RESUMO
AIM OF THE STUDY: In vitro evaluation of the effects of plasminogen activator inhibitor-1 (PAI-1) transfected-conditioned media (P-CM) on the differentiation of human periodontal ligament stem cells (hPDLSCs) and human periapical follicular stem cells (hPAFSCs). MATERIALS AND METHODS: The hPDLSCs and hPAFSCs received from impacted third molars were treated with P-CM and viability, as well as differentiation of the cells were evaluated. Plasmids were constructed according to standard techniques, and all sequences were validated by proper enzyme digestion and sequencing. Chinese hamster ovarian (CHO) cells were transfected with pcDNA3.1-hPAI-1 plasmid to obtain P-CM, followed by western blotting and PAI-1-specific ELISA kit to evaluate the proteins of P-CM. The cell viability of hPDLSCs and hPAFSCs were analyzed using MTT assay after 48 h of incubation. Alizarin red S staining was performed to evaluate the differentiation of hPDLSCs and hPAFSCs. The reverse transcription-polymerase chain reaction was used to observe the expression levels of osteogenic/cementogenic marker genes. The human cytokine antibody array was applied for further analysis of cytokine expression in P-CM. RESULTS: P-CM significantly promoted the differentiation of hPDLSCs and hPAFSCs and upregulated the expression of osteogenic/cementogenic marker genes in vitro. Furthermore, rhPAI-1 promoted mineralized nodules formation of hPDLSCs and hPAFSCs, and we identified that other proteins, RANTES and IL-6, were highly expressed in P-CM. CONCLUSIONS: P-CM promoted the differentiation of hPDLSCs and hPAFSCs by upregulating the expression of RANTES and IL-6, and interaction between PAI-1 and RANTES/IL-6 signaling may be involved in P-CM-induced osteogenic/cementogenic differentiation.
Assuntos
Osteogênese , Inibidor 1 de Ativador de Plasminogênio , Células-Tronco/metabolismo , Transfecção/métodos , Animais , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Citocinas/metabolismo , Humanos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Tecido Periapical/citologia , Ligamento Periodontal/citologia , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
OBJECTIVES: To compare the sealing ability and biocompatibility of Biodentine with mineral trioxide aggregate (MTA) when used as root-end filling materials. METHODOLOGY: The Cell Counting Kit-8 (CCK-8) assay was used to compare the cytotoxicity of MTA and Biodentine. Twenty-one extracted teeth with a single canal were immersed in an acidic silver nitrate solution after root-end filling. Then, the volume and depth of silver nitrate that infiltrated the apical portion of the teeth were analyzed using micro-computed tomography (micro-CT). Seventy-two roots from 3 female beagle dogs were randomly distributed into 3 groups and apical surgery was performed. After six months, the volume of the bone defect surrounding these roots was analyzed using micro-CT. RESULTS: Based on the results of the CCK-8 assay, MTA and Biodentine did not show statistically significant differences in cytotoxicity (P>0.05). The volume and the depth of the infiltrated nitrate solution were greater in the MTA group than in the Biodentine group (P<0.05). The volume of the bone defect was larger in the MTA group than in the Biodentine group. However, the difference was not significant (P>0.05). The volumes of the bone defects in the MTA and Biodentine groups were smaller than the group without any filling materials (P<0.05). CONCLUSIONS: MTA and Biodentine exhibited comparable cellular biocompatibility. Biodentine showed a superior sealing ability to MTA in root-end filling. Both Biodentine and MTA promoted periradicular bone healing in beagle dog periradicular surgery models.
Assuntos
Compostos de Alumínio/farmacologia , Compostos de Cálcio/farmacologia , Óxidos/farmacologia , Tecido Periapical/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Materiais Restauradores do Canal Radicular/farmacologia , Tratamento do Canal Radicular/métodos , Silicatos/farmacologia , Cicatrização/efeitos dos fármacos , Adolescente , Animais , Regeneração Óssea/efeitos dos fármacos , Contagem de Células , Células Cultivadas , Cães , Combinação de Medicamentos , Humanos , Masculino , Teste de Materiais , Osteogênese/efeitos dos fármacos , Tecido Periapical/citologia , Tecido Periapical/diagnóstico por imagem , Ligamento Periodontal/diagnóstico por imagem , Reprodutibilidade dos Testes , Fatores de Tempo , Raiz Dentária/diagnóstico por imagem , Raiz Dentária/efeitos dos fármacos , Raiz Dentária/cirurgia , Resultado do Tratamento , Microtomografia por Raio-X , Adulto JovemRESUMO
Biological phenotypes of mesenchymal stem cells (MSCs) are regulated by a series of biochemical elements, including microRNAs, hormones and growth factors. Our previous study illustrated a significant role of miR-141-3p during the osteogenic differentiation of stem cells from apical papilla (SCAPs). Nevertheless, the functions of miR-141-3p in regulating the proliferative ability and senescence of SCAPs have not been determined. This study identified that overexpression of miR-141-3p inhibited the proliferative ability of SCAPs. Meanwhile, the senescence of SCAPs was ahead of time. Conversely, transfection of miR-141-3p inhibitor promoted the proliferative ability of SCAPs and delayed their senescence. Yes-associated protein (YAP) was predicted as the downstream target gene of miR-141-3p by online softwares (miRDB, miRTarBase, miRWalk, and TargetScan), and was further verified by dual-luciferase reporter gene assay. Additionally, knockdown of YAP inhibited the proliferation and accelerated the senescence of SCAPs. Collectively, these findings proposed a novel direction that miR-141-3p impeded proliferative ability and promoted senescence of SCAPs through post-transcriptionally downregulating YAP.
Assuntos
Proteínas de Ciclo Celular/genética , Proliferação de Células/genética , Senescência Celular/genética , Papila Dentária/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/fisiologia , Fatores de Transcrição/genética , Adolescente , Diferenciação Celular/genética , Células Cultivadas , Regulação da Expressão Gênica , Células HEK293 , Humanos , Osteogênese/genética , Tecido Periapical/citologia , Tecido Periapical/metabolismo , Adulto JovemRESUMO
ABSTRACT Objectives: To compare the sealing ability and biocompatibility of Biodentine with mineral trioxide aggregate (MTA) when used as root-end filling materials. Methodology: The Cell Counting Kit-8 (CCK-8) assay was used to compare the cytotoxicity of MTA and Biodentine. Twenty-one extracted teeth with a single canal were immersed in an acidic silver nitrate solution after root-end filling. Then, the volume and depth of silver nitrate that infiltrated the apical portion of the teeth were analyzed using micro-computed tomography (micro-CT). Seventy-two roots from 3 female beagle dogs were randomly distributed into 3 groups and apical surgery was performed. After six months, the volume of the bone defect surrounding these roots was analyzed using micro-CT. Results: Based on the results of the CCK-8 assay, MTA and Biodentine did not show statistically significant differences in cytotoxicity (P>0.05). The volume and the depth of the infiltrated nitrate solution were greater in the MTA group than in the Biodentine group (P<0.05). The volume of the bone defect was larger in the MTA group than in the Biodentine group. However, the difference was not significant (P>0.05). The volumes of the bone defects in the MTA and Biodentine groups were smaller than the group without any filling materials (P<0.05). Conclusions: MTA and Biodentine exhibited comparable cellular biocompatibility. Biodentine showed a superior sealing ability to MTA in root-end filling. Both Biodentine and MTA promoted periradicular bone healing in beagle dog periradicular surgery models.
Assuntos
Humanos , Animais , Masculino , Adolescente , Cães , Óxidos/farmacologia , Tecido Periapical/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Materiais Restauradores do Canal Radicular/farmacologia , Tratamento do Canal Radicular/métodos , Cicatrização/efeitos dos fármacos , Silicatos/farmacologia , Compostos de Cálcio/farmacologia , Compostos de Alumínio/farmacologia , Osteogênese/efeitos dos fármacos , Tecido Periapical/citologia , Tecido Periapical/diagnóstico por imagem , Ligamento Periodontal/diagnóstico por imagem , Fatores de Tempo , Raiz Dentária/cirurgia , Raiz Dentária/efeitos dos fármacos , Raiz Dentária/diagnóstico por imagem , Regeneração Óssea/efeitos dos fármacos , Teste de Materiais , Contagem de Células , Células Cultivadas , Reprodutibilidade dos Testes , Resultado do Tratamento , Combinação de Medicamentos , Microtomografia por Raio-XRESUMO
INTRODUCTION: This study evaluated the expression of CD90 (mesenchymal stem cell) and Sox2 (progenitor stem cell) markers in persistent apical periodontitis (PAP) (n = 16) and primary periapical lesions (PPLs) (n = 10). METHODS: All samples were classified histologically according to the intensity of inflammatory cell infiltrate in the periapical lesion. Immunohistochemistry was used to detect CD90 and Sox2 in PAP and PPLs. The Spearman correlation coefficient and the Mann-Whitney U test were used to analyze data at the 5% significance level. RESULTS: CD90 expression was found in mesenchymal cells and vascular endothelial cells of 68.5% of all cases of PAP. There was no correlation between CD90 expression and histopathological diagnosis (P = .053) or inflammatory cell infiltrate intensity (P = .112). CD90 staining was predominantly found in the vascular endothelial cells of 30% (n = 3) of PPLs. CD90 expression was significantly higher in PAP than in PPLs (Mann-Whitney U test, P < .05). Sox2 expression was found in all cases of PAP. Eventually, all mesenchymal and chronic inflammatory cells exhibited Sox2 expression. There was no correlation between Sox2 expression and histopathological diagnoses (P = .749), inflammatory cell infiltrate intensity (P = .510), or acute or chronic inflammatory cell infiltrate (P = .256). Sox2 expression was found in 100% of PPLs. There was no difference in Sox2 expression between PAP and PPLs (P = .477). CONCLUSIONS: Mesenchymal stem cells may contribute to the immunosuppressive environment in PAP. Additionally, distinct stem cell sources may be associated with the chronic nature of PAP as well as with the development of PPLs.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Periodontite Periapical/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Antígenos Thy-1/metabolismo , Biomarcadores , Células Endoteliais/metabolismo , Periodontite Periapical/patologia , Tecido Periapical/citologia , Tecido Periapical/metabolismo , Tecido Periapical/patologia , Células-TroncoRESUMO
In the process of tooth root development, stem cells from the apical papilla (SCAPs) can differentiate into odontoblasts and form root dentin, however, molecules regulating SCAPs differentiation have not been elucidated. Zinc fingers and homeoboxes 2 (ZHX2) is a novel transcriptional inhibitor. It is reported to modulate the development of nerve cells, liver cells, B cells, red blood cells, and so on. However, the role of ZHX2 in tooth root development remains unclear. In this study, we explored the potential role of ZHX2 in the process of SCAPs differentiation. The results showed that overexpression of ZHX2 upregulated the expression of osteo/odontogenic related genes and ALP activity, inhibited the proliferation of SCAPs. Consistently, ZHX2 knockdown reduced SCAPs mineralization and promoted SCAPs proliferation. These results indicated that ZHX2 plays a critical role in the proliferation and osteo/odontogenic differentiation of SCAPs.
Assuntos
Proteínas de Homeodomínio/metabolismo , Odontogênese/fisiologia , Osteoblastos/citologia , Osteogênese/fisiologia , Tecido Periapical/citologia , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Adolescente , Proliferação de Células/fisiologia , Células Cultivadas , Feminino , Humanos , Masculino , Osteoblastos/fisiologia , Tecido Periapical/fisiologia , Células-Tronco/fisiologiaRESUMO
Regenerative endodontic procedures are stem cell-based treatments for immature teeth with pulp necrosis. The translation of regenerative endodontic procedures into treating mature teeth depends, among other factors, on the availability and delivery of mesenchymal stem cells (MSCs) into the root canal system. The aim of this clinical study was to evaluate whether evoked bleeding from the periapical tissues elicits the influx of MSCs into the root canal system in mature teeth with apical lesions. Participants included in this study (N = 20) were referred for endodontic treatment of mature teeth with apical lesions. Following chemomechanical debridement, intracanal bleeding from the periapical tissues was achieved, and intracanal blood samples were collected. A positive blood aspirate was also collected in the cartridges during local anesthesia. Total RNA was isolated and used as a template in quantitative reverse transcription polymerase chain reactions using MSC-specific arrays. Data were analyzed with the Wilcoxon signed-rank test, and correlation between gene expression and sex or age was tested with Spearman's rank correlation coefficient test. In addition, MSCs were isolated from an intracanal bleeding sample and subjected to flow cytometry and quantitative osteogenesis assay. Last, the presence and distribution of MSCs within periradicular lesions were evaluated with immunohistochemistry (n = 4). The MSC markers CD73, CD90, CD105, and CD146 were significantly upregulated, with median fold change values of 2.9, 31.7, 4.6, and 6.8, respectively. Conversely, the negative marker for MSCs, CD45, was significantly downregulated (median, -2.7). There was no correlation with age, sex, tooth type, or treatment for any of the evaluated genes. Isolated intracanal cells coexpressed MSC markers and demonstrated robust mineralizing differentiation potential. Finally, immunohistochemical analysis revealed that MSCs were found compartmentalized mainly within vasculature structures located in periapical lesions. Collectively, findings indicate that the evoked-bleeding technique delivers MSCs into the root canal system in mature teeth with apical lesions.
Assuntos
Transplante de Células-Tronco Mesenquimais/métodos , Tecido Periapical/citologia , Tratamento do Canal Radicular/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Cavidade Pulpar/cirurgia , Feminino , Citometria de Fluxo , Humanos , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Osteogênese , Periodontite/cirurgia , Reação em Cadeia da Polimerase em Tempo Real , Adulto JovemRESUMO
INTRODUCTION: Previous studies describe contrasting molecular profiles of active and inactive periapical granulomas characterized by distinct expression of cytokines, osteoclastogenic factors, and wound healing markers. Although the molecular mechanisms underlying such a dichotomy remain unknown, in this study we investigated the potential involvement of mesenchymal stem cells (MSCs) in determining human and murine periapical lesion activity and outcomes. METHODS: Periapical granulomas (n = 83) and control samples (n = 24) were comparatively assessed for the expression levels of 11 mesenchymal stem cell (MSC) markers using real-time polymerase chain reaction. Experimental periapical lesions induced in mice were evaluated for MSC marker expression and the effects of AMD3100 treatment on lesion outcomes. RESULTS: MCS marker expression was prevalent in periapical granulomas compared with that in controls, whereas CD29, CD73, CD90, CD146, CD166, NANOG, Stro-1, and CXCR4 expressions were higher in inactive than in active lesions. Experimental periapical lesion inactivity was also associated with an increased expression of MSC markers. The inhibition of MSC mobilization to the periapex by AMD3100 resulted in increased lesion sizes; decreased expression of MSCs and wound healing markers; and increased expression of interleukin 1 beta (IL-17ß), interleukin 17 (IL-17), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and nuclear factor kappa-B ligand (RANKL). CONCLUSIONS: Our results show that MSC markers are overexpressed in inactive human and experimental periapical lesions and that MSC mobilization results in the attenuation of experimental lesion progression associated with immunosuppressive and prohealing mechanisms.
Assuntos
Imunossupressores/farmacologia , Células-Tronco Mesenquimais/fisiologia , Granuloma Periapical/patologia , 5'-Nucleotidase/análise , Molécula de Adesão de Leucócito Ativado/análise , Adulto , Animais , Antígenos de Superfície/análise , Benzilaminas , Biomarcadores/análise , Antígeno CD146/análise , Ciclamos , Modelos Animais de Doenças , Compostos Heterocíclicos/uso terapêutico , Proteínas de Homeodomínio/análise , Humanos , Integrina beta1/análise , Interferon gama/análise , Interleucina-17/análise , Interleucina-1beta/análise , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Pessoa de Meia-Idade , Granuloma Periapical/tratamento farmacológico , Granuloma Periapical/fisiopatologia , Tecido Periapical/citologia , Tecido Periapical/efeitos dos fármacos , Tecido Periapical/fisiologia , Ligante RANK/análise , Receptores CXCR4/análise , Receptores CXCR4/antagonistas & inibidores , Antígenos Thy-1/análise , Fator de Necrose Tumoral alfa/análise , Cicatrização/fisiologiaRESUMO
The dental pulp space can become infected due to a breach in the surrounding hard tissues. This leads to inflammation of the pulp (pulpitis), soft tissue breakdown, and finally to bone loss around the root apex (apical periodontitis). The succession of the molecular events leading to apical periodontitis is currently not known. The main inflammatory mediator associated with neutrophil chemotaxis is interleukin-8 (IL-8), and with bone resorption the dyad of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). The levels of RANKL, OPG and IL-8 were studied in periapical tissue fluid of human teeth (n = 48) diagnosed with symptomatic irreversible pulpitis (SIP) and asymptomatic apical periodontitis (AAP). SIP represents the starting point, and AAP an established steady state of the disease. Periapical tissue fluid samples were collected using paper points and then evaluated using enzyme-linked immunosorbent assays (ELISAs). Target protein levels per case were calibrated against the corresponding total protein content, as determined fluorometrically. RANKL was expressed at significantly higher levels in SIP compared to AAP (P < 0.05), whereas OPG was under the detection limit in most samples. In contrast, IL-8 levels were significantly lower in SIP compared to AAP (P < 0.05). Spearman's correlation analysis between RANKL and IL-8 revealed a significantly (P < 0.05) negative correlation between the two measures (rho = -.44). The results of this study suggest that, in the development of apical periodontitis, periapical bone resorption signaling, as determined by RANKL, occurs prior to inflammatory cell recruitment signaling, as determined by IL-8.
Assuntos
Interleucina-8/metabolismo , Periodontite Periapical/patologia , Tecido Periapical/patologia , Pulpite/patologia , Ligante RANK/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Reabsorção Óssea/patologia , Polpa Dentária/patologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Inflamação/imunologia , Inflamação/patologia , Interleucina-8/biossíntese , Masculino , Pessoa de Meia-Idade , Osteoprotegerina/biossíntese , Osteoprotegerina/metabolismo , Periodontite Periapical/imunologia , Tecido Periapical/citologia , Tecido Periapical/imunologia , Pulpite/imunologia , Ligante RANK/biossíntese , Adulto JovemRESUMO
The standard treatment modality for teeth with irreversibly damaged dental pulp is root canal therapy, which involves complete removal of the soft tissue and obturation with a synthetic material. So far, research studies show that the combination of stem cells with a suitable scaffold material and transplantation into the root canal may result in the generation of pulplike tissue and the formation of tubular dentin. Because of the technical challenges associated with such a procedure, cell-free alternatives that take advantage of the dental pulp's inherent regenerative capacity because of endogenous stem cell populations and bioactive dentin matrix components need to be considered and explored. Following the tissue engineering approach, this includes (1) a bioactive scaffold, (2) growth and differentiation factors from dentin, and (3) the recruitment of stem cells from resident populations within the pulp or from the periapical region. If this concept proved to be successful, cell-free therapies may be a safer, more practical, feasible, and affordable approach to dental pulp regeneration.
Assuntos
Polpa Dentária/fisiologia , Regeneração/fisiologia , Engenharia Tecidual/métodos , Fatores Quimiotáticos/uso terapêutico , Polpa Dentária/citologia , Dentina/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Tecido Periapical/citologia , Tratamento do Canal Radicular/métodos , Células-Tronco/fisiologia , Alicerces TeciduaisRESUMO
INTRODUCTION: Regenerative endodontics is a valuable treatment modality for immature teeth with pulpal necrosis. A common feature in regenerative cases is the use of intracanal medicaments. Although these medicaments are chosen because of their antibacterial properties, their enduring effect on dentin (conditioning) and the subsequent impact on stem cell survival has never been evaluated. In this study, we hypothesized that triple antibiotic paste (TAP), double antibiotic paste (DAP), or Ca(OH)2 has an indirect adverse effect on the survival of stem cells of apical papilla (SCAP) by dentin conditioning. METHODS: Human dentin disks were created with a standardized root canal diameter of 3.2 mm. The disks were then exposed to either TAP or DAP (at concentrations of 1 mg/mL or 1000 mg/mL), Ca(OH)2 (Ultracal), or Hank's balanced salt solution for 7 or 28 days. Next, the medicaments were removed with copious irrigation, followed by placement of SCAP in a Matrigel scaffold in the lumen of the disks. The bioengineered constructs were cultured for 7 days, followed by determination of cellular viability by using the CellTiter-Glo luminescence assay. Data were analyzed using 1-way analysis of variance with Bonferroni post hoc test. RESULTS: Exposure of dentin to TAP or DAP at 1000 mg/mL resulted in no viable SCAP, whereas the use of these medicaments at 1 mg/mL had no adverse effect on cell viability. In contrast, Ca(OH)2 treatment significantly increased SCAP survival and proliferation when compared with the control group. CONCLUSIONS: Dentin conditioning with TAP and DAP at commonly used clinical concentration (approximately 1000 mg/mL) alters dentin in such a way as to prevent SCAP survival. This lethal indirect effect of both TAP and DAP can be largely avoided if these medicaments are used at the 1 mg/mL concentration. Conversely, dentin conditioning with Ca(OH)2 promotes SCAP survival and proliferation.
Assuntos
Antibacterianos/farmacologia , Dentina/efeitos dos fármacos , Tecido Periapical/citologia , Irrigantes do Canal Radicular/farmacologia , Células-Tronco/efeitos dos fármacos , Antibacterianos/administração & dosagem , Materiais Biocompatíveis/química , Hidróxido de Cálcio/farmacologia , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ciprofloxacina/administração & dosagem , Ciprofloxacina/farmacologia , Colágeno/química , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Humanos , Soluções Isotônicas , Laminina/química , Metronidazol/administração & dosagem , Metronidazol/farmacologia , Minociclina/administração & dosagem , Minociclina/farmacologia , Tecido Periapical/efeitos dos fármacos , Proteoglicanas/química , Irrigantes do Canal Radicular/administração & dosagem , Fatores de Tempo , Alicerces Teciduais/químicaRESUMO
Mesenchymal stem cells can be obtained with ease from dental/oral tissue, making them an attractive source of autologous stem cells. They offer a biological solution for restoring damaged dental tissues such as vital pulp engineering, regeneration of periodontal ligament lost in periodontal disease, and for generation of complete or partial tooth structures to form biological implants. Dental mesenchymal stem cells share properties with mesenchymal stem cells from bone marrow and there is a considerable potential for these cells to be used in different stem-cell-based therapies, such as bone and muscle regeneration. In addition, their immunosuppressive-immunomodulatory properties make these cells a suitable source for treating immunodisorders like systematic lupus erythematosus. In addition, gingival tissue might also be a very good source of epithelial cells used in the treatment of severe ocular surface disorders. Being such an accessible source for different stem cells, the tooth and the attached gingival tissue (usually discarded in the clinics) represent an ideal source of autologous or allogeneic stem cells that can be used in the treatment of many clinical conditions in dentistry and medicine.
Assuntos
Células-Tronco/citologia , Dente/citologia , Polpa Dentária/citologia , Regeneração Tecidual Guiada/métodos , Humanos , Tecido Periapical/citologia , Doenças Periodontais/terapia , Ligamento Periodontal/citologia , Transplante de Células-Tronco , Doenças Dentárias/terapia , Dente Decíduo/citologiaRESUMO
INTRODUCTION: We investigated the biological effects of conditioned medium (CM) from periapical follicle cells (PAFCs) of root-developing tooth on the proliferation and differentiation of stem cells from the apical papilla (SCAP) in vitro. METHODS: Human SCAP and PAFCs were isolated and expanded. CM from PAFCs was prepared with the primary cells. Cell cycle analysis, methyl-thiazol-diphenyltetrazolium assay, alkaline phosphatase activity, mineralization behavior, and gene expression of odontoblast phenotype SCAP cultured with or without CM from PAFCs were evaluated. RESULTS: In the CM-treated group, the cell growth, alkaline phosphatase activity, and mineralization of SCAP were up-regulated. The expression of dentin sialophosphoprotein, alkaline phosphatase, and osteocalcin mRNA progressively increased in SCAP treated with CM from PAFCs. CONCLUSIONS: Our findings suggest that CM from PAFCs is able to provide a favorable odontogenic microenvironment to induce differentiation of SCAP along the odontoblast lineage.
Assuntos
Meios de Cultivo Condicionados/farmacologia , Papila Dentária/citologia , Saco Dentário/citologia , Odontogênese/fisiologia , Tecido Periapical/citologia , Células-Tronco/fisiologia , Ápice Dentário/citologia , Adolescente , Fosfatase Alcalina/análise , Calcificação Fisiológica/fisiologia , Técnicas de Cultura de Células , Ciclo Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem da Célula , Sobrevivência Celular/fisiologia , Células Cultivadas , Microambiente Celular/fisiologia , Papila Dentária/efeitos dos fármacos , Saco Dentário/efeitos dos fármacos , Proteínas da Matriz Extracelular/análise , Humanos , Odontoblastos/efeitos dos fármacos , Odontoblastos/fisiologia , Osteocalcina/análise , Tecido Periapical/efeitos dos fármacos , Fenótipo , Fosfoproteínas/análise , Sialoglicoproteínas/análise , Ápice Dentário/efeitos dos fármacos , Regulação para CimaRESUMO
OBJECTIVE: To assess histological features and the expression of STRO-1 and BMP-4 in dental pulp and periapical tissues in vital or necrotic rat immature teeth. DESIGN: The lower left first molars of male Wistar rats ageing four weeks (n=24) had their pulps exposed to the oral environment for 3, 6, 9 and 12 weeks (animals ageing 7, 10, 13 and 16 weeks-old, respectively; n=24). The right lower first molars served as control untouched teeth. After sample harvesting the jaws were dissected and processed for histology and immunodetection of STRO-1 and BMP-4. RESULTS: Necrotic teeth had root development arrested, while control animals showed development of dental tissues. Immunohistochemistry showed that detection of BMP-4 was restricted to vital pulps. For both groups, STRO-1 expression was evident around blood vessels walls. Neither BMP-4 nor STRO-1 was observed in the apical papilla region. CONCLUSION: STRO-1-positive precursor cells were not detected in the apical papilla. BMP-4 expression has not been detected during infection.
Assuntos
Antígenos de Superfície/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Necrose da Polpa Dentária/induzido quimicamente , Polpa Dentária/metabolismo , Células-Tronco Mesenquimais/metabolismo , Tecido Periapical/metabolismo , Ápice Dentário/patologia , Animais , Polpa Dentária/citologia , Necrose da Polpa Dentária/metabolismo , Imuno-Histoquímica , Masculino , Tecido Periapical/citologia , Ratos , Ratos WistarRESUMO
Immunoregulatory mechanisms within periapical lesions (PLs) are as of yet unexplored. Considering the crucial role of DCs in controlling the immune response within PLs, the immunomodulatory properties of mesenchymal stem cells (MSCs), and the colocalization of MSCs and DCs in situ, we wondered whether MSCs from PLs modulate the development and functions of DCs. Using a model of monocyte-derived DCs, we showed that PL-MSCs inhibited differentiation of DCs via soluble factors, of which IL-6 had a minor effect, but did not impair their subsequent maturation induced by pro-inflammatory cytokines. However, upon maturation such DCs favored the production of Th2/Th17 cytokines by allogenic CD4(+) lymphocytes in coculture, compared with mature DCs differentiated without PL-MSCs. PL-MSC-differentiated DCs, cultivated with pro-inflammatory cytokines and PL-MSCs, although phenotypically mature, exhibited poor allostimulatory activity, induced anergy, Th2 polarization, differentiation of suppressive CD4(+) CD25(high) CD39(+) Treg-cell subsets via IDO-1-, ILT-3-, and ILT-4-dependent mechanisms, and increased production of TGF-ß in the coculture. In contrast, DCs cultivated with PL-MSCs only during maturation stimulated proliferation and Th1 polarization of CD4(+) T cells in an IL-12-independent manner. In conclusion, PL-MSCs significantly modulate the development and functions of DCs, depending on the phase of DCs development during which the interaction occurs.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células-Tronco Mesenquimais/imunologia , Tecido Periapical/citologia , Tecido Periapical/imunologia , Separação Celular , Técnicas de Cocultura , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Teste de Cultura Mista de Linfócitos , Monócitos/citologia , Monócitos/imunologia , Doenças Periapicais/imunologiaRESUMO
INTRODUCTION: Under physiological conditions, matrix metalloproteinases (MMPs) are involved in the turnover of periapical tissue, and their activity is tightly regulated by tissue inhibitors of metalloproteinases (TIMPs). Disturbances in the balance between MMPs and TIMPs may result in excessive tissue destruction. In addition, the extracellular metalloproteinase inducer (EMMPRIN) capable of inducing MMPs may also play a role in the pathologic processes. This study aimed to investigate the effects of interleukin (IL)-17 on the mRNA expression and protein production of MMP-1, MMP-2, MMP-9, MMP-13, TIMP-1, and EMMPRIN through human periodontal ligament cells. METHODS: The cells were stimulated with IL-17 (1, 10, and 50 ng/mL) for different time periods. The mRNA levels of MMP-1, MMP-2, MMP-9, MMP-13, TIMP-1, and EMMPRIN were evaluated via quantitative real-time polymerase chain reaction analysis, whereas the protein secretion into the culture medium was assessed via enzyme-linked immunosorbent assay and zymography analysis. RESULTS: IL-17 significantly up-regulated MMP-1 and MMP-13 mRNA expression but down-regulated MMP-2, MMP-9, and TIMP-1 mRNA expression. Furthermore, IL-17 (50 ng/mL) increased the secreted protein level of MMP-1 and MMP-13 and conversely reduced the level of MMP-2, MMP-9, and TIMP-1. However, IL-17 exerted no effect on EMMPRIN mRNA or protein secretion. CONCLUSIONS: This study first reported the ability of IL-17 to regulate MMP and TIMP-1 production through human periodontal ligament cells, a phenomenon that may contribute to periapical tissue destruction.
Assuntos
Basigina/efeitos dos fármacos , Fibroblastos/enzimologia , Interleucina-17/farmacologia , Metaloproteinases da Matriz/efeitos dos fármacos , Ligamento Periodontal/enzimologia , Inibidores de Proteases/análise , Inibidor Tecidual de Metaloproteinase-1/efeitos dos fármacos , Técnicas de Cultura de Células , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Mediadores da Inflamação/farmacologia , Metaloproteinase 1 da Matriz/efeitos dos fármacos , Metaloproteinase 13 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Tecido Periapical/citologia , Tecido Periapical/enzimologia , Ligamento Periodontal/citologia , RNA Mensageiro/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de TempoRESUMO
OBJECTIVE: To isolate and characterize the Beagle stem cells from apical papilla. METHODS: Apical papilla was severed from the end of freshly extracted Beagle's young permanent upper anterior teeth, and digested by collagenase type I and dispase for cell culture. The isolated cells were investigated for stem cell properties by analyzing their colony-forming efficiency, growth characteristics and the expression of mesenchymal stem cell markers; and evaluating their multidifferentiation potentials including osteogenic, adipogenic, and chondrogenic potentials. Additionally, the cells were transplanted subcutaneously into immunocompromised mice to observe the mineral tissue formation. RESULTS: Our study showed that a clonogenic, rapidly proliferative population of cells existed in Beagle's apical papilla, and these cells had a significantly higher colony-forming rate than the stem cells from apical papilla derived from humans (P<0.001). These cells had multilineage differentiation ability including osteogenic, adipogenic and chondrogenic potentials. Mineralized nodules were formed after osteogenic induction, lipid droplets were found after adipogenic induction, and the pellets showed positive immunohistochemical staining for collagen II after chondrogenic induction. These cells also expressed the mesenchymal stem cell markers including STRO-1 and CD146, while negative for CK. Moreover, these cells transplanted with hydroxyapatite in immunocompromised mice could form mineral tissue and pulp-dentin complex-like tissue. CONCLUSION: There are stem cells from the apical papilla which have high proliferation ability and multilineage differentiation potential existing in the Beagle's apical papilla.
Assuntos
Papila Dentária/citologia , Células-Tronco Mesenquimais/citologia , Tecido Periapical/citologia , Engenharia Tecidual , Animais , Células Cultivadas , Cães , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Camundongos SCIDRESUMO
This article focuses on the biological characterization and discussion of the potential application of oral-derived adult stem cells for craniofacial tissue engineering applications. The authors reviewed experimental (in vitro and in vivo) and clinical reports regarding the isolation, characterization, modulation, and translational clinical application of human precursor cell populations derived from postnatal dental tissues. Five different human dental stem/progenitor cell populations have been isolated and characterized. These postnatal populations present mesenchymal stem cell-like characteristics and enjoy forceful capabilities regarding the differentiation into odontogenic/osteogenic lineages, supporting evidence--in preclinical and clinical trials--for the regeneration of oral/dental tissues.
Assuntos
Células-Tronco Adultas , Polpa Dentária/citologia , Ligamento Periodontal/citologia , Engenharia Tecidual , Germe de Dente/citologia , Regeneração Óssea , Humanos , Células-Tronco Mesenquimais/citologia , Odontogênese , Tecido Periapical/citologia , Crânio/cirurgia , Dente Decíduo/citologiaRESUMO
INTRODUCTION: We previously reported the presence of mesenchymal stem/progenitor cells (MSCs) in inflamed pulp tissue. Here we asked whether MSCs also exist in inflamed periapical tissues resulting from endodontic infection. The objectives of this study were to detect the expression of MSC markers in periapical inflammatory tissues and to characterize isolated cells from these tissues. METHODS: Human periapical inflammatory tissues were collected and processed to detect MSC marker expression by immunohistochemistry. Cells were isolated and tested for cell surface marker expression by using flow cytometry and examined for multiple differentiation potential into osteogenic and adipogenic pathways. In vivo formation of mineralized tissues was assessed in a mouse model. RESULTS: Immunohistochemistry showed positive staining for MSC markers STRO-1, CD90, and CD146. Isolated cells at passage 0 appeared as typical fibroblastic cells, and a few cells formed colony-forming unit-fibroblasts (CFU-Fs). After passaging, the CFU-F forming ability diminished dramatically, and the population doubling was up to 26. Flow cytometry data showed that these cells at passage 2 expressed low levels of STRO-1 and CD146 and moderate to high levels of CD90, CD73, and CD105. At passage 6, the levels of these markers decreased. When incubated in specific differentiation medium, cells demonstrated a strong osteogenic but weak adipogenic capacity. After in vivo cell transplantation, mineralized tissues formed in immunocompromised mice. CONCLUSIONS: Human periapical inflammatory tissues expressed MSC markers, suggesting the presence of MSCs. Isolated cells exhibited typical mesenchymal cell immunophenotype with a capacity to form mineralized matrix in vitro and in vivo.
Assuntos
Células-Tronco Mesenquimais , Osteogênese , Periodontite Periapical/patologia , 5'-Nucleotidase , Adipogenia , Adolescente , Adulto , Idoso , Animais , Antígenos CD , Antígenos de Superfície , Antígeno CD146 , Diferenciação Celular , Endoglina , Fibroblastos/transplante , Citometria de Fluxo , Proteínas Ligadas por GPI , Humanos , Imunofenotipagem , Camundongos , Pessoa de Meia-Idade , Tecido Periapical/citologia , Tecido Periapical/patologia , Cultura Primária de Células , Receptores de Superfície Celular , Antígenos Thy-1 , Adulto JovemRESUMO
Multipotent mesenchymal stem cells from bone marrow are expected to be a somatic stem cell source for the development of new cell-based therapy in regenerative medicine. However, dental clinicians are unlikely to carry out autologous cell/tissue collection from patients (i.e., marrow aspiration) as a routine procedure in their clinics; hence, the utilization of bone marrow stem cells seems impractical in the dental field. Dental tissues harvested from extracted human teeth are well known to contain highly proliferative and multipotent stem cell compartments and are considered to be an alternative autologous cell source in cell-based medicine. This article provides a short overview of the ongoing studies for the potential application of dental stem cells and suggests the utilization of 2 concepts in future regenerative medicine: (1) dental stem cell-based therapy for hepatic and other systemic diseases and (2) tooth replacement therapy using the bioengineered human whole tooth, called the "test-tube dental implant." Regenerative therapies will bring new insights and benefits to the fields of clinical medicine and dentistry.