RESUMO
BACKGROUND: Decellularized extracellular matrix (dECM) has been proposed as a useful source of biomimetic materials for regenerative medicine due to its biological properties that regulate cell behaviors. The present study aimed to investigate the influence of decellularized ECM derived from dental pulp stem cells (DPSCs) on gingival fibroblast (GF) cell behaviors. Cells were isolated from dental pulp and gingival tissues. ECM was derived from culturing dental pulp stem cells in growth medium supplemented with ascorbic acid. A bioinformatic database of the extracellular matrix was constructed using Metascape. GFs were reseeded onto dECM, and their adhesion, spreading, and organization were subsequently observed. The migration ability of the cells was determined using a scratch assay. Protein expression was evaluated using immunofluorescence staining. RESULTS: Type 1 collagen and fibronectin were detected on the ECM and dECM derived from DPSCs. Negative phalloidin and nuclei were noted in the dECM. The proteomic database revealed enrichment of several proteins involved in ECM organization, ECM-receptor interaction, and focal adhesion. Compared with those on the controls, the GFs on the dECM exhibited more organized stress fibers. Furthermore, cultured GFs on dECM exhibited significantly enhanced migration and proliferation abilities. Interestingly, GFs seeded on dECM showed upregulation of FN1, ITGB3, and CTNNB1 mRNA levels. CONCLUSIONS: ECM derived from DSPCs generates a crucial microenvironment for regulating GF adhesion, migration and proliferation. Therefore, decellularized ECM from DPSCs could serve as a matrix for oral tissue repair.
Assuntos
Adesão Celular , Movimento Celular , Polpa Dentária , Matriz Extracelular , Fibroblastos , Gengiva , Células-Tronco , Polpa Dentária/citologia , Humanos , Gengiva/citologia , Matriz Extracelular/metabolismo , Proliferação de Células , Células Cultivadas , Fibronectinas/metabolismoRESUMO
Mesenchymal stem cell derived extracellular vesicles (MSC EVs) are paracrine modulators of macrophage function. Scientific research has primarily focused on the immunomodulatory and regenerative properties MSC EVs derived from bone marrow. The dental pulp is also a source for MSCs, and their anatomical location and evolutionary function has primed them to be potent immunomodulators. In this study, we demonstrate that extracellular vesicles derived from dental pulp stem cells (DPSC EVs) have pronounced immunomodulatory effect on primary macrophages by regulating the NFκb pathway. Notably, the anti-inflammatory activity of DPSC-EVs is enhanced following exposure to an inflammatory stimulus (LPS). These inhibitory effects were also observed in vivo. Sequencing of the naïve and LPS preconditioned DPSC-EVs and comparison with our published results from marrow MSC EVs revealed that Naïve and LPS preconditioned DPSC-EVs are enriched with anti-inflammatory miRNAs, particularly miR-320a-3p, which appears to be unique to DPSC-EVs and regulates the NFκb pathway. Overall, our findings highlight the immunomodulatory properties of DPSC-EVs and provide vital clues that can stimulate future research into miRNA-based EV engineering as well as therapeutic approaches to inflammation control and disease treatment.
Assuntos
Polpa Dentária , Vesículas Extracelulares , Imunomodulação , Inflamação , NF-kappa B , Polpa Dentária/citologia , Polpa Dentária/imunologia , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/imunologia , Humanos , Animais , Inflamação/imunologia , Inflamação/metabolismo , NF-kappa B/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , MicroRNAs/genética , Lipopolissacarídeos/farmacologia , Camundongos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/imunologia , Células Cultivadas , Transdução de Sinais , Células-Tronco/imunologia , Células-Tronco/metabolismo , MasculinoRESUMO
OBJECTIVE: To investigate the biological regulatory function of Gremlin1 (GREM1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta (YWHAH) in dental pulp stem cells (DPSCs), and determine the underlying molecular mechanism involved. METHODS: Alkaline phosphatase (ALP) activity, alizarin red staining, scratch migration assays and in vitro and in vivo osteo-/dentinogenic marker detection of bone-like tissue generation in nude mice were used to assess osteo-/dentinogenic differentiation. Coimmunoprecipitation and polypeptide microarray assays were employed to detect the molecular mechanisms involved. RESULTS: The data revealed that knockdown of GREM1 promoted ALP activity, mineralisation in vitro and the expression of osteo-/dentinogenic differentiation markers and enhanced osteo-/ dentinogenesis of DPSCs in vivo. GREM1 bound to YWHAH in DPSCs, and the binding site was also identified. Knockdown of YWHAH suppressed the osteo-/dentinogenesis of DPSCs in vitro, and overexpression of YWHAH promoted the osteo-/dentinogenesis of DPSCs in vitro and in vivo. CONCLUSION: Taken together, the findings highlight the critical roles of GREM1-YWHAH in the osteo-/dentinogenesis of DPSCs.
Assuntos
Diferenciação Celular , Polpa Dentária , Peptídeos e Proteínas de Sinalização Intercelular , Osteogênese , Células-Tronco , Animais , Humanos , Camundongos , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/genética , Células Cultivadas , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Dentinogênese/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos Nus , Osteogênese/genética , Células-Tronco/metabolismoRESUMO
Premixed calcium silicate-based materials have recently been developed and are recommended for a wide range of endodontic procedures, including vital pulp therapy. This study investigated the in vitro biocompatibility and pro-mineralization effect and in vivo reparative dentin formation of EndoSequence Root Repair Material, EndoSequence BCRRM, Bio-C Repair, and Well-pulp PT. Both fresh and set extracts had no detrimental effect on the growth of human dental pulp stem cells. The fresh extracts had a higher calcium concentration than the set extracts and induced considerably greater mineralized nodule formation. EndoSequence Root Repair Material had the longest setting time, whereas Bio-C Repair had the shortest. When these materials were applied to exposed rat molar pulps, mineralized tissue deposition was found at the exposure sites after 2 weeks. These results indicate that the premixed calcium silicate-based materials tested could have positive benefits for direct pulp capping procedures.
Assuntos
Materiais Biocompatíveis , Compostos de Cálcio , Polpa Dentária , Silicatos , Células-Tronco , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/citologia , Silicatos/farmacologia , Compostos de Cálcio/farmacologia , Humanos , Células-Tronco/efeitos dos fármacos , Materiais Biocompatíveis/farmacologia , Ratos , Animais , Teste de Materiais , Células Cultivadas , Técnicas In Vitro , Masculino , Fosfatos de Cálcio , Combinação de Medicamentos , ÓxidosRESUMO
BACKGROUND: Previous studies have reported the link between hypoxic conditions and NLRP3 inflammasome-mediated pulpal inflammation in the progression of pulpitis. However, the underlying mechanism has not been fully elucidated. This study aimed to investigate the role of HIF-1α in the regulation of NLRP3 inflammasome pathway via NF-κB signaling under hypoxic conditions with or without LPS in human dental pulp fibroblasts (HDPFs) during the progression of pulpitis. METHODS: HIF-1α plasmids or siRNAs were used to upregulate or downregulate HIF-1α in HDPFs, respectively. The effect of hypoxia with or without LPS on the NF-κB signaling and NLRP3 inflammasome pathway was analyzed by immunofluorescence staining, qRT-PCR, western blotting and ELISA. RESULTS: The hypoxic conditions alone induced ASC oligomerization and NLRP3/CASP1 inflammasome pathway activation via NF-κB signaling in a time-dependent manner in HDPFs. The upregulation of HIF-1α further promoted hypoxia-induced ASC oligomerization and NLRP3/CASP1 inflammasome pathway activation via NF-κB signaling compared to the hypoxia-induced group. In comparison, downregulation of HIF-1α inhibited ASC oligomerization and NLRP3/CASP1 inflammasome pathway activation via NF-κB signaling compared to the hypoxia-induced group. Additionally, LPS plus hypoxia further promoted HIF-1α expression and NLRP3/ASC/CASP1 inflammasome pathway activation via NF-κB signaling compared to the hypoxia-induced group. CONCLUSIONS: HIF-1α served as a positive regulator of NLRP3/ASC/CASP1 inflammasome pathway activation via NF-κB signaling in HDPFs in the sterile pulpal inflammation and caries-related pulpitis microenvironment. The finding of a novel functional HIF-1α-NF-κB-NLRP3 axis provides insight into the link between the hypoxic microenvironment and pulpal inflammation, thus supporting a promising therapeutic strategy for the control of pulpal inflammation.
Assuntos
Polpa Dentária , Fibroblastos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Inflamassomos , NF-kappa B , Proteína 3 que Contém Domínio de Pirina da Família NLR , Transdução de Sinais , Humanos , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fibroblastos/metabolismo , NF-kappa B/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamassomos/metabolismo , Hipóxia/metabolismo , Lipopolissacarídeos/farmacologia , Pulpite/metabolismo , Células Cultivadas , Western Blotting , Ensaio de Imunoadsorção EnzimáticaRESUMO
This study aimed to determine the effect of ozone on the expression of Bax and Bcl-2 genes in dental pulp cells. Additionally, the programmed cell death protein 1, programmed death-ligand 1, and CD200 antigens were determined in lymphocytes to assess their surface expression. Dental pulp cells were cultured from extracted healthy third molars and characterized as dental pulp stromal cells. Gene expression of Bcl-2 and Bax was analyzed at 0 s, 6 s, and 12 s of ozone exposure using real-time PCR. Lymphocytes from dental pulp were subjected to ozone exposure for 12 s and PD-1, PD-L1, and CD200/CD200R expression was analyzed by flow cytometry. Upon exposure to ozone for 6 s, the Bcl-2 expression decreased significantly to -0.09, and at 12 s, it increased significantly to 0.3. Bax gene expression level increased significantly to 0.188 after 6 s exposure, and at 12 s, to 0.16. Lymphocytes exposed to ozone for 12 s showed minimal changes in PD-1, PD-L1, and CD200/CD200R expression levels, indicating that oxidative stress does not impact the signaling pathways regulating these molecules. The significant upregulation of Bcl-2 at 12 s highlights the cells' effort to protect themselves from prolonged oxidative stress, possibly tipping the balance toward cell survival and tissue repair. However, the absence of changes in PD-1 and PD-L1 expression on lymphocytes under oxidative stress suggests that these molecules are not sensitive to oxidative stress in this context.
Assuntos
Antígenos CD , Apoptose , Antígeno B7-H1 , Polpa Dentária , Ozônio , Receptor de Morte Celular Programada 1 , Humanos , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Apoptose/efeitos dos fármacos , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/genética , Antígenos CD/metabolismo , Antígenos CD/genética , Células Cultivadas , Estresse Oxidativo , Projetos Piloto , Regulação da Expressão Gênica/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/imunologia , Linfócitos/efeitos dos fármacos , Adulto Jovem , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Adulto , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Influence on stem cells' angiogenesis and osteogenesis of NAD(P)H Quinone Dehydrogenase 1(NQO1) has been established, but its impact on dental pulp stem cells (DPSCs) is unexplored. An important strategy for the treatment of arteriosclerosis is to inhibit calcium deposition and to promote vascular repair and angiogenesis. This study investigated the function and mechanism of NQO1 on angiogenesis and osteogenesis of DPSCs, so as to provide a new ideal for the treatment of arteriosclerosis. METHODS: Co-culture of human DPSCs and human umbilical vein endothelial cells (HUVECs) was used to detect the angiogenesis ability. Alkaline phosphatase (ALP) activity, alizarin red staining (ARS), and transplantation of HA/tricalcium phosphate with DPSCs were used to detect osteogenesis. RESULTS: NQO1 suppressed in vitro tubule formation, migration, chemotaxis, and in vivo angiogenesis, as evidenced by reduced CD31 expression. It also enhanced ALP activity, ARS, DSPP expression and osteogenesis and boosted mitochondrial function in DPSCs. CoQ10, an electron transport chain activator, counteracted the effects of NQO1 knockdown on these processes. Additionally, NQO1 downregulated MAPK signaling, which was reversed by CoQ10 supplementation in DPSCs-NQO1sh. CONCLUSIONS: NQO1 inhibited angiogenesis and promoted the osteogenesis of DPSCs by suppressing MAPK signaling pathways and enhancing mitochondrial respiration.
Assuntos
Polpa Dentária , Células Endoteliais da Veia Umbilical Humana , Sistema de Sinalização das MAP Quinases , NAD(P)H Desidrogenase (Quinona) , Neovascularização Fisiológica , Osteogênese , Humanos , Osteogênese/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , Neovascularização Fisiológica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Técnicas de Cocultura , Células-Tronco/metabolismo , Células-Tronco/citologia , Células Cultivadas , Ubiquinona/análogos & derivados , Ubiquinona/farmacologia , Ubiquinona/metabolismo , Animais , Diferenciação Celular , AngiogêneseRESUMO
BACKGROUND: Although Tideglusib cytotoxicity studies and its effects on human dental pulp-derived stem cells (DPSCs) have been examined in previous studies, there is no study investigating the expression of type 1 collagen and type 3 collagen by Tideglusib. AIM: The purpose of this study is to examine the effect of Wnt signaling activation using Tideglusib execution on human DPSC to determine its potential efficacy in collagen expression. METHODS: Stem cell isolation was performed from five human third molar wisdom tooth pulps. DPSCs identified in only one sample were treated with 50 nM Tideglusib for 24 h and 1 week. Axin-2, type 1 and type 3 collagen expressions were evaluated by Western blot analysis. DPSCs without treatment served as a negative control. The Mann-Whitney U test was used for statistical analysis. RESULTS: The levels of type 1 collagen and Axin-2 in the test group were significantly higher than those in the control group at 24 h (P = 0.000, P = 0.001, respectively). Compared to the control group, a slight increase in type 3 collagen expression was observed in the test group at 24 h (P value = 0.063). Application of 50 nM Tideglusib for 1 week revealed marked decreases in type 1 and type 3 collagen expressions (P = 0.029, P = 0.038, respectively). In contrast, there was a significant increase in the level of Axin-2 (P = 0.000) compared to the control group. CONCLUSION: The fact that Wnt signaling pathway activation obtained by Tideglusib application on DPSCs confirmed by the finding in the increase of Axin-2 at short and long-term evaluation periods which is resulted in the increase in the type 1 collagen expression at 24 h and decrease at 1 week together with the decrease in type 3 collagen expression at 1 week warrants further studies to evaluate the effect of Tideglusib on extracellular matrix expression.
Assuntos
Colágeno Tipo III , Colágeno Tipo I , Polpa Dentária , Células-Tronco , Humanos , Polpa Dentária/citologia , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/metabolismo , Colágeno Tipo I/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Colágeno Tipo III/metabolismo , Proteína Axina/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Células CultivadasRESUMO
Dental pulp inflammation is a common and significant factor related to poor dental prognosis. Current treatment strategies primarily concentrate on managing the inflammatory response, with specific targets for intervention still under investigation. Triggering receptors expressed on myeloid cells (TREMs) are a group of receptor molecules extensively present on myeloid cell surfaces, crucial in the regulation of inflammatory process. Our analysis of transcriptomic sequencing data from clinical pulp samples of dataset GSE77459 and animal models revealed up-regulation of Trem1 during pulpitis. Administration of the Trem1-blocking peptide LP17 led to lower (more than 1-fold) levels of several pro-inflammatory factors and inhibition of M1 macrophage polarization both in vivo and in vitro. This study of the expression patterns and functions of Trem1 in the development of dental pulp inflammation provides novel insights into the therapeutic strategies for clinical pulpitis.
Assuntos
Macrófagos , Pulpite , Receptor Gatilho 1 Expresso em Células Mieloides , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismo , Receptor Gatilho 1 Expresso em Células Mieloides/antagonistas & inibidores , Animais , Pulpite/metabolismo , Macrófagos/metabolismo , Camundongos , Humanos , Modelos Animais de Doenças , Polpa Dentária/metabolismo , Polpa Dentária/citologia , Regulação para CimaRESUMO
In response to pro-inflammatory cytokines such as interleukin (IL)-1ß, dental pulp fibroblasts produce various inflammatory mediators, including IL-6, IL-8, CC chemokine ligand 20 (CCL20), and CXC chemokine ligand 10 (CXCL10), leading to the progression of pulpitis. IL-17/IL-17A (IL-17A) is a pro-inflammatory cytokine secreted by T helper (Th) 17 cells following their recruitment to inflamed sites; however, the roles of IL-17A during pulpitis remain unclear. The purpose of this study was to investigate the effect of IL-17A on IL-6, IL-8, CCL20 and CXCL10 production by human dental pulp fibroblasts (HDPFs) in vitro. IL-17A at a concentration of 100 ng/ml induced the production of 10 times more IL-8 and 4 times more CXCL10, but not IL-6 and CCL20, compared to controls. Co-stimulation of HDPFs with IL-17A and IL-1ß synergistically enhanced the production of IL-6, CCL20, IL-8 and CXCL10. IL-1ß increased expression of IL-17 receptor/IL-17RA (IL-17R) on HDPFs. Moreover, the cell signal pathways of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) were more potently activated by simultaneous stimulation with IL-17A and IL-1ß. These findings suggest that IL-17A participates in the progression of dental pulp inflammation through the enhanced production of inflammatory mediators in HDPFs.
Assuntos
Quimiocina CXCL10 , Polpa Dentária , Fibroblastos , Interleucina-17 , Interleucina-1beta , Interleucina-6 , Interleucina-8 , Humanos , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Polpa Dentária/efeitos dos fármacos , Interleucina-17/farmacologia , Interleucina-17/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Interleucina-1beta/metabolismo , Quimiocina CXCL10/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Mediadores da Inflamação/metabolismo , Quimiocina CCL20/metabolismo , Pulpite/metabolismo , Células Cultivadas , NF-kappa B/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Receptores de Interleucina-17/metabolismoRESUMO
The dental pulp is the only soft tissue structure within the tooth, serving functions such as sensation and nutrition. However, the dental pulp is highly susceptible to necrosis due to external factors. Currently, root canal therapy is the most commonly used treatment for pulp necrosis. Nevertheless, teeth treated with root canal therapy are prone to secondary infections and adverse outcomes like vertical root fractures. Regenerative endodontic therapy has emerged as a solution, aiming to replace damaged tooth structures, including dentin, root structure, and the pulp-dentin complex cells. This approach demonstrates significant advantages in addressing clinical symptoms and achieving regeneration of the root and even the pulp. Since the discovery of dental pulp stem cells, regenerative endodontic therapy has gained new momentum. Advances in cell transplantation and cell homing techniques have rapidly developed, showing promising potential for clinical applications.
Assuntos
Polpa Dentária , Regeneração , Transplante de Células-Tronco , Polpa Dentária/fisiologia , Polpa Dentária/citologia , Humanos , Regeneração/fisiologia , Transplante de Células-Tronco/métodos , Endodontia Regenerativa/métodos , Células-Tronco/citologia , Tratamento do Canal Radicular/métodos , Engenharia Tecidual/métodos , Necrose da Polpa Dentária/terapiaRESUMO
Introduction: Microbial pathogens invade various human organs, including the oral cavity. Candida albicans (C.a) and Streptococcus mutans (S.m) served respectively as representative oral pathogenic fungi and bacteria to stimulate dental pulp stem cells (DPSCs) and to screen the DPSC subcluster that specifically responded to fungal infection. Methods: DPSCs were obtained from the impacted third molars of six healthy subjects. Then, cells were mixed and divided into three samples, two of which were stimulated with C.a and S.m, respectively; the third sample was exposed to cell medium only (Ctrl). Single-cell mRNA sequencing analysis of treated DPSCs was performed. Results: DPSCs were composed of four major clusters of which one, DPSC.7, exhibited unique changes compared to those of other subclusters. The DPSC.7 cell percentage of the C.a sample was twice those of the Ctrl and S.m samples. DPSC.7 cells expressed genes associated with the response to reactive oxygen species (ROS) response. DPSC.7 subgroup cells established characteristic aggregation under the stimulation of different pathogens in UMAP. The MAPK/ERK1/2 and NF-κB pathways were up-regulated, DUSP1/5/6 expressions were suppressed, FOS synthesis was activated, the immune-related pathway was induced, and the levels of cytokines, including IL-6 and CCL2, were up-regulated in DPSC.7 cells when stimulated with C.a. Conclusions: Our study analyzed the cellular and molecular properties of DPSCs infected by oral fungi and bacteria with single-cell RNA sequencing. A subcluster of DPSCs responded specifically to infections with different pathogens, activating the MAPK and NF-κB pathways to induce immune responses via the ROS pathway. This suggests novel treatment strategies for fungal infections.
Assuntos
Candida albicans , Polpa Dentária , Espécies Reativas de Oxigênio , Células-Tronco , Streptococcus mutans , Humanos , Polpa Dentária/microbiologia , Polpa Dentária/citologia , Polpa Dentária/imunologia , Células-Tronco/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Streptococcus mutans/genética , NF-kappa B/metabolismo , Adulto Jovem , Análise de Célula Única , Transdução de Sinais , Células Cultivadas , Citocinas/metabolismoRESUMO
The transplantation of collagen hydrogels encapsulating human dental pulp stem cell (DPSC)-derived chondrogenic cells is potentially a novel approach for the regeneration of degenerated nucleus pulposus (NP) and cartilage. Grafted cell migration allows cells to disperse in the hydrogels and the treated tissue from the grafted location. We previously reported the cell migration in type I and type II hydrogels. It is important to explore further how cell culture time affect the cell motility. In this study, we observed the decreased motility of DPSC-derived chondrogenic cells after culturing for 2 weeks compared with cells cultured for 2 days in these gels. The Alamarblue assay showed the cell proliferation during the two-week cell culture period. The findings suggest that the transitions of cell motility and proliferation during the longer culture time. The result indicates that the early culture stage is an optimal time for cell transplantation. In a degenerated disc, the expression of IL-1ß and TNFα increased significantly compared with healthy tissue and therefore may affect grafted cell migration. The incorporation of IL-1ß and TNFα into the collagen hydrogels decreased cell motility. The study indicates that the control of IL-1ß and TNFα production may help to maintain cell motility after transplantation.
Assuntos
Movimento Celular , Colágeno , Polpa Dentária , Hidrogéis , Células-Tronco , Polpa Dentária/citologia , Humanos , Células-Tronco/citologia , Células-Tronco/metabolismo , Colágeno/metabolismo , Células Cultivadas , Proliferação de Células , Condrogênese , Interleucina-1beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Adulto , Adolescente , Citocinas/metabolismo , Técnicas de Cultura de Células/métodos , Adulto JovemRESUMO
OBJECTIVES: This study sought to determine effects of Thai propolis extract mixed in mineral trioxide aggregate (MTA) on matrix metalloproteinase-2 (MMP-2) expression and its activity in inflamed human dental pulp cells (HDPCs). MATERIALS AND METHODS: Interleukin-1ß-primed HDPCs were treated with either the eluate of MTA mixed with distilled water, of MTA mixed with 0.75 mg/ml of the propolis extract, or of Dycal®, 0.75 mg/ml of the propolis extract, or 0.2% (v/v) of chlorhexidine for 24 or 72 h. The viability of HDPCs was determined by the PrestoBlue® cytotoxic assay. HDPCs' lysates were analyzed for MMP-2 mRNA expression by RT-qPCR, while their supernatants were measured for MMP-2 activity by gelatin zymography. RESULTS: At 24 and 72 h, a non-toxic dose of the propolis extract at 0.75 mg/ml by itself or mixed in MTA tended to reduce MMP-2 expression upregulated by MTA, while it further decreased the MMP-2 activity as compared to that of MTA mixed with distilled water. The MMP-2 activity of interleukin-1ß-primed HDPCs treated with the eluate of the propolis extract mixed in MTA was significantly lower than that of interleukin-1ß-primed HDPCs at 24 h (p=0.012). As a control, treatment with chlorhexidine significantly inhibited MMP-2 expression induced by MTA and MMP-2 activity enhanced by interleukin-1ß (p<0.05). Treatment with Dycal® caused a significant increase in HDPC's death, resulting in a significant decrease in MMP-2 expression and activity (p<0.05). CONCLUSIONS: MTA mixed with Thai propolis extract can reduce MMP-2 mRNA expression and activity when compared to MTA mixed with distilled water in inflamed HDPCs.
Assuntos
Compostos de Alumínio , Compostos de Cálcio , Polpa Dentária , Metaloproteinase 2 da Matriz , Óxidos , Própole , Silicatos , Compostos de Alumínio/farmacologia , Compostos de Cálcio/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Clorexidina/farmacologia , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/citologia , Combinação de Medicamentos , Interleucina-1beta , Teste de Materiais , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Óxidos/farmacologia , Própole/farmacologia , Própole/química , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , RNA Mensageiro/efeitos dos fármacos , Silicatos/farmacologia , Tailândia , Fatores de Tempo , HumanosRESUMO
BACKGROUND: Limited treatment options exist for damaged nerves and despite impressive advances in tissue engineering, scientists and clinicians have yet to fully replicate nerve development and recruitment. Innervation is a critical feature for normal organ function. While most organs are innervated prior to birth, a rare example of postnatal nerve recruitment occurs in the natural development of secondary teeth during adolescence. Many animals undergo postnatal shedding of deciduous teeth with development and eruption of secondary teeth, a process requiring recruitment of nerve and vasculature to each tooth pulp for viability. Here, the investigators created a novel model for the study of postnatal innervation by exploiting the natural phenomenon of tooth-driven nerve recruitment. METHODS: The investigators theorized that developing teeth possess a special capacity to induce innervation which could be harnessed in a clinical setting for nerve regeneration, and hyptothesized that a transplant model could be created to capture this phenomenon. In this descriptive study, a rat model of autologous tooth transplantation and de novo nerve recruitment was developed by surgically transferring whole developing molars to the autologous tibia. RESULTS: Downstream histological analysis performed 6 to 14 weeks after surgery demonstrated integration of molar into tibia in 81% of postoperative rats, with progressive pulpal expression of nerve marker ß-tubulin III suggestive of neuronal recruitment. CONCLUSIONS: These findings provide a novel model for the study of organ transplantation and support the theory that developing dental tissues may retain nerve-inductive properties postnatally.
Assuntos
Transplante Autólogo , Animais , Ratos , Polpa Dentária/inervação , Polpa Dentária/citologia , Dente Molar , Modelos Animais , Regeneração Nervosa/fisiologia , Tíbia/cirurgia , Ratos Sprague-DawleyRESUMO
BACKGROUND: Different materials have been used as wound dressings after vital pulp therapies. Some of them have limitations such as delayed setting, difficult administration, slight degree of cytotoxicity, crown discoloration and high cost. Therefore, to overcome these disadvantages, composite scaffolds have been used in regenerative dentistry. This study aims to construct and characterize the physicochemical behavior of a novel injectable alginate hydrogel loaded with different bioactive glass nanoparticles in various concentrations as a regenerative pulpotomy filling material. METHODS: Alginate hydrogels were prepared by dissolving alginate powder in alcoholic distilled water containing mesoporous bioactive glass nanoparticles (MBG NPs) or boron-doped MBG NPs (BMBG NPs) at 10 and 20 wt% concentrations. The mixture was stirred and incubated overnight in a water bath at 50 0 C to ensure complete solubility. A sterile dual-syringe system was used to mix the alginate solution with 20 wt% calcium chloride solution, forming the hydrogel upon extrusion. Then, constructed hydrogel specimens from all groups were characterized by FTIR, SEM, water uptake percentage (WA%), bioactivity and ion release, and cytotoxicity. Statistical analysis was done using One-Way ANOVA test for comparisons between groups, followed by multiple pairwise comparisons using Bonferroni adjusted significance level (p < 0.05). RESULTS: Alginate/BMBG loaded groups exhibited remarkable increase in porosity and pore size diameter [IIB1 (168), IIB2 (183) (µm)]. Similarly, WA% increased (~ 800%) which was statistically significant (p < 0.05). Alginate/BMBG loaded groups exhibited the strongest bioactive capability displaying prominent clusters of hydroxyapatite precipitates on hydrogel surfaces. Ca/P ratio of precipitates in IIA2 and IIB1 (1.6) were like Ca/P ratio for stoichiometric pure hydroxyapatite (1.67). MTT assay data revealed that the cell viability % of human gingival fibroblast cells have declined with increasing the concentration of both powders and hydrogel extracts in all groups after 24 and 48 h but still higher than the accepted cell viability % of (Ë70%). CONCLUSIONS: The outstanding laboratory performance of the injectable alginate/BMBGNPs (20 wt%) composite hydrogel suggested it as promising candidate for pulpotomy filling material potentially enhancing dentin regeneration in clinical applications.
Assuntos
Alginatos , Materiais Biocompatíveis , Boro , Dentina , Hidrogéis , Nanopartículas , Alginatos/química , Humanos , Boro/química , Materiais Biocompatíveis/química , Dentina/efeitos dos fármacos , Porosidade , Sobrevivência Celular/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Teste de Materiais , Espectroscopia de Infravermelho com Transformada de Fourier , Polpa Dentária/citologia , Polpa Dentária/efeitos dos fármacos , Microscopia Eletrônica de Varredura , Endodontia Regenerativa/métodos , Vidro/química , Fibroblastos/efeitos dos fármacos , Cerâmica/química , Água/químicaRESUMO
Human dental tissue mesenchymal stem cells (DT-MSCs) constitute an attractive alternative to bone marrow-derived mesenchymal stem cells (BM-MSCs) for potential clinical applications because of their accessibility and anti-inflammatory capacity. We previously demonstrated that DT-MSCs from dental pulp (DP-MSCs), periodontal ligaments (PDL-MSCs), and gingival tissue (G-MSCs) show immunosuppressive effects similar to those of BM, but to date, the DT-MSC-mediated immunoregulation of T lymphocytes through the purinergic pathway remains unknown. In the present study, we compared DP-MSCs, PDL-MSCs, and G-MSCs in terms of CD26, CD39, and CD73 expression; their ability to generate adenosine (ADO) from ATP and AMP; and whether the concentrations of ADO that they generate induce an immunomodulatory effect on T lymphocytes. BM-MSCs were included as the gold standard. Our results show that DT-MSCs present similar characteristics among the different sources analyzed in terms of the properties evaluated; however, interestingly, they express more CD39 than BM-MSCs; therefore, they generate more ADO from ATP. In contrast to those produced by BM-MSCs, the concentrations of ADO produced by DT-MSCs from ATP inhibited the proliferation of CD3+ T cells and promoted the generation of CD4+CD25+FoxP3+CD39+CD73+ Tregs and Th17+CD39+ lymphocytes. Our data suggest that DT-MSCs utilize the adenosinergic pathway as an immunomodulatory mechanism and that this mechanism is more efficient than that of BM-MSCs.
Assuntos
5'-Nucleotidase , Adenosina , Apirase , Polpa Dentária , Células-Tronco Mesenquimais , Ligamento Periodontal , Linfócitos T , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/imunologia , Humanos , Adenosina/metabolismo , Polpa Dentária/citologia , Polpa Dentária/imunologia , Polpa Dentária/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , 5'-Nucleotidase/metabolismo , Apirase/metabolismo , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , Trifosfato de Adenosina/metabolismo , Células Cultivadas , Gengiva/citologia , Gengiva/metabolismo , Gengiva/imunologia , Antígenos CD/metabolismo , Imunomodulação , Diferenciação Celular , Proliferação de Células , Dipeptidil Peptidase 4/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Proteínas Ligadas por GPIRESUMO
MicroRNA-27a-5p (miR-27a-5p) was significantly upregulated in dental pulp inflammation, yet its underlying mechanisms remain unclear. This study investigated the effect of miR-27a-5p on the expression of proinflammatory cytokines in human dental pulp cells (hDPCs) stimulated by lipopolysaccharide (LPS). LPS-stimulated hDPCs showed concurrent increases in the expression of miR-27a-5p and proinflammatory cytokines (IL-6, IL-8, and MCP1), and the increased expression was suppressed by NF-κB inhibitor BAY 11-0785. Transfection of the miR-27a-5p mimic downregulated the expression of proinflammatory cytokines, NF-κB activity, and the expression of NF-κB signaling activators (TAB1, IRAK4, RELA, and FSTL1) in LPS-stimulated hDPCs. Luciferase reporter assays revealed that miR-27a-5p bound directly to the 3'-UTR of TAB1. siTAB1 downregulated NF-κB activity and proinflammatory cytokine expression. Downregulation of proinflammatory cytokine expression, NF-κB activity, and NF-κB signaling activator expression (TAB1, IRAK4, and RELA) was also found in LPS-stimulated rat incisor pulp tissue explants following transfection with the miR-27a-5p mimic ex vivo. MiR-27a-5p, whose expression was induced by NF-κB signaling, negatively regulated the synthesis of proinflammatory cytokines via targeting NF-κB signaling. In particular, TAB1, a potent NF-κB activator, was targeted by miR-27a-5p. These results provide insights into the negative regulatory effects of miR-27a-5p, particularly those targeting the TAB1-NF-κB signaling pathway, on pulp inflammation.
Assuntos
Citocinas , Polpa Dentária , Lipopolissacarídeos , MicroRNAs , NF-kappa B , Transdução de Sinais , MicroRNAs/genética , MicroRNAs/metabolismo , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , NF-kappa B/metabolismo , Citocinas/metabolismo , Ratos , Animais , Regulação para Baixo/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Células Cultivadas , Regiões 3' não Traduzidas , Regulação da Expressão Gênica/efeitos dos fármacos , MasculinoAssuntos
Citocinas , Polpa Dentária , Edição de Genes , Organoides , Regeneração , Retina , Células-Tronco , Humanos , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Organoides/metabolismo , Organoides/citologia , Retina/metabolismo , Retina/citologia , Edição de Genes/métodos , Células-Tronco/metabolismo , Células-Tronco/citologia , Regeneração/genética , Citocinas/metabolismo , Animais , Técnicas de Cultura de Células em Três Dimensões/métodosRESUMO
OBJECTIVE: Several materials have been developed to preserve pulp vitality. They should have ideal cytocompatibility characteristics to promote the activity of stem cells of human exfoliated deciduous teeth (SHED) and thus heal pulp tissue. OBJECTIVE: To evaluate the cytotoxicity of different dilutions of bioceramic material extracts in SHED. METHODOLOGY: SHED were immersed in αMEM + the material extract according to the following experimental groups: Group 1 (G1) -BBio membrane, Group 2 (G2) - Bio-C Repair, Group 3 (G3) - MTA Repair HP, Group 4 (G4) - TheraCal LC, and Group 5 (G5) - Biodentine. Positive and negative control groups were maintained respectively in αMEM + 10% FBS and Milli-Q Water. The methods to analyze cell viability and proliferation involved MTT and Alamar Blue assays at 24, 48, and 72H after the contact of the SHED with bioceramic extracts at 1:1 and 1:2 dilutions. Data were analyzed by the three-way ANOVA, followed by Tukey's test (p<0.05). RESULTS: At 1:1 dilution, SHED in contact with the MTA HP Repair extract showed statistically higher cell viability than the other experimental groups and the negative control (p<0.05), except for TheraCal LC (p> 0.05). At 1:2 dilution, BBio Membrane and Bio-C showed statistically higher values in intra- and intergroup comparisons (p<0.05). BBio Membrane, Bio-C Repair, and Biodentine extracts at 1:1 dilution showed greater cytotoxicity than 1:2 dilution in all periods (p<0.05). CONCLUSION: MTA HP Repair showed the lowest cytotoxicity even at a 1:1 dilution. At a 1:2 dilution, the SHED in contact with the BBio membrane extract showed high cell viability. Thus, the BBio membrane would be a new non-cytotoxic biomaterial for SHED. Results offer possibilities of biomaterials that can be indicated for use in clinical regenerative procedures of the dentin-pulp complex.