RESUMO
Regenerative approaches using mesenchymal stem cells (MSCs) have been evaluated to promote the complete formation of all missing periodontal tissues, e.g., new cementum, bone, and functional periodontal ligaments. MSCs derived from bone marrow have been applied to bone and periodontal defects in several forms, including bone marrow aspirate concentrate (BMAC) and cultured and isolated bone marrow mesenchymal stem cells (BM-MSCs). This study aimed to evaluate the periodontal regeneration capacity of BMAC and cultured BM-MSCs in the wound healing of fenestration defects in rats. METHODOLOGY: BM-MSCs were obtained after bone marrow aspiration of the isogenic iliac crests of rats, followed by cultivation and isolation. Autogenous BMAC was collected and centrifuged immediately before surgery. In 36 rats, fenestration defects were created and treated with suspended BM-MSCs, BMAC or left to spontaneously heal (control) (N=6). Their regenerative potential was assessed by microcomputed tomography (µCT) and histomorphometry, as well as their cell phenotype and functionality by the Luminex assay at 15 and 30 postoperative days. RESULTS: BMAC achieved higher bone volume in 30 days than spontaneous healing (p<0.0001) by enhancing osteoblastic lineage commitment maturation, with higher levels of osteopontin (p=0.0013). Defects filled with cultured BM-MSCs achieved higher mature bone formation in early stages than spontaneous healing and BMAC (p=0.0241 and p=0.0143, respectively). Moreover, significantly more cementum-like tissue formation (p<0.0001) was observed with new insertion of fibers in specimens treated with BM-MSCs within 30 days. CONCLUSION: Both forms of cell transport, BMAC and BM-MSCs, promoted bone formation. However, early bone formation and maturation were achieved when cultured BM-MSCs were used. Likewise, only cultured BM-MSCs were capable of achieving complete periodontal regeneration with inserted fibers in the new cementum-like tissue.
Assuntos
Medula Óssea , Células-Tronco Mesenquimais , Animais , Células da Medula Óssea , Regeneração Óssea , Ligamento Periodontal , Ratos , Microtomografia por Raio-XRESUMO
Abstract Regenerative approaches using mesenchymal stem cells (MSCs) have been evaluated to promote the complete formation of all missing periodontal tissues, e.g., new cementum, bone, and functional periodontal ligaments. MSCs derived from bone marrow have been applied to bone and periodontal defects in several forms, including bone marrow aspirate concentrate (BMAC) and cultured and isolated bone marrow mesenchymal stem cells (BM-MSCs). This study aimed to evaluate the periodontal regeneration capacity of BMAC and cultured BM-MSCs in the wound healing of fenestration defects in rats. Methodology: BM-MSCs were obtained after bone marrow aspiration of the isogenic iliac crests of rats, followed by cultivation and isolation. Autogenous BMAC was collected and centrifuged immediately before surgery. In 36 rats, fenestration defects were created and treated with suspended BM-MSCs, BMAC or left to spontaneously heal (control) (N=6). Their regenerative potential was assessed by microcomputed tomography (µCT) and histomorphometry, as well as their cell phenotype and functionality by the Luminex assay at 15 and 30 postoperative days. Results: BMAC achieved higher bone volume in 30 days than spontaneous healing (p<0.0001) by enhancing osteoblastic lineage commitment maturation, with higher levels of osteopontin (p=0.0013). Defects filled with cultured BM-MSCs achieved higher mature bone formation in early stages than spontaneous healing and BMAC (p=0.0241 and p=0.0143, respectively). Moreover, significantly more cementum-like tissue formation (p<0.0001) was observed with new insertion of fibers in specimens treated with BM-MSCs within 30 days. Conclusion: Both forms of cell transport, BMAC and BM-MSCs, promoted bone formation. However, early bone formation and maturation were achieved when cultured BM-MSCs were used. Likewise, only cultured BM-MSCs were capable of achieving complete periodontal regeneration with inserted fibers in the new cementum-like tissue.
RESUMO
Parathyroid hormone (PTH) plays a key role in the development and homeostasis of mineralized tissues such as bone and dentine. We have reported that PTH (1-34) administration can increase dentine formation in mice and that this hormone modulates in vitro mineralization of odontoblast-like cells. The purpose of the present study was to investigate whether PTH (1-34) participates in the proliferative and apoptotic signaling of odontoblast-like cells (MDPC23). MDPC23 cells were exposed to 50 ng/ml hPTH (1-34) or vehicle for 1 (P1), 24 (P24), or 48 (P48) hours, and the cell proliferation, apoptosis, and cell number were evaluated. To examine whether changes in the proliferative and apoptotic signaling in response to PTH involve protein kinases A (PKA) and/or C (PKC), MDPC23 cells were exposed to PTH with or without PKC or PKA signaling pathway inhibitors. Overall, the results showed that the PKA pathway acts in response to PTH exposure maintaining levels of cell proliferation, while the PKC pathway is mainly involved for longer exposure to PTH (24 or 48 h), leading to the reduction of cell proliferation and increase of apoptosis. The exposure to PTH reduced the cell number in relation to the control group in a time-dependent manner. In conclusion, PTH modulates odontoblast-like cell proliferative and apoptotic response in a time-dependent manner. Both PKC and PKA pathways participate in PTH-induced modulation in an antagonist mode.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Odontoblastos/metabolismo , Hormônio Paratireóideo/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais/fisiologia , Animais , Apoptose/fisiologia , Linhagem Celular , Proliferação de Células/fisiologia , Humanos , CamundongosRESUMO
Parathyroid hormone participates in the metabolism of mineralized tissue. Its role in the formation of dentine is, as yet, incompletely understood. In the present study we analyzed the effect of transient (1 and 24-h/cycle) or continuous hPTH (1-34) treatment in odontoblast-like cells (MDPC-23) to the following parameters: mineral deposition detected by alizarin red, mRNA expression of the type I collagen (COL1), alkaline phosphatase (ALP), biglycan (BGN), matrix metalloproteinase 2 (MMP-2) and dentine sialophosphoprotein (DSPP) quantified by qRT-PCR. MMP-2 and ALP activities were quantified by zymography and colorimetric assay, respectively. The results showed that compared to Control group: intermittent PTH administration (1 and 24-h/cycle) decreased the mineral deposition and ALP activity. DSPP gene expression was not detected in both control and PTH treated cells. The PTH administration for 24-h/cycle increased the ALP, BGN and COL1 mRNA expression and continuous PTH treatment increased BGN and COL1 mRNA expression. Zymography assays showed that compared to Control group: PTH treatment for 1-h/cycle increased the total MMP-2 secretion and the continuous treatment decreased the secreted levels of MMP-2 active-form. Taken together, the results shown that PTH may regulate the odontoblast-like cells-induced secretion, and potentially this hormone can affect in vivo odontoblasts functions.
Assuntos
Odontoblastos/efeitos dos fármacos , Hormônio Paratireóideo/administração & dosagem , Fosfatase Alcalina/efeitos dos fármacos , Animais , Antraquinonas , Biglicano/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/efeitos dos fármacos , Colorimetria , Corantes , Esquema de Medicação , Proteínas da Matriz Extracelular/efeitos dos fármacos , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Camundongos , Hormônio Paratireóideo/farmacologia , Fosfoproteínas/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialoglicoproteínas/efeitos dos fármacos , Sais de Tetrazólio , TiazóisRESUMO
AIM: This investigation evaluated the bone healing in peri-implant defects treated with periosteum-derived cells (PCs) and guided bone regeneration (GBR). MATERIAL AND METHODS: PCs were harvested from six beagle dogs and characterized in vitro with regard to their osteogenic properties. The animals were subjected to teeth extraction in the mandible, and after 3 months of healing, implant sites were drilled, bone dehiscences were created and implants were placed. Dehiscences were randomly assigned to: PCs+GBR, GBR, PCs and non-treated defects. After 3 months, the implants/adjacent tissues were processed. Bone-to-implant contact (BIC) bone fill (BF) within implant threads, and bone area (BA) in a zone lateral to the implant were obtained. RESULTS: In vitro analyses confirmed the osteogenic potential of PCs. Histometrically, no statistically significant differences were observed among the PCs+GBR, GBR and PCs groups for both BF and BIC (p>0.05), whereas these groups showed statistically higher values, as compared with the non-treated group (p<0.05). With respect to BA, the PCs+GBR and GBR groups presented significantly higher means, as compared with the PCs and non-treated groups (p<0.05). CONCLUSION: Although successful outcomes have been promoted by using the combined approach, PCs in conjunction with membranes did not provide additional benefit during peri-implant bone regeneration, when compared with the therapeutic approaches used alone.
Assuntos
Perda do Osso Alveolar/cirurgia , Regeneração Óssea/fisiologia , Implantes Dentários , Regeneração Tecidual Guiada Periodontal/métodos , Mandíbula/cirurgia , Periósteo/transplante , Processo Alveolar/patologia , Animais , Diferenciação Celular/fisiologia , Separação Celular , Implantação Dentária Endóssea , Cães , Mandíbula/patologia , Membranas Artificiais , Microscopia Eletrônica de Varredura , Osseointegração/fisiologia , Osteogênese/fisiologia , Periósteo/citologia , Distribuição Aleatória , Engenharia Tecidual/métodos , Alicerces Teciduais , Coleta de Tecidos e Órgãos , Transplante Autólogo , Resultado do TratamentoRESUMO
BACKGROUND: Human postnatal stem cells have been identified in periodontal ligaments (PDLs). In this study, the in vitro biologic properties of CD105(+) enriched cell subsets from PDLs harvested from deciduous (DePDL) and permanent (PePDL) teeth are comparatively assessed. METHODS: PDL tissue was obtained from 12 teeth (six primary and six permanent) from which CD105(+) CD34(-) CD45(-) cells were isolated by magnetic cell sorting. To identify and quantitatively compare the stem cell markers, DePDL and PePDL cells were assessed for CD166 surface antigen expression by flow cytometry, real-time polymerase chain reaction, and immunostaining for Stro-1 and Oct-4, osteogenic and adipogenic differentiation, and proliferation rate by trypan blue method. RESULTS: Magnetic cell sorting isolated cell populations containing 23.87% (+/- 11.98%) and 11.68% (+/- 6.27%) of CD105(+) expressing cells from PePDL and DePDL, respectively. Flow cytometric analysis demonstrated a higher proportion of CD105(+) cells coexpressing CD166 surface antigen in PePDL, whereas immunostaining and real-time polymerase chain reaction analysis demonstrated that both cell subsets expressed Stro-1 and Oct-4. DePDL-CD105(+) subsets were more proliferative compared to PePDL subsets, and both cell populations showed multipotential capabilities to differentiate in vitro to osteoblast/cementoblast- and adipocyte-like cells. However, a higher expression of adipogenic-related genes was observed in DePDL cells, whereas PePDL-CD105(+) cell subset presented a more homogeneous osteoblast/cementoblast response. CONCLUSION: These findings demonstrate that highly purified mesenchymal progenitor cell subsets can be obtained from the PDLs of both deciduous and permanent teeth, and further indicate phenotype dissimilarities that may have an impact on their clinical applications.