RESUMO
Dental stem cells have many applications in medicine, dentistry and stem cell biology in general due to their easy accessibility and low morbidity. A common surgical manoeuvre after a tooth extraction is the dental socket curettage which is necessary to clean the alveolus and favour alveolar bone healing. This procedure can cause very low morbidity compared to bone marrow collection procedures and the collected material is normally discarded. In order to investigate if the tissue obtained by dental socket curettage after a tooth extraction was a feasible alternative source to isolate human stem cells, we isolated and characterized two different stem cell populations based on STRO-1 and CD146 expression. We were able to collect and grow cells from dental socket of vital and non-vital teeth. Both populations were proliferative, clonogenic and expressed STRO-1, CD146, CD90, NG2, PDGFR-ß, which are markers found in stem cells, presented in vitro multiline-differentiation into osteogenic, chondrogenic, and adipogenic tissue, and in vivo transplanted cells formed mineralized tissue. Interestingly, STRO-1+ clonogenic cells presented better multidifferentiation than CD146+ cells. Our results showed that mesenchymal stem cells can be isolated from the tiny tissue collected by dental socket curettage after vital and non-vital tooth extraction and suggest that STRO-1 is an important marker to be used to sort cells with multidifferentiation capacity.
Assuntos
Diferenciação Celular , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Alvéolo Dental/citologia , Animais , Antígenos de Superfície/análise , Antígeno CD146/análise , Proliferação de Células , Células Cultivadas , Humanos , Separação Imunomagnética , Masculino , Camundongos Endogâmicos BALB C , Camundongos NusRESUMO
O objetivo deste estudo foi investigar o papel do fator de crescimento derivado de plaquetas-BB (PDGF-BB) na concentração de 300ng/ml na taxa de proliferação e adesão de células derivadas da granulação óssea humana a fragmentos radiculares periodontalmente comprometidos. Na primeira etapa do estudo, foi estabelecida cultura primária de células da granulação óssea de dois pacientes adultos, sistemicamente saudáveis, não fumantes. Após a expansão celular, as células foram caracterizadas para determinação do fenótipo por meio de ensaios de viabilidade celular, MTT, ensaio de atividade de fosfatase alcalina, ensaio de mineralização e caracterização imunohistoquímica por meio de citometria de fluxo (segunda etapa). Na terceira etapa do estudo, os efeitos da adição de PDGF-BB recombinante humano na concentração de 300ng/ml na taxa de proliferação e adesão de células derivadas da granulação óssea a superfícies radiculares periodontalmente comprometidas foram investigados. A taxa de proliferação celular estimulada pelo PDGF-BB (grupo teste) ou pelo meio de cultura (grupo controle) foi investigada por meio de contagem de células viáveis nos frascos de cultura após 1, 3, 5 e 7 dias do cultivo celular. Foram obtidos 30 fragmentos dentários a partir de dentes extraídos por razões periodontais. Os fragmentos foram raspados com curetas Gracey e condicionados com solução em gel de EDTA a 24% durante 3 minutos, lavados com solução de soro fisiológico, secos e posicionados em placas de 24 poços. Foram incubadas sobre os fragmentos tratados 1x104 células GO por 24 horas, seguido por fixação e preparo para análise por microscopia eletrônica de varredura (MEV). O número de células aderidas sobre os fragmentos foi analisado nas fotomicrografias. O padrão de crescimento das células GO foi compatível com células ósseas, com modificação do padrão do crescimento com o aumento do número de passagens. Houve atividade de fosfatase alcalina em meio osteogênico e convencional, com pico máximo aos 7 dias e atividade de mineralização estimulada ou não por meio osteogênico, com pico máximo aos 21 dias. A análise por meio de citometria de fluxo demonstrou que as células GO não expressaram CD105 e CD166 na 14a passagem, indicando sua diferenciação celular avançada nesse período. A adição de rhPDGF-BB resultou em mudança na taxa de proliferação celular, observando-se pico máximo de crescimento aos 7 dias, com diferenças estatisticamente significantes (p < 0.005; ANOVA post hoc Tukey) em relação aos períodos de 1, 3 e 5 dias. O ensaio de MTT demonstrou maior viabilidade celular no período de 48 hs, comparativamente aos períodos de 24 e 72 horas, quando a densidade óptica celular diminuiu de forma significativa (p< 0.05; Friedmann pósteste Dunn). No ensaio de adesão celular, pode-se observar que a adição de rhPDGFBB aumentou significativamente o número de células aderidas aos fragmentos dentários (p< 0.05; teste t não pareado com correção Welch), com alteração da morfologia celular. Esses resultados sugerem que as células GO tem características compatíveis com linhagem de células osteoblásticas, de fenótipo mais diferenciado após a 12a passagem. A adição de rhPDGF-BB (300ng/ml) resulta em aumento da taxa de proliferação das células GO e do número de células aderidas a fragmentos radiculares, indicando que, nesta concentração, o fator de crescimento é citocompatível, favorecendo a proliferação e adesão celular.(AU)
The goal of this study was to investigate the effects of recombinant human platelet derived growth factor (rhPDGF-BB) at the concentration of 300ng/ml in the proliferation and adhesion of human bone granulation cells to periodontally diseased root fragments. At the first stage of the study, the granulation tissue existent in healing sockets (21 days after its creation) was collected from two systemically healthy nonsmoking adults to the establishment of primary culture. The in vitro properties of bone granulation (BG) cell lineage were characterized by cell viability, MTT, alkaline phosphatase activity and mineralization assays. The effects of culture medium (control) and rhPGDF-BB 300ng/ml (test) in the proliferation and adhesion of BG cells were investigated. The rate of BG cells proliferation was investigated by the number of viable cells present at 1, 3, 5 and 7 days after platting. Thirty root fragments were obtained from teeth extracted for periodontal reasons. Root fragments were scaled and root planed, conditioned with EDTA 24% for 3 minutes, rinsed in saline solution, air-dryed and positioned in 24-well plates. Each fragment was seeded with 104 BG cells, fixated after 24 hours and prepared for analysis in SEM. The number of cells adhered to the fragments was analysed in photomicrographies. BG cells growth pattern was compatible with osteogenic cell lineage, showing modification with the increasing number of cell passage. GO cells expressed alkaline phosphatase activity in conventional and osteogenic culture medium, with maximum peak at 7 days, as well as mineralization activity stimulated or not by osteogenic or non-osteogenic culture medium, with maximum peak at 21 days. The analysis by flow cytometer showed that BG cells have not expressed CD105 and CD106 at the 14th passage, indicating its advanced cell differentiation. The addition of rhPDGF-BB resulted in modification of proliferation rate, with maximum peak observed at 7 days, significantly different from 1-, 3- and 5-day periods (p< 0.005; ANOVA post hoc Tukey). MTT assay showed greater cell viability after 48 hours than after 24 and 72 hours, when optical density has significantly diminished (p< 0.05; Friedmann post hoc Dunn). At cell adhesion assay, it could be observed that the adhesion of rhPDGF-BB has significantly increased the number of cells adhered to root fragments (p< 0.05; unpaired t test with Welchs correction), and alterations in cell morphology. These results suggest that BG cells present in vitro characteristics compatible with osteoblastic cell lineages, with a more differentiated phenotype after the 12th passage. The addition of rhPDGF-BB (300 ng/ml) results in increase of the rate of BG cell proliferation and in the number of cells adhered to root fragments, indicating that, at this concentration, the growth factor is compatible with BG cells and favors cells proliferation and adhesion.(AU)
Assuntos
Humanos , Masculino , Feminino , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Tecido de Granulação/citologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Raiz Dentária/citologia , Alvéolo Dental/citologia , Análise de Variância , Regeneração Óssea/efeitos dos fármacos , Contagem de Células , Células Cultivadas , Citometria de Fluxo , Imuno-Histoquímica , Microscopia Eletrônica de Varredura , Reprodutibilidade dos Testes , Estatísticas não ParamétricasRESUMO
The aim of the present research was to analyze ultrastructural and immunohistochemical aspects of the alveolar repair after the extraction of molars of alendronate (ALN)-treated rats. Wistar rats received 2.5mg/kg body wt/day of ALN during 14 days previously and 7, 14 and 21 days after the extraction of the second mandibular molar. Specimens were fixed in 2% glutaraldehyde + 2.5% formaldehyde under microwave irradiation, decalcified in 4.13% EDTA and paraffin embedded for TRAP histochemistry and immunohistochemistry for OPN, BSP and endoglin, or embedded in Spurr epoxy resin for TEM analysis. Additional specimens had their soft tissues removed and were processed for scanning electron microscopy. The ALN group presented latent TRAP-positive osteoclasts and nonresorbed alveolar crests with bacterial infection. Mild bone necrosis signs were observed at all time points studied. Ultrastructurally, empty osteocyte lacunae were observed and bone trabeculae surface presented hyalinized aspect. A significant delay in alveolar repair occurred, as well as decreased angiogenesis. ALN treatment provoked mild signs of bone necrosis, despite the high dose employed. The present findings add new information about the ultrastructural aspect of the early repair of rats under ALN treatment and highlight for giving attention when oral surgeries are performed in patients using this drug.
Assuntos
Alendronato/administração & dosagem , Conservadores da Densidade Óssea/administração & dosagem , Extração Dentária , Alvéolo Dental/citologia , Alvéolo Dental/fisiologia , Cicatrização , Animais , Histocitoquímica , Imuno-Histoquímica , Microscopia Eletrônica de Varredura , Ratos WistarRESUMO
A óxido nítrico sintase induzível (iNOS) é uma enzima responsável pela síntese do óxido nítrico, envolvido na regulação de vários processos fisiológicos, com destaque para relevantes efeitos sobre o tecido ósseo. Entretanto, o papel da iNOS no reparo ósseo alveolar permanece pouco conhecido. Assim, o objetivo deste estudo foi caracterizar o papel da iNOS no processo de reparo ósseo alveolar pósexodontia em condições homeostáticas (controle) e infecciosas em camundongos. Para isso, foram utilizados animais das linhagens WT (C57Bl/6) e iNOSKO, divididos em 2 grupos, condição homeostática (exodontia do incisivo superior direito) e alveolite experimental (induzida pela inoculação de uma suspensão de cultura bacteriana), e analisados de acordo com os diferentes períodos experimentais (0, 7, 14 e 21 dias pós-exodontia), através da análise histomorfométrica e molecular. Na análise histomorfométrica, avaliou-se a densidade de volume das estruturas referentes ao coágulo sanguíneo, tecido conjuntivo e tecido ósseo. Na análise molecular, quantificou-se a expressão de mRNA codificando genes de marcadores do metabolismo ósseo; marcadores de osteoclastos; citocinas e quimiocinas, através de reações de RealTimePCR. A expressão de iNOS esteve presente durante todo o processo de reparo ósseo alveolar nos camundongos C57Bl/6, porém de maneira mais expressiva no período de 7 dias pós-exodontia, e se mostrou aumentada pela indução de alveolite. A análise histomorfométrica demonstrou discretas alterações no processo de reparo ósseo alveolar, na ausência de iNOS, tanto em condições homeostáticas quanto infecciosas. Em condições homeostáticas, a ausência de iNOS não impactou de forma significativa o processo de reparo ósseo alveolar pósexodontia, mas se mostrou associada à modulação de vasos sanguíneos. Já em condições infecciosas, a ausência de iNOS se mostrou associada à modulação de células inflamatórias, osteoblastos e osteoclastos. De forma geral, conclui-se que embora...
The inducible nitric oxide synthase (iNOS) is an enzyme responsible for the synthesis of nitric oxide, a reactive molecule involved in the regulation of several physiological processes, highlighting relevant effects on bone tissue. However, the role of iNOS in alveolar bone repair remains unknown. The aim of this study was to characterize the role of iNOS in alveolar bone healing process after dental extraction in homeostatic and infectious conditions in mice. With this aim, WT (C57Bl/6) and iNOSKO mice strains were divided into 2 groups, homeostatic condition (extraction of the upper right incisor) and experimental alveolitis (induced by inoculation of a suspension of bacterial culture), and analyzed according to the different experimental periods (0, 7, 14 and 21 days post-extraction), through molecular and histomorphometric analysis. In histomorphometric analysis, the volume density of structures related to blood clot, tissue and bone were evaluated. The molecular analysis quantified the expression of mRNA encoding genes of defined as bone metabolism markers; osteoclast markers, as well cytokines and chemokines through RealTimePCR reactions. The expression of iNOS was detected during the all process of the alveolar bone repair in C57Bl/6 mice, with an expression peak at 7 days post-extraction time point, and was significantly enhanced by alveolitis induction. Histomorphometric analysis showed small changes in alveolar bone repair process in the absence of iNOS, in both homeostatic as infectious conditions. Under homeostatic conditions, the absence of iNOS did not impact significantly the process of alveolar bone healing after dental extraction, but it was associated with modulation of blood vessels formation. In the infectious conditions scenario, the absence of iNOS was associated with modulation of inflammatory cells, osteoblasts and osteoclasts counts. In general, it is concluded that even the enzyme iNOS module some aspects of alveolar bone...
Assuntos
Animais , Masculino , Camundongos , Alvéolo Dental/patologia , Homeostase/fisiologia , Óxido Nítrico Sintase/fisiologia , Regeneração Óssea/fisiologia , Alvéolo Seco/patologia , Alvéolo Dental/citologia , Citocinas/fisiologia , Fibroblastos/patologia , Biomarcadores , Reação em Cadeia da Polimerase , Fatores de TempoRESUMO
O objetivo deste estudo foi estabelecer cultura primária de células derivadas da granulação óssea (GO) de seres humanos para determinar seu padrão de crescimento in vitro e determinar os efeitos biológicos de três membranas reabsorvíveis feitas de colágeno (BioGide®, GenDerm®, CollaTape®) em culturas de fibroblastos gengivais humanos (FGH) e células da granulação óssea (GO). Foram coletadas amostras de tecido ósseo presente no alvéolo de cicatrização de dois pacientes adultos saudáveis sistemicamente com indicação de cirurgia periodontal regenerativa pela técnica do enxerto ósseo em neoformação. Imediatamente após a coleta, as amostras foram transportadas ao laboratório de cultura de células para estabelecimento da cultura primária. As células foram cultivadas em atmosfera úmida, contendo 5% CO2 a 37oC. A curva de crescimento das células foi determinada por meio de contagem de células viáveis. Após a caracterização da curva de crescimento, foram realizadas a caracterização da amostra por meio de determinação da atividade de fosfatase alcalina e de mineralização. Posteriormente, os efeitos de três diferentes tipos de membranas colágenas sobre a proliferação de células GO e FGH foram investigados por meio do teste MTT. As amostras foram divididas em oito grupos: (1) células FGH em meio DMEM (C-FGH); (2) células FGH em meio DMEM condicionado com membrana GenDerm (GD-FGH); (3) células FGH em meio DMEM condicionado com membrana BioGide (BG-FGH); (4) células FGH em meio DMEM condicionado com membrana ColaTape (CT-FGH); (5) células GO em meio DMEM (C-GO); (6) células GO em meio DMEM condicionado com membrana GenDerm (GD-GO); (7) células GO em meio DMEM condicionado com membrana BioGide (BG-GO); (8) células GO em meio DMEM condicionado com membrana CollaTape (CT-GO). O teste de proliferação celular mostrou que houve aumento significativo (p< 0.05; ANOVA para medidas repetidas) do número de células vitais presentes na cultura nos dias 3 (90,8%)...
The aim of this study was to establish primary culture of cells derived from human bone granulation tissue (GO) in order to determine its growth pattern in vitro and the biological effects of three absorbable collagen membranes (BioGide®, GenDerm®, CollaTape®) in human gingival fibroblasts (FGH) and human bone granulation (GO) cell cultures. Samples of bone tissue present at healing sockets of two systemically healthy adults with indication of periodontal regenerative therapy by the newly forming bone were collected. Immediately after, samples were transported to the laboratory of cell culture to the establishment of primary cultures. Cells were cultivated in humid atmosphere with 95% CO2 at 37oC. Cells growth pattern were determined by counting of viable cells. After characterization of growth pattern, samples were characterized according to alkaline phosphatase activity and mineralization detected by alizarin red. Afterwards, the effects of three different types of collagen membranes on GO and FGH cells were investigated by MTT test. Samples were divided into eight groups: (1) FGH cells in DMEM (C-FGH); (2) FGH in DMEM conditioned by GenDerm® membrane (GD-FGH); (3) FGH in DMEM conditioned with BioGide® (BG-FGH); (4) FGH in DMEM conditioned by CollaTape® (CT-FGH); (5) GO cells in DMEM (C-GO); (6) GO cells in DMEM conditioned by GenDerm® (GD-GO); (7) GO cells in DMEM conditioned by BioGide® (BG-GO); (8) GO cells in DMEM conditioned by CollaTape® (CT-GO). Cell proliferation test showed a significant (p< 0.05; ANOVA for repeated measures) increase in the number of vital cells present in the culture at days 3 (90.8%), 5 (132.50%), 7 (137.50%) and 10 (227.50%) compared to control (dia 0). It was observed alkaline phosphatase activity and mineralization in vitro. There was an increase in the number of FGH and GO viable cells at all groups (p< 0.05; ANOVA for repeated measures). Greater proliferative effect at FGH and GO cells at GD and CT...
Assuntos
Humanos , Alvéolo Dental/citologia , Colágeno/química , Materiais Biocompatíveis/química , Proliferação de Células , Fosfatase Alcalina , Sobrevivência Celular , Membranas , Fatores de TempoRESUMO
This study investigated the response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane. Osteoblastic cells were cultured in osteogenic conditions either on P(VDF-TrFE)/BT or polytetrafluoroethylene (PTFE) for up to 14 days. At 7 and 14 days, the mRNA expression of Runt-related transcription factor 2 (RUNX2), Type I collagen (COL I), Osteopontin (OPN), Alkaline phosphatase (ALP), Bone sialoprotein (BSP), and Osteocalcin (OC), key markers of the osteoblastic phenotype, and of Bcl2-associated X protein (Bax), B-cell CLL/lymphoma 2 (Bcl-2), and Survivin (SUR), associated with the control of the apoptotic cell death, was assayed by real-time PCR. In situ ALP activity was qualitatively evaluated by means of Fast red staining. Surface characterization was also qualitatively and quantitatively assayed in terms of topography, roughness, and wettability. Cells grown on P(VDF-TrFE)/BT exhibited a significantly higher mRNA expression for all markers compared to the ones on PTFE, except for Bcl-2, which was not detected for both groups. Additionally, Fast red staining was noticeably stronger in cultures on P(VDF-TrFE)/BT at 7 and 14 days. At micron- and submicron scale, SEM images and roughness analysis revealed that PTFE and P(VDF-TrFE)/BT exhibited a smooth topography and a similar roughness, respectively. PTFE membrane displayed higher contact angles compared with P(VDF-TrFE)/BT, as indicated by wettability assay. The novel P(VDF-TrFE)/BT membrane supports the acquisition of the osteoblastic phenotype in vitro, while up-regulating the expression of apoptotic markers. Further in vivo experiments should be carried out to confirm the capacity of P(VDF-TrFE)/BT membrane in promoting bone formation in guided bone regeneration.
Assuntos
Compostos de Bário/química , Osso e Ossos/efeitos dos fármacos , Membranas Artificiais , Polivinil/química , Titânio/química , Alvéolo Dental/citologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Compostos de Bário/farmacologia , Regeneração Óssea/efeitos dos fármacos , Regeneração Óssea/genética , Regeneração Óssea/fisiologia , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes bcl-2/efeitos dos fármacos , Regeneração Tecidual Guiada , Humanos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Polivinil/farmacologia , Titânio/farmacologiaRESUMO
The enamel-related periodontium (ERP) in rat incisors is related to bone resorption. In these teeth the face of the socket related to the enamel is continuously removed at the inner side and newly formed at the outer side. CSF-1, RANKL and OPG are regulatory molecules essential for osteoclastogenesis. To verify the effects of impeded eruption on bone remodeling, the tooth eruption was prevented by immobilization of lower rat incisor and CSF-1, RANKL and OPG distribution in the ERP was analyzed after 18 days of immobilization and in normal eruption. The region of the alveolar crest of the rat incisor was used. Immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) were performed. The immunostaining of the dental follicle was quantified using Leica QWin software. Positive-TRAP osteoclasts were counted, and both groups were compared. In the normal incisor, the number of osteoclasts was significantly greater than in the immobilized tooth. In the dental follicle, there was no significant difference in the immunostaining intensity for CSF-1 and OPG between the groups (p > 0.05), but for RANKL the immobilized incisor group showed immunostaining intensity smaller than the normal incisor group (p < 0.01). These findings suggest that changes in the ERP, in the immobilized incisor, modify the RANKL/OPG ratio, in the presence of CSF-1, altering the metabolism of cells that participate in the bone remodeling.