gold standard material for pulp capping and has been widely used in clinical dentistry. Calcium hydroxide promotes proliferation, migration and osteogenic differentiation of dental pulp stem cells (DPSCs). However, the underlying mechanism is not clear. Our study investigated the role of Wnt/ß-catenin pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation and mineralization of human DPSCs. METHODOLOGY: Protein and gene expression was detected by western blot (WB), immunofluorescence staining and quantitative real-time PCR (qPCR). Cell viability was analysed using the Cell Counting Kit-8 (CCK-8) assay. Wound-healing assay was used to analyse cell migration. The expression of alkaline phosphatase (ALP) was detected using ALP staining. Mineralization was analysed by alizarin red staining. RESULTS: Calcium hydroxide increased the protein expression of phosphorylated-GSK3ß/GSK3ß, ß-catenin and the gene expression of LEF-1. Inhibition of Wnt/ß-catenin abolished calcium hydroxide-induced proliferation and migration of DPSCs in 24 h. However, incubation with calcium hydroxide for 7 days and 14 days reduced Wnt/ß-catenin signalling. Inhibition of Wnt/ß-catenin promoted calcium hydroxide-induced osteogenic differentiation and mineralization in DPSCs. CONCLUSION: Wnt/ß-catenin pathway plays a dual role in calcium hydroxide-regulated DPSC behaviour. Incubation with calcium hydroxide promoted rapid proliferation and migration of DPSCs, while prolonged incubation negatively regulated osteogenic differentiation and mineralization.
Assuntos
Osteogênese , beta Catenina , Humanos , Hidróxido de Cálcio/farmacologia , Polpa Dentária , Diferenciação Celular , Proliferação de Células , Células-TroncoRESUMO
Magnetic materials are now commonly used in dental clinics. These materials generally produce a static magnetic field (SMF). While it is known that SMF can affect cells' behaviors such as proliferation, migration, and differentiation, the mechanisms underlying these effects are still unclear. Our study investigates the role of the mitogen-activated protein (MAP) kinase pathway in SMF-induced proliferation, migration, osteogenic/odontogenic differentiation, and mineralization in human dental pulp stem cells (DPSCs). Human DPSCs were exposed to SMF of 1 mT and the phosphorylated MAP kinases were detected by Western blot analysis. Three MAP kinases inhibitors were pre-cultured with DPSCs and exposed to SMF for 24 h. Cell viability was analyzed using Cell Counting Kit-8. Cell migration was tested by a wound healing assay. Osteogenic/odontogenic differentiation was detected by ALP staining assay, ALP and DSPP Western blot analysis. Mineralization was studied by alizarin red staining analysis. SMF activated phosphorylation of c-Jun N-terminal kinase (JNK), P38 and extracellular signal-regulated kinase (ERK). The inhibition of JNK, P38, and ERK signaling decreased SMF-induced proliferation and migration. ERK and P38 play more important roles in SMF-induced ALP staining and protein expression. JNK was vital for SMF-induced DSPP expression. JNK, P38, and ERK all involved in SMF-mediated mineralization. Our study demonstrated that the MAPK pathway regulated SMF-induced proliferation, migration, osteogenic/odontogenic differentiation, and mineralization in human DPSCs.
RESUMO
INTRODUCTION: Fluid shear stress (FSS) is the most common stress produced by mastication, speech, or tooth movement. However, how FSS regulates human periodontal ligament (PDL) cell proliferation and migration as well as the underlying mechanism remains unknown. METHODS: FSS (6 dyn/cm2) was produced in a flow chamber. Cell proliferation was tested by the 5-ethynyl-2'-deoxyuridine assay. Cell migration was tested by the wound healing assay. Gene and protein expression of platelet-derived growth factors (PDGFs) and matrix metalloproteinase (MMP)-2 were measured by reverse transcription-polymerase chain reaction and western blot analyses. RESULTS: We investigated the effect of 4 h of 6 dyn/cm2 FSS on proliferation and migration of PDL cells. FSS promoted PDL cell proliferation but inhibited migration. The gene and protein expression of PDGF receptor (PDGFR)-α and ß both decreased in response to FSS. Activating and inhibiting the PDGFRs did not affect the FSS-induced increase in cell proliferation. However, activating PDGFRs with PDGF-BB, which bound both PDGFR-α and ß, and PDGF-CC and DD, which had high affinities for PDGFR-α and PDGFR-ß, individually rescued FSS-inhibited migration. FSS also inhibited MMP-2 gene expression, which was the most important factor for matrix turnover and migration of PDLs. PDGF-BB, CC, and DD increased the FSS-induced decline in MMP-2 expression. These results indicate that MMP-2 is regulated by FSS and contributes to the FSS-induced decrease in cell migration. CONCLUSIONS: Our study suggests a role for PDGFR-α and ß in short-term FSS-regulated cell proliferation and migration. These results will help provide the scientific foundation for revealing the mechanisms clinical tooth movement and PDL regeneration.
RESUMO
It is well known that the differentiation of stem cells is affected by the cell culture medium, the scaffold surface and electrochemical signals. However, stimulation of patterned biomaterials seeded with stem cell cultures has not been explored. Herein the effect of electrical stimulation on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBMSCs) cultured on solid and nanoporous micropyramid patterned Si surfaces was evaluated. It was found that both stimulation and scaffold patterning significantly enhanced osteo-differentiation. The stimulated nanoporous micropyramid scaffolds were more promising compared to the stimulated solid micropyramid surfaces, as they significantly promoted the osteogenic differentiation of rBMSCs via BMP/Smad signaling pathway. Particularly, as compared to the unstimulated patterned biomaterials, the stimulated patterned scaffolds allowed for a significant increase in core binding factor alpha l, alkaline phosphatase, the alpha l chain of type I Col, osteocalcin, and osteonectin, all of which are characteristic for osteo-differentiation. The proposed combination of electrical stimulation with scaffold patterning may provide novel promising strategies for bone tissue engineering and regenerative medicine.
Assuntos
Materiais Biocompatíveis/farmacologia , Células-Tronco Mesenquimais/citologia , Nanoestruturas/química , Osteogênese/fisiologia , Silício/química , Fosfatase Alcalina/metabolismo , Animais , Materiais Biocompatíveis/química , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Estimulação Elétrica , Regulação da Expressão Gênica , Células-Tronco Mesenquimais/fisiologia , Osteoblastos/citologia , Osteoblastos/fisiologia , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Ratos Sprague-Dawley , Proteínas Smad/genética , Proteínas Smad/metabolismo , Alicerces TeciduaisRESUMO
The dental pulp stem cells (DPSCs) are a population of mesenchymal stem cells, which have multilineage potential and high proliferation. DPSCs are regarded as a promising tool for tissue regeneration of dentine, dental pulp, bone, cartilage, and muscle. Recently, magnetic materials have become commonly applied in dental clinics. Static magnetic field has been reported to regulate the proliferation, migration, or differentiation of stem cells. However, whether static magnetic fields affect DPSCs is still unknown. In our study, we investigated the effect of static magnetic field on the proliferation, migration, and differentiation of DPSCs. The results indicated that static magnetic field rearranged the cytoskeleton of DPSCs. A static magnetic field of 1 mT increased DPSC proliferation, as well as the gene expression of several growth factors such as FGF-2, TGF-ß, and VEGF. Moreover, the static magnetic field promoted the migration of DPSCs by regulating MMP-1 and MMP-2 gene expression. Static magnetic field of 1 mT also induced osteo/odontogenesis and mineralization in DPSCs. Otherwise, the static magnetic field recruited YAP/TAZ to the nucleus, inhibited the phosphorylation of YAP/TAZ, and upregulated the two YAP/TAZ-regulated genes, CTGF and ANKRD1. Cytoskeleton inhibitor, cytochalasin D, obviously inhibited the nuclear localization of YAP/TAZ. When YAP/TAZ were knocked-down, the static magnetic field-induced mineralization of DPSCs was diminished. Our findings provide an insight into the effect of static magnetic field on DPSCs and provide the foundation for the future tissue regeneration.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Diferenciação Celular , Movimento Celular , Polpa Dentária/citologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Campos Magnéticos , Fosfoproteínas/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Adolescente , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Odontogênese/efeitos dos fármacos , Odontogênese/genética , Células-Tronco/efeitos dos fármacos , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAP , Adulto JovemRESUMO
Shear stress is one of the main stress type produced by speech, mastication or tooth movement. The mechano-response of human periodontal ligament (PDL) cells by shear stress and the mechanism are largely unknown. In our study, we investigated the effects of fluid shear stress on proliferation, migration and osteogenic potential of human PDL cells. 6dyn/cm(2) of fluid shear stress was produced in a parallel plate flow chamber. Our results demonstrated that fluid shear stress rearranged the orientation of human PDL cells. In addition, fluid shear stress inhibited human PDL cell proliferation and migration, but increased the osteogenic potential and expression of several growth factors and cytokines. Our study suggested that shear stress is involved in homeostasis regulation in human PDL cells. Inhibiting proliferation and migration potentially induce PDL cells to respond to mechanical stimuli in order to undergo osteogenic differentiation.
Assuntos
Líquido Extracelular/metabolismo , Osteogênese , Ligamento Periodontal/citologia , Resistência ao Cisalhamento , Estresse Mecânico , Fenômenos Biomecânicos , Diferenciação Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , HumanosRESUMO
INTRODUCTION: Calcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. METHODS: Human DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. RESULTS: Calcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide-induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal-regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. CONCLUSIONS: Our study showed that the MAP kinase pathway was involved in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs.
Assuntos
Calcificação Fisiológica/efeitos dos fármacos , Hidróxido de Cálcio/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Polpa Dentária/citologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Adolescente , Fosfatase Alcalina/metabolismo , Western Blotting , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Células-Tronco/fisiologia , Adulto JovemRESUMO
Regenerative medicine treatments that combine the use of cells and materials may open new options for tissue/organ repair and regeneration. The microenvironment of mesenchymal stem cells (MSCs) strictly regulates their self-renewal and functions. In this study, when rat bone marrow derived MSCs (rBMSCs) and rat adipose tissue derived MSCs (rAMSCs) in passages 2-4 were cultured on different substrates, they presented the cellular functions to be dependent of substrate stiffness. The cells attached better on the softer substrate than on the stiffer one. The substrate stiffness had no significant influence on the proliferation of those cells. However, the substrate stiffness significantly promoted the osteogenic differentiation of the two kinds of stem cells. Furthermore, rBMSCs cultured on the same stiffness expressed more osteoblast-related markers than rAMSCs. In addition, combined biomaterials and biochemical reagents treatment yielded a stronger effect on osteogenic differentiation of MSCs than either treatment alone. These results have significant implications for further extending our capabilities in engineering functional tissue substitutes.
Assuntos
Tecido Adiposo/citologia , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Fosfatase Alcalina/metabolismo , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Células Cultivadas , DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Osteocalcina/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Matrix metalloproteinase (MMP)-1, 2, with their endogenous inhibitors, tissue inhibitor of metalloproteinase (TIMP)-1, 2 are critical for extracellular matrix remodeling in human periodontal ligament (PDL) and their expression are sensitive to mechanical stresses. Shear stress as the main type of mechanical stress in tooth movement is involved in matrix turnover. However, how shear stress regulates MMPs and TIMPs system is still unclear. In this study, we investigated the effect of fluid shear stress on expression of MMP-1, 2 and TIMP-1, 2 in human PDL cells and the possible roles of mitogen-activated protein kinases in this process. Three levels of fluid shear stresses (6, 9 and 12 dyn/cm(2)) were loaded on PDL cells for 2, 4, 8 and 12h. The results indicated that fluid shear stress rearranged cytoskeleton in PDL cells. Fluid shear stress increased expression of MMP-1, 2, TIMP-1 and suppressed TIMP-2 expression. MAP kinases including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 were activated rapidly by fluid shear stress. The ERK inhibitor blocked fluid shear stress induced MMP-1 expression and P38 inhibitor reduced fluid shear stress stimulated MMP-2 expression. Our study suggested that fluid shear stress involved in PDL remodeling via regulating MMP-1, 2 and TIMP-1, 2 expression. ERK regulated fluid shear stress induced MMP-1 expression and P38 play a role in fluid shear stress induced MMP-2 upregulation.