RESUMO
Periodontal disease, a chronic disease caused by bacterial infection, eventually progresses to severe inflammation and bone loss. Regulating excessive inflammation of inflamed periodontal tissues is critical in treating periodontal diseases. The periodontal ligament (PDL) is primarily a connective tissue attachment between the root and alveolar bone. PDL fibroblasts (PDLFs) produce pro-inflammatory cytokines in response to bacterial infection, which could further adversely affect the tissue and cause bone loss. In this study, we determined the ability of Litsea japonica leaf extract (LJLE) to inhibit pro-inflammatory cytokine production in PDLFs in response to various stimulants. First, we found that LJLE treatment reduced lipopolysaccharide (LPS)-induced pro-inflammatory cytokine (interleukin-6 and interleukin-8) mRNA and protein expression in PDLFs without cytotoxicity. Next, we observed the anti-inflammatory effect of LJLE in PDLFs after infection with various oral bacteria, including Fusobacterium nucleatum, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia. These anti-inflammatory effects of LJLE were dose-dependent, and the extract was effective following both pretreatment and posttreatment. Moreover, we found that LJLE suppressed the effect of interleukin-1 beta-induced pro-inflammatory cytokine production in PDLFs. Taken together, these results indicate that LJLE has anti-inflammatory activity that could be exploited to prevent and treat human periodontitis by controlling inflammation.
Assuntos
Anti-Inflamatórios/farmacologia , Fibroblastos/efeitos dos fármacos , Interleucina-1beta/antagonistas & inibidores , Lipopolissacarídeos/antagonistas & inibidores , Litsea/química , Extratos Vegetais/farmacologia , Adulto , Anti-Inflamatórios/química , Dente Pré-Molar/citologia , Dente Pré-Molar/cirurgia , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Fibroblastos/citologia , Fibroblastos/imunologia , Fibroblastos/microbiologia , Fusobacterium nucleatum/química , Fusobacterium nucleatum/crescimento & desenvolvimento , Fusobacterium nucleatum/patogenicidade , Voluntários Saudáveis , Humanos , Interleucina-1beta/farmacologia , Interleucina-6/antagonistas & inibidores , Interleucina-6/biossíntese , Interleucina-6/imunologia , Interleucina-8/antagonistas & inibidores , Interleucina-8/biossíntese , Interleucina-8/imunologia , Lipopolissacarídeos/farmacologia , Dente Molar/citologia , Dente Molar/cirurgia , Ligamento Periodontal/citologia , Ligamento Periodontal/cirurgia , Extratos Vegetais/química , Folhas de Planta/química , Porphyromonas gingivalis/química , Porphyromonas gingivalis/crescimento & desenvolvimento , Porphyromonas gingivalis/patogenicidade , Cultura Primária de Células , Tannerella forsythia/química , Tannerella forsythia/crescimento & desenvolvimento , Tannerella forsythia/patogenicidade , Treponema denticola/química , Treponema denticola/crescimento & desenvolvimento , Treponema denticola/patogenicidadeRESUMO
OBJECTIVE: To compare the proliferation and osteoblastic differentiation of dental pulp stem cell (DPSC) isolated from normal and inflamed pulps of different degrees in Beagle immature premolars, and provide evidence for the use of inflammatory DPSC (IDPSC). METHODS: This study evaluated 14 Beagle's young premolars (21 roots). In the experiment group, irreversible pulpitis was induced by pulp exposure and the inflamed pulps were extracted 2 weeks and 6 weeks after the pulp chamber opening.For the control group, normal pulps were extracted immediately after the exposure. HE staining and real-time PCR were performed to confirm the inflammation. The cells were isolated from the inflamed and normal pulps (IDPSC and DPSC). Cell proliferation and osteoblastic differentiation potentials of the two cells were compared. RESULTS: Inflammation cells infiltration was observed in the inflamed pulps by HE staining. The expression of inflammatory factor was much higher in the 6 week inflamed pulp. IDPSC had higher potential of cell proliferation and osteoblastic differentiation potentials. Furthermore, the osteoblastic differentiation potentials of IDPSC from 2 week inflamed pulp were higher than those from 6 week inflamed pulp. CONCLUSION: The potential of cell proliferation and osteoblastic differentiation of DPSC was enhanced at early stage of irreversible pulpitis, and reduced at late stage in Beagle immature premolars.
Assuntos
Polpa Dentária/citologia , Polpa Dentária/fisiopatologia , Inflamação/fisiopatologia , Pulpite/fisiopatologia , Células-Tronco Adultas/citologia , Células-Tronco Adultas/fisiologia , Animais , Dente Pré-Molar/citologia , Dente Pré-Molar/fisiopatologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Cães , Osteoblastos/citologia , Osteoblastos/fisiologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The intention of this study was to investigate the effect of modified 3D culture conditions on dental pulp cells (DPCs). DPCs were isolated from extracted primary molar, premolar, and wisdom teeth. Tooth samples were divided into three groups as control group; plated into methyl cellulose medium without any supplementation, growth factor (GF) group; supplemented with bone morphogenetic proteins (BMP2, BMP4), transforming growth factor—β1 (TGF—β1) and growth factor+conditioned medium (GF+CM) group; supplemented with both growth factors and pulp conditioned medium. The DPCs were tested for colony forming ability, proliferation capacity and morphology. The highest colony forming ability was detected in the GF and GF+CM groups of DPCs isolated from wisdom teeth. The proliferation capacity was higher in GF+CM group of DPCs isolated from primary molars, and in GF and GF+CM groups of DPCs isolated from wisdom teeth. Scanning electron microscope (SEM) observation of the wisdom teeth samples showed cell—cell interactions in the GF and GF+CM groups. Our results indicate that growth factors and pulp conditioned medium in methyl cellulose culture created proper environment to follow the behavior of dental cells three—dimensionally.
Assuntos
Dente Pré-Molar/citologia , Proteína Morfogenética Óssea 2/farmacologia , Comunicação Celular/fisiologia , Polpa Dentária/citologia , Dente Serotino/citologia , Fator de Crescimento Transformador beta1/farmacologia , Adolescente , Adulto , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Criança , Ensaio de Unidades Formadoras de Colônias , Humanos , Microscopia Eletrônica de Varredura , Adulto JovemRESUMO
PURPOSE: The aim of the study was to compare the viability of periodontal ligament-derived stem/progenitor cells (PDLSCs) from 2 different sources. MATERIALS AND METHODS: Periodontal ligament (PDL) tissue was obtained from 20 surgically extracted human third molars and 20 healthy premolars extracted for orthodontic reasons. Periodontal ligament-derived stem/progenitor cells were isolated from 2 different PDL tissue sources and characterized by colony forming unit assay, cell surface marker characterizations, and their osteogenic differentiation potential. To determine cell viability within 2 groups, the colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) metabolic activity assay was used. Data were statistically analyzed using independent t-test by SPSS 16 software (SPSS Inc, Chicago, IL). RESULTS: According to the MTT assay, the mean viability rate ± standard deviation of PDLSCs in the impacted third molar sample cells was 0.355 ± 0.411 and for erupted premolar sample cells was 0.331 ± 0.556. Based on One-Sample Kolmogorov-Smirnov test, P value for impacted and erupted teeth was 0.954 and 0.863, respectively. No statistical difference was seen between 2 groups. (P value > 0.05) CONCLUSIONS: Our results demonstrated that if surgical aseptic technique is a method employed to maintain asepsis, PDLSCs obtained from impacted and erupted tooth root would have the same viability rate.
Assuntos
Ligamento Periodontal/citologia , Células-Tronco/fisiologia , Raiz Dentária/citologia , Dente Impactado/patologia , Adolescente , Adulto , Dente Pré-Molar/citologia , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Forma Celular , Sobrevivência Celular/fisiologia , Colorimetria/métodos , Corantes , Fibroblastos/fisiologia , Humanos , Células-Tronco Mesenquimais/fisiologia , Dente Serotino/patologia , Osteogênese/fisiologia , Sais de Tetrazólio , Tiazóis , Erupção Dentária/fisiologia , Adulto JovemRESUMO
BACKGROUND: LHX8 (LIM-homeobox gene 8) is known as an important regulating factor in tooth morphogenesis. Odontoma is a mixed odontogenic tumor where epithelium and mesenchyme differentiated together, resulting in anomalous tooth structures. In this study, gene and protein expressions of LHX8 were analyzed in human odontoma-derived mesenchymal cells (HODC) compared to adult dental mesenchymal stem cells (aDSC), as well as morphological and histological characteristics of odontoma were analyzed. METHODS: aDSCs were isolated from normal teeth, and HODCs were isolated from surgically removed odontoma mass. Morphological and histological evaluations were performed to compare between compound odontomas and normal premolars. RT-PCR and real-time PCR were performed to identify LHX8 mRNA expression in the HODCs and aDSCs. LHX8 protein expression levels were observed by immunoblotting and immunofluorescent staining. RESULTS: The compound odontoma was composed of multiple tooth-like structures, which contained disorganized but recognizable enamel matrix, dentin, pulp, and cementum. LHX8 mRNA and LHX8 protein expressions were all higher in HODCs compared to those in aDSCs examined by RT-PCR, immunoblot, and immunofluorescent staining. Especially, real-time PCR showed 2.77-fold higher LHX8 expression in HODCs than in normal periodontal ligament stem cells (PDLSCs), while alveolar bone marrow stem cells (ABMSCs) expressed 0.12-fold LHX8 than PDLSCs. CONCLUSIONS: Based on these observations, LHX8 might play an important role in odontoma formation. This is the first report regarding the comparison of LHX8 expression between HODC and normal aDSCs and its overexpression in human samples. The specific mechanism of LHX8 in odontoma morphogenesis awaits further study.
Assuntos
Células-Tronco Adultas/citologia , Proteínas de Homeodomínio/análise , Células-Tronco Mesenquimais/citologia , Odontoma/patologia , Dente/citologia , Dedos de Zinco , Adolescente , Adulto , Processo Alveolar/citologia , Dente Pré-Molar/citologia , Células da Medula Óssea/citologia , Criança , Cemento Dentário/patologia , Esmalte Dentário/patologia , Polpa Dentária/patologia , Dentina/patologia , Feminino , Imunofluorescência , Humanos , Immunoblotting , Proteínas com Homeodomínio LIM , Masculino , Dente Serotino/citologia , Ligamento Periodontal/citologia , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TranscriçãoRESUMO
OBJECTIVE: Recent studies revealed a highly innervated layer in close proximity to the root surface in the periodontal membrane of human teeth. Persistence of the epithelial cells of Malassez along root surfaces without resorption has also been demonstrated. It is hypothesized that resorption is connected to apoptosis of the epithelial cells of Malassez. The purpose of this study is to localize cells undergoing apoptosis in the periodontal membrane of human primary and permanent teeth. MATERIALS AND METHODS: Human primary and permanent teeth were examined immunohistochemically for apoptosis and epithelial cells of Malassez in the periodontal membrane. All teeth examined were extracted in connection with treatment. RESULTS: Apoptosis was seen in close proximity to the root surface and within the epithelial cells of Malassez. This pattern of apoptotis is similar in the periodontal membrane in primary and permanent teeth. CONCLUSIONS: The inter-relationship between apoptotis and root resorption cannot be concluded from the present study. Apoptosis seen in close proximity to the root surface presumably corresponds to the highly innervated layer of the periodontal membrane. The function of this layer still needs to be elucidated.
Assuntos
Apoptose/fisiologia , Ligamento Periodontal/citologia , Dente Decíduo/citologia , Dente/citologia , Adolescente , Dente Pré-Molar/citologia , Caspase 3/análise , Criança , Células Epiteliais/citologia , Humanos , Imuno-Histoquímica , Inclusão em Parafina , Erupção Ectópica de Dente/patologia , Raiz Dentária/citologia , Adulto JovemRESUMO
This study aimed to assess the viability of dental cells following time-dependent carbamide peroxide teeth-whitening treatments using an in-vitro dentin perfusion assay model. 30 teeth were exposed to 5% or 16% CP gel (4 h daily) for 2-weeks. The enamel organic content was measured with thermogravimetry. The time-dependent viability of human dental pulp stem cells (HDPSCs) and gingival fibroblast cells (HGFCs) following either indirect exposure to 3 commercially available concentrations of CP gel using an in-vitro dentin perfusion assay or direct exposure to 5% H2O2 were investigated by evaluating change in cell morphology and by hemocytometry. The 5% and 16% CP produced a significantly lower (p < 0.001) enamel protein content (by weight) when compared to the control. The organic content in enamel varied accordingly to the CP treatment: for the 16% and 5% CP treatment groups, a variation of 4.0% and 5.4%, respectively, was observed with no significant difference. The cell viability of HDPSCs decreased exponentially over time for all groups. Within the limitation of this in-vitro study, we conclude that even low concentrations of H2O2 and CP result in a deleterious change in enamel protein content and compromise the viability of HGFCs and HDPSCs. These effects should be observed in-vivo.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Polpa Dentária/citologia , Clareadores Dentários/farmacologia , Dente Pré-Molar/citologia , Dente Pré-Molar/efeitos dos fármacos , Peróxido de Carbamida/farmacologia , Células Cultivadas , Esmalte Dentário/citologia , Esmalte Dentário/efeitos dos fármacos , Polpa Dentária/efeitos dos fármacos , Dentina/citologia , Dentina/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Dente Molar/citologia , Dente Molar/efeitos dos fármacosRESUMO
Dental pulp stem cells (DPSCs) are adult multipotent stem cells of neuroectodermal origin; they provide an encouraging perspective in the domain of nerve tissue engineering. DPSCs could be transplanted in biodegradable electrospun neuro-supportive scaffold (optimized in various 3D geometries like coating on the surface of titanium implant, hollow/solid tubes, etc.) for enhanced in vivo recovery of peripheral nerves. Herein, we describe the fabrication of uniform bead-free nanofibrous scaffold which supports DPSCs, proliferation, and their subsequent neural differentiation and thus could be utilized for enhanced regeneration of peripheral nervous system.
Assuntos
Técnicas de Cultura de Células/métodos , Polpa Dentária/citologia , Regeneração Nervosa/fisiologia , Sistema Nervoso Periférico/fisiologia , Células-Tronco/citologia , Alicerces Teciduais/química , Dente Pré-Molar/citologia , Diferenciação Celular , Separação Celular , Humanos , Nanofibras/química , Nanofibras/ultraestrutura , Neurônios/citologiaRESUMO
Impaired periodontal healing is a common complication of diabetes mellitus (DM), frequently related to hyperglycemia. MicroRNAs 221 and 222 have been studied as biomarkers for inflammatory diseases, including diabetes, but their role in the periodontal ligament (PL) is unknown. The effects of high glucose on human PL cells death were studied, as well as the expression of microRNA-221 and microRNA-222, potentially modulated by DM. Cells were obtained from the premolar teeth of young humans and cultured for 7 days under different glucose concentrations (5 or 30 mM). MicroRNAs-221/222 expressions were evaluated by real-time RT-PCR and apoptosis by TUNEL assays. Caspase-3 expression was studied by western blotting and immunocytochemistry. High glucose increased apoptosis and caspase-3 protein expression by about 3×. MicroRNA-221 and microRNA-222 expressions decreased by nearly 40% under high glucose. MicroRNA-221 and microRNA-222 inhibition using antagomiRs increased apoptosis by 2-3×, while the expression of caspase-3, a validated target for these microRNAs, was increased by 50%. The overexpression of both microRNAs using miR mimics in high glucose cells did no effect on apoptosis but increased caspase-3 expression by 30%. In conclusion, high glucose induces apoptosis of human PL cells potentially through a reduction of microRNA-221 and microRNA-222 expression and elevation of caspase-3.
Assuntos
Apoptose , Glucose/metabolismo , MicroRNAs/genética , Ligamento Periodontal/citologia , Adolescente , Dente Pré-Molar/citologia , Caspase 3/metabolismo , Células Cultivadas , Criança , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Reação em Cadeia da Polimerase em Tempo Real , TransfecçãoRESUMO
An acid-base resistant zone (ABRZ) has been shown to be created under a hybrid layer in a self-etching adhesive system at the adhesive/dentin interface. The purpose of this study was to assess the nanostructure of the ABRZ by applying all-in-one adhesive systems. Human premolar dentin was treated with one of two all-in-one adhesive systems; Clearfil Tri-S Bond and G-Bond according to the manufacturers' instructions. After placement of a resin composite, the bonded interface was vertically sectioned and subjected to an acid-base challenge. Following this, the nanostructure of the ABRZ was examined by SEM and TEM. The SEM observations of the adhesive-dentin interface after the acid-base challenge indicated that a hybrid layer less than 1 mum thick was created, and a ABRZ was formed beneath the hybrid layer for each adhesive system. The TEM observations indicated that the ABRZ contained mineral components in both adhesive systems, however, the thickness of the ABRZ was material dependent. The application of the all-in-one adhesive systems created an ABRZ at the underlying dentin, which reinforced normal dentin against dental caries. Therefore, this zone was named 'Super Dentin'. Formation of 'Super Dentin' is a new approach in caries prevention.
Assuntos
Dente Pré-Molar/química , Dente Pré-Molar/citologia , Adesivos Dentinários/química , Dentina/química , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Cimentos de Resina/química , Absorção , Adesividade , Cristalização/métodos , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Teste de Materiais , Tamanho da Partícula , Porosidade , Propriedades de SuperfícieRESUMO
A characteristic feature of mammalian dentition is the evolutionary reduction of tooth number and replacement. Because mice do not replace teeth, here we used Sorex araneus, the common shrew, as a model to investigate the loss of tooth replacement. Historically, shrews have been reported to initiate the development of several, milk or deciduous teeth but these soon become rudimentary and only the replacement teeth erupt. Shrews thus offer a living example of a derived mammalian pattern where the deciduous tooth development is being suppressed. Based on histological and gene expression analyses of serial sections, we suggest that S. araneus has discernible tooth replacement only in the premolar 4 (P4) position. Both generations of teeth express Shh in the enamel knot and in the inner enamel epithelium. Nevertheless, the deciduous P4 (dP4) is reduced in size during embryogenesis and is eventually lost without becoming functional. Analysis of growth shows that P4 replaces the dP4 in a "double-wedge" pattern indicative of competitive replacement where the suppression of the deciduous tooth coincides with the initiation of its replacement. Because activator-inhibitor mechanisms have been implicated in adjacent mouse molars and in transgenic mice with continuous tooth budding, we suggest that evolutionary suppression of deciduous teeth may involve early activation of replacement teeth, which in turn begin to suppress their deciduous predecessors.
Assuntos
Evolução Biológica , Musaranhos/genética , Dente Decíduo , Animais , Dente Pré-Molar/citologia , Dente Pré-Molar/crescimento & desenvolvimento , Dente Pré-Molar/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Imageamento Tridimensional , Hibridização In Situ , Modelos Animais , Odontogênese/genética , Musaranhos/anatomia & histologia , Musaranhos/crescimento & desenvolvimento , Dente/anatomia & histologia , Dente/crescimento & desenvolvimento , Dente/metabolismoRESUMO
Mesenchymal stem cells (MSCs) are adult multipotent stem cells that are capable of self-renewal and differentiation into multiple cell lineages. Methods for cell therapy using MSCs have been developed in equine medicine. Recently, human dental pulp stem cells (DPSCs) have drawn much attention owing to their trophic factor producing ability and minimally invasive collection methods. However, there have been no reports on equine dental pulp-derived cells (eDPCs). Therefore, the aim of this study was to isolate and characterize the eDPCs from discarded wolf teeth. Plastic-adherent spindle-shaped cells were isolated from wolf teeth. The doubling time of the isolated eDPCs was approximately 1 day. Differentiation assays using induction medium eDPCs differentiated into osteogenic, chondrogenic and adipogenic lineages. The eDPCs expressed mesenchymal makers (CD11a/18, CD44, CD90 CD105 and MHC class I and II), but did not express hematopoietic markers (CD34 and CD45). Taken together, the results show that eDPCs can be isolated from discarded wolf teeth, and they satisfy the minimal criteria for MSCs. Thus, these eDPCs can be referred to as equine DPSCs (eDPSCs). These eDPSCs may become a new source for cell therapy.
Assuntos
Dente Pré-Molar/citologia , Polpa Dentária/citologia , Cavalos , Células-Tronco Mesenquimais/citologia , Animais , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , FemininoRESUMO
INTRODUCTION: Apical papilla represents a source of an enriched mesenchymal stem cell (MSC) population (stem cells of the apical papilla [SCAPs]) that modulates root development and may participate in regenerative endodontic procedures in immature teeth with pulp necrosis. The characteristics and phenotype of this tissue in the presence of inflammation are largely unknown. The purpose of this study was to characterize a human apical papilla sample that was isolated from an immature tooth with pulp necrosis and apical periodontitis. METHODS: Inflamed periapical tissue that included part of the apical papilla (apical papilla clinical sample [CS]) was collected from an immature mandibular premolar previously diagnosed with pulp necrosis and apical periodontitis during an apexification procedure. Harvested cells from this tissue (SCAP CS) were compared with inflamed periapical progenitor cells (IPAPCs) and normal SCAP (SCAP-RP89) in flow cytometry and quantitative osteogenesis experiments. Part of the issue was further processed for immunohistochemistry and compared with apical papilla and coronal pulp sections from normal immature teeth as well as inflamed periapical tissues from mature teeth. RESULTS: Similar to SCAP-RP89, 96.6% of the SCAP CS coexpressed the MSC markers CD73, CD90, and CD105, whereas only 66.3% of IPAPCs coexpressed all markers. The SCAP CS showed a significantly greater mineralization potential than both SCAP-RP89 and IPAPCs. Finally, immunohistochemical analysis revealed moderate infiltration of cells expressing the inflammatory markers CD45/68 in the apical papilla CS and prominent CD24, CD105, and von Willebrand factor expression. CONCLUSIONS: Under inflammatory conditions, human apical papilla was found moderately inflamed with retained SCAP vitality and stemness and increased osteogenic and angiogenesis potential.
Assuntos
Papila Dentária/citologia , Necrose da Polpa Dentária/patologia , Células-Tronco Mesenquimais/citologia , Periodontite Periapical/patologia , Ápice Dentário/citologia , Dente Pré-Molar/citologia , Dente Pré-Molar/patologia , Criança , Papila Dentária/patologia , Feminino , Citometria de Fluxo , Humanos , Células-Tronco Mesenquimais/fisiologia , Microscopia Confocal , Células-Tronco/citologia , Células-Tronco/fisiologia , Ápice Dentário/patologiaRESUMO
In the field of oral and maxillofacial surgery, tissue-engineering techniques have been found useful in regenerating lost tissues. Periodontal disease causes severe destruction of periodontal tissue, including the alveolar bone. In this study we attempted to regenerate canine periodontal tissue defects by grafting autologous cultured membrane derived from the periosteum. Under appropriate culture conditions, periosteal cells produce enough extracellular matrix to form sheets. Periosteum specimens were peeled from the mandibular body of adult hybrid dogs and were cultured until cells formed membrane. ALP activity was measured to determine an optimal time for grafting. The cultured periosteum (CP) was grafted and sutured on a mechanically made Class III furcation defect in the 4th mandibular premolars. After 3 months, the samples were harvested and observed radiologically and histologically. In cases of CP, the bone defects were regenerated and filled with newly formed hard tissue, whereas in the controls the defects remained. These results show that our novel treatment is effective in regenerating alveolar bone for the treatment of periodontal disease.
Assuntos
Periodonto , Periósteo , Regeneração , Engenharia Tecidual , Animais , Dente Pré-Molar/anormalidades , Dente Pré-Molar/citologia , Cães , Matriz Extracelular/fisiologia , Feminino , Mandíbula/anormalidades , Mandíbula/citologia , Doenças Periodontais/terapia , Periodonto/citologia , Periodonto/fisiologia , Periósteo/citologia , Periósteo/fisiologia , Regeneração/fisiologia , Técnicas de Cultura de Tecidos , Transplante AutólogoRESUMO
The aim of this study was to evaluate the viability of periodontal ligament cells of avulsed teeth in three different storage media. Forty-five mature premolars extracted for orthodontic therapeutic purposes were randomly and equally divided into three groups according to the storage medium: milk (control), rice water and egg white. After placing extracted teeth for 30 min in storage media, the scrapings of the periodontal ligament (PDL) were collected in Falcon tubes containing collagenase in 2.5 mL of phosphate buffer saline and were incubated for 30 min and centrifuged for 5 min at 800 rpm. Cell viability was analyzed by Trypan blue exclusion. Rice water had a significantly higher number of viable cells compared to egg white and milk. There was no statistically significant difference between egg white and milk. Rice water may be able to maintain PDL cell viability of avulsed teeth better than egg white or milk.
Assuntos
Ligamento Periodontal/citologia , Dente Pré-Molar/citologia , HumanosRESUMO
Microenvironmental conditions can interfere with the functional role and differentiation of mesenchymal stem cells (MSCs). Recent studies suggest that an inflammatory microenvironment can significantly impact the osteogenic potential of periodontal ligament stem cells (PDLSCs), but the precise effects and mechanisms involved remain unclear. Here, we show for the first time that interleukin-1ß (IL-1ß) has dual roles in the osteogenesis of PDLSCs at concentrations ranging from physiologically healthy levels to those found in chronic periodontitis. Low doses of IL-1ß activate the BMP/Smad signaling pathway to promote the osteogenesis of PDLSCs, but higher doses of IL-1ß inhibit BMP/Smad signaling through the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling, inhibiting osteogenesis. These results demonstrate that crosstalk between NF-κB, MAPK and BMP/Smad signaling mediates this dual effect of IL-1ß on PDLSCs. We also show that the impaired osteogenesis of PDLSCs results in more inflammatory cytokines and chemokines being released, inducing the chemotaxis of macrophages, which further clarifies the role of PDLSCs in the pathogenesis of periodontitis.
Assuntos
Proteína Morfogenética Óssea 2/genética , Interleucina-1beta/genética , Células-Tronco Mesenquimais/metabolismo , NF-kappa B/genética , Osteoblastos/metabolismo , Proteína Smad1/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Adolescente , Dente Pré-Molar/citologia , Dente Pré-Molar/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular , Feminino , Regulação da Expressão Gênica , Genes Reporter , Humanos , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , Luciferases/genética , Luciferases/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , NF-kappa B/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , Cultura Primária de Células , Transdução de Sinais , Proteína Smad1/metabolismo , Extração Dentária , Adulto Jovem , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: The critical challenge in tissue engineering is to establish an optimal combination of stem cells, signaling morphogenetic molecules, and extracellular matrix scaffold/microenvironment. The extracellular matrix components of teeth may be reconstituted as an inductive microenvironment in an ectopic tooth transplantation bioassay. Thus, the isolation and identification of the chemical components of the inductive microenvironment in pulp/dentin regeneration will accelerate progress towards the goal of tissue engineering of the tooth. METHODS: The teeth demineralized in 0.6 M hydrochloric acid were sequentially extracted by 4.0 M guanidine hydrochloride (GdnHCl), pH 7.4, and 0.5 M ethylenediaminetetraacetic acid (EDTA), pH 7.4. The extracted teeth were transplanted into an ectopic site in severe combined immunodeficiency (SCID) mice with mobilized dental pulp stem cells (MDPSCs). The unextracted tooth served as a positive control. Furthermore, the soluble components for the inductive microenvironment, the GdnHCl extracts, or the EDTA extracts together with or without MDPSC conditioned medium (CM) were reconstituted systematically with autoclaved teeth in which the chemical components were completely inactivated and only the physical microenvironment was preserved. Their pulp/dentin regenerative potential and angiogenic potential were compared 28 days after ectopic tooth transplantation by histomorphometry and real-time RT-PCR analysis. RESULTS: Expression of an odontoblastic marker, enamelysin, and a pulp marker, thyrotropin-releasing hormone degrading enzyme (TRH-DE), was lower, and expression of a periodontal cell marker, anti-asporin/periodontal ligament-associated protein 1 (PLAP-1), was higher in the transplant of the EDTA-extracted teeth compared with the GdnHCl-extracted teeth. The autoclaved teeth reconstituted with the GdnHCl extracts or the EDTA extracts have weak regenerative potential and minimal angiogenic potential, and the CM significantly increased this potential. Combinatorial effects of the EDTA extracts and the CM on pulp/dentin regeneration were demonstrated in vivo, consistent with their in-vitro effects on enhanced proliferation, migration, and odontoblastic differentiation. CONCLUSIONS: The EDTA-extracted teeth demonstrated significantly lower pulp/dentin regenerative potential compared with the GdnHCl-extracted teeth. The EDTA soluble chemical components when reconstituted with the physical structure of autoclaved teeth serve as an inductive microenvironment for pulp/dentin regeneration, promoting cell proliferation, migration, and odontoblastic differentiation.
Assuntos
Dente Pré-Molar/transplante , Meios de Cultivo Condicionados/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Odontoblastos/efeitos dos fármacos , Aminopeptidases/genética , Aminopeptidases/metabolismo , Animais , Dente Pré-Molar/citologia , Dente Pré-Molar/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Microambiente Celular , Meios de Cultivo Condicionados/isolamento & purificação , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Metaloproteinase 20 da Matriz/genética , Metaloproteinase 20 da Matriz/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos SCID , Odontoblastos/citologia , Odontoblastos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Cultura Primária de Células , Ácido Pirrolidonocarboxílico/análogos & derivados , Ácido Pirrolidonocarboxílico/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Transdução de Sinais , Suínos , Engenharia Tecidual , Transplante HeterólogoRESUMO
Effective pulp-capping materials must have antibacterial properties and induce dentin bridge formation; however, many current materials do not satisfy clinical requirements. Accordingly, the effects of an experiment pulp-capping material (Exp) composed of an antibacterial resin monomer (MAE-DB) and Portland cement (PC) on the viability, adhesion, migration, and differentiation of human dental pulp stem cells (hDPSCs) were examined. Based on a Cell Counting Kit-8 assay, hDPSCs exposed to Exp extracts showed limited viability at 24 and 48 h, but displayed comparable viability to the control at 72 h. hDPSC treatment with Exp extracts enhanced cellular adhesion and migration according to in vitro scratch wound healing and Transwell migration assays. Exp significantly upregulated the expression of osteogenesis-related genes. The hDPSCs cultured with Exp exhibited higher ALP activity and calcium deposition in vitro compared with the control group. The novel material showed comparable cytocompatibility to control cells and promoted the adhesion, migration, and osteogenic differentiation of hDPSCs, indicating excellent biocompatibility. This new direct pulp-capping material containing MAE-DB and PC shows promise as a potential alternative to conventional materials for direct pulp capping.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Cimentos Dentários/farmacologia , Polpa Dentária/efeitos dos fármacos , Metacrilatos/farmacologia , Osteogênese/efeitos dos fármacos , Compostos de Amônio Quaternário/farmacologia , Células-Tronco/efeitos dos fármacos , Adolescente , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Dente Pré-Molar/citologia , Dente Pré-Molar/cirurgia , Bioensaio , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Polpa Dentária/citologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Dente Molar/citologia , Dente Molar/cirurgia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/genética , Osteonectina/genética , Osteonectina/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Cultura Primária de Células , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Células-Tronco/citologia , Extração Dentária , Cicatrização/efeitos dos fármacos , Adulto JovemRESUMO
BACKGROUND: The NLRP3 inflammasome plays an important role in the cellular defense against invading pathogens and is reported to be expressed in human dental pulp fibroblasts (HDPFs). However, the role of the NLRP3 inflammasome in HDPFs during pulpal infection and inflammation remains unclear. OBJECTIVES: To elucidate the function of the NLRP3 inflammasome and the mechanisms that lead to its expression and activation in HDPFs. METHODS: The test model used lipopolysaccharide (LPS) and adenosine triphosphate (ATP) to simulate an inflammatory environment. Lentiviral vectors encoding short hairpin RNAs were used to knock down NLRP3 and caspase-1 in HDPFs. Specific inhibitors were used to determine whether the toll-like receptor 4 (TLR4), myeloid differentiating factor 88 (MyD88), or nuclear factor-kappa B (NF-κB) pathways were involved in the regulation of NLRP3 expression. Reactive oxygen species (ROS) production was measured by fluorescent microscopy and flow cytometry using the total ROS/superoxide detection kit. Gene and protein expression were quantified by real-time polymerase chain reaction and Western blot, while cytokine release was measured by an enzyme-linked immunosorbent assay. RESULTS: LPS up-regulated NLRP3 and IL-1ß expression while ATP induced the activation of caspase-1 and the release of IL-1ß in LPS-primed HDPFs. The knockdown of NLRP3 or caspase-1 expression significantly inhibited IL-1ß secretion. Pretreatment with a TLR4 inhibitor, a MyD88 inhibitory peptide, or an I Kappa B alpha (IκBα) phosphorylation inhibitor significantly inhibited LPS-induced NLRP3 and IL-1ß expression. ATP potently promoted ROS generation in HDPFs; N-acetyl cysteine inhibited ROS production, caspase-1 activation and IL-1ß secretion induced by ATP. CONCLUSIONS: Our results demonstrated that the NLRP3 inflammasome in HDPFs is crucial for IL-1ß secretion in response to LPS plus ATP. LPS engaged the TLR4/MyD88/NF-κB pathway to enhance NLRP3 and pro-IL-1ß expression in HDPFs. ATP promoted the generation of ROS and activated the NLRP3 inflammasome in a ROS-dependent manner.
Assuntos
Proteínas de Transporte/imunologia , Fibroblastos/efeitos dos fármacos , Proteínas I-kappa B/imunologia , Inflamassomos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Fator 88 de Diferenciação Mieloide/imunologia , Receptor 4 Toll-Like/imunologia , Acetilcisteína/farmacologia , Trifosfato de Adenosina/farmacologia , Dente Pré-Molar/citologia , Dente Pré-Molar/efeitos dos fármacos , Dente Pré-Molar/imunologia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Caspase 1/genética , Caspase 1/imunologia , Polpa Dentária/citologia , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/imunologia , Fibroblastos/citologia , Fibroblastos/imunologia , Regulação da Expressão Gênica , Humanos , Proteínas I-kappa B/antagonistas & inibidores , Proteínas I-kappa B/genética , Inflamassomos/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Fator 88 de Diferenciação Mieloide/antagonistas & inibidores , Fator 88 de Diferenciação Mieloide/genética , Inibidor de NF-kappaB alfa , Proteína 3 que Contém Domínio de Pirina da Família NLR , Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/genética , Extração DentáriaRESUMO
Normal human cementum-derived cells (HCDCs), expanded in vitro, formed mineralized matrix when attached to a ceramic carrier and transplanted subcutaneously into immunodeficient mice. The mineralized matrix elaborated by transplanted HCDC exhibited several features identical to cementum in situ and was significantly different from bone deposited by similarly transplanted human bone marrow stromal cells (BMSCs). No bone marrow formation and very few or no tartrate-resistant acid phosphatase (TRAP)-positive cells (osteoclasts and osteoclastic precursors) were found in HCDC transplants. In contrast, in BMSC transplants both hematopoiesis and TRAP-positive cells were routinely observed. Furthermore, compared with BMSC-derived matrix, HCDC-derived matrix was less cellular, numerous empty lacunae were present, and fewer cells were found on the cementum matrix/ceramic carrier interface. The organization of collagen fibers in HCDC-derived matrix, as visualized by using the Picrosirus red staining method, was similar to cementum, with typical unorganized bundles of collagen fibers. In contrast, bone matrix elaborated by transplanted BMSC had lamellar structure, identical to mature bone in situ. Finally, cementocytes embedded in the cementum-like matrix were immunopositive for fibromodulin and lumican, whereas osteocytes within the bonelike matrix were negative. This pattern is consistent with the cementum and bone in situ, respectively. These results indicate that human cementum cells are phenotypically distinct from bone cells and provide further validation of the combined in vitro/in vivo model of human cementogenesis recently developed in our laboratory.