RESUMO
BACKGROUND: Melatonin has many roles, including bone remodeling and osseointegration of dental implants. The topical application of melatonin facilitated bone regeneration in bone defects. We evaluated the effects of topical application of melatonin on vertical bone augmentation in rat calvaria secluded spaces. MATERIAL AND METHODS: In total, 12 male Fischer rats were used and two plastic caps were fixed in the calvarium. One plastic cap was filled with melatonin powder and the other was left empty. RESULTS: Newly generated bone at bone defects and within the plastic caps was evaluated using micro-CT and histological sections. New bone regeneration within the plastic cap was increased significantly in the melatonin versus the control group. CONCLUSIONS: Melatonin promoted vertical bone regeneration in rat calvaria in the secluded space within the plastic cap.
Assuntos
Regeneração Óssea/efeitos dos fármacos , Melatonina/farmacologia , Crânio/efeitos dos fármacos , Crânio/fisiologia , Animais , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
The 75 kDa transmembrane protein, p75(NTR), is a marker of mesenchymal stem cells (MSCs). Isolated MSCs are capable of differentiating into osteoblasts, but the molecular function of p75(NTR) in MSCs and osteoblasts is poorly understood. The aim of this study was to examine the function of p75(NTR) in the human MG63 osteoblast cell line compared to the murine MC3T3E-1 pre-osteoblast cell line. MG63 cells and MC3T3-E1 cells expressing exogenous p75(NTR) protein (denoted as p75-MG63 and p75GFP-E1, respectively) were generated to compare osteogenic differentiation and cell proliferation abilities. Overexpression of p75(NTR) induced alkaline phosphatase activity and the mRNA expression of osteoblast-related genes such as osterix and bone sialoprotein in both p75-MG63 and p75GFP-E1. Interestingly, exogenous p75(NTR) stimulated cell proliferation and cell cycle progression in p75GFP-E1, but not in p75-MG63. To elucidate any different effects of p75(NTR) expression on osteogenic differentiation and cell proliferation, we examined the mRNA expression of tropomyosin receptor kinase (trk) genes (trkA, trkB, trkC) and Nogo receptor (NgR), which are binding partners of p75(NTR). Although trkA, trkB, and trkC were detected in both p75-MG63 and p75GFP-E1, only NgR was detected in p75-MG63. We then used the K252a inhibitor of the trks to identify the signaling pathway for osteogenic differentiation and cell proliferation. Inhibition of trks by K252a suppressed p75(NTR)-mediated osteogenic differentiation of p75GFP-E1, whereas deletion of the GDI domain in P75(NTR) from the p75-MG63 produced enhanced cell proliferation compared to p75-MG63. These results suggest that p75(NTR) signaling associated with trk receptors promotes both cell proliferation and osteoblast differentiation, but that p75(NTR)-mediated proliferation may be suppressed by signaling from the p75(NTR)/NgR complex.
Assuntos
Proliferação de Células , Osteoblastos/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Fosfatase Alcalina/metabolismo , Carbazóis/farmacologia , Linhagem Celular , Humanos , Alcaloides Indólicos/farmacologia , Osteoblastos/citologia , Osteogênese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Receptor de Fator de Crescimento Neural/genética , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Transdução de Sinais , Fator de Transcrição Sp7 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
We have been studying an easy bracket debonding method using heating of an orthodontic adhesive containing thermal expansion microcapsules. However, heating with a high-temperature heater brings obvious risks of burns around the oral cavity. Thus, we examined safer and more effective bracket debonding methods. The purpose of this in vitro study was to examine the reduction in debonding strength and the time taken using a bracket bonded with an orthodontic adhesive containing thermal expansion microcapsules and a CO2 laser as the heating method while maintaining safety. Ceramic brackets were bonded to bovine permanent mandibular incisors using bonding materials containing various microcapsule contents (0, 30, and 40 wt%), and the bond strengths were measured after laser irradiation for 4, 5, and 6 s and compared with nonlaser-treated groups. Subsequently, the temperature in the pulp chamber during laser irradiation was measured. After laser irradiation for 5 or 6 s, the bond strengths of the adhesive containing 40 wt% microcapsules were significantly decreased to â¼0.40 - 0.48-fold (4.6-5.5 MPa) compared with the nonlaser groups. The mean temperature rise of the pulp chamber was 4.3 °C with laser irradiation for 6 s, which was less than that required to induce pulp damage. Based on these results, we conclude that the combined use of a CO2 laser and an orthodontic adhesive containing thermal expansion microcapsules can be effective and safe for debonding ceramic brackets with less enamel damage or tooth pain.
Assuntos
Colagem Dentária/métodos , Cimentos Dentários/farmacologia , Descolagem Dentária/métodos , Porcelana Dentária/farmacologia , Temperatura Alta , Lasers de Gás , Braquetes Ortodônticos , Animais , Cápsulas , Bovinos , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/efeitos da radiação , Resistência ao Cisalhamento , Fatores de TempoRESUMO
While the role of p75(NTR) signaling in the regulation of nerve-related cell growth and survival has been well documented, its actions in osteoblasts are poorly understood. In this study, we examined the effects of p75(NTR) on osteoblast proliferation and differentiation using the MC3T3-E1 pre-osteoblast cell line. Proliferation and osteogenic differentiation were significantly enhanced in p75(NTR)-overexpressing MC3T3-E1 cells (p75GFP-E1). In addition, expression of osteoblast-specific osteocalcin (OCN), bone sialoprotein (BSP), and osterix mRNA, ALP activity, and mineralization capacity were dramatically enhanced in p75GFP-E1 cells, compared to wild MC3T3-E1 cells (GFP-E1). To determine the binding partner of p75(NTR) in p75GFP-E1 cells during osteogenic differentiation, we examined the expression of trkA, trkB, and trkC that are known binding partners of p75(NTR), as well as NgR. Pharmacological inhibition of trk tyrosine kinase with the K252a inhibitor resulted in marked reduction in the level of ALPase under osteogenic conditions. The deletion of the GDI binding domain in the p75(NTR)-GFP construct had no effect on mineralization. Taken together, our studies demonstrated that p75(NTR) signaling through the trk tyrosine kinase pathway affects osteoblast functions by targeting osteoblast proliferation and differentiation.
Assuntos
Diferenciação Celular , Osteoblastos/citologia , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Calcificação Fisiológica , Carbazóis , Técnicas de Cultura de Células , Proliferação de Células , Regulação da Expressão Gênica , Alcaloides Indólicos , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Camundongos , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Ligação Proteica , Proteína Quinase C/antagonistas & inibidores , RNA Mensageiro/biossíntese , Receptor trkC/genética , Receptor trkC/metabolismo , Receptores de Fator de Crescimento Neural/genética , Transdução de Sinais , Fator de Transcrição Sp7 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Oxytalan fibers are extracellular matrix components consisting of pure microfibrils. However, the mechanism whereby oxytalan fibers develop is not fully understood. We have previously reported that in human periodontal ligament (PDL) fibroblasts subjected to stretching stress, bundles of oxytalan fibers coalesce under the control of fibulin-5. Latent transforming growth factor-ß binding protein 2 (LTBP-2) is known to bind to fibulin-5. The purpose of this study was to clarify the role of LTBP-2 in the coalescence of oxytalan fibers. We subjected PDL fibroblasts to stretching in order to examine the effects of LTBP-2 on the coalescence of oxytalan fibers in cell/matrix layers. Interaction of LTBP-2 with fibulin-5 was examined by immunoprecipitation assay, and changes in LTBP-2 deposition upon stretching were investigated by Western blotting and immunofluorescence assays. We used small interfering RNA against LTBP-2 in PDL cell culture and examined the appearance of oxytalan fibers on the basis of immunofluorescence. Stretching induced coalescence of oxytalan fibers, but did not affect LTBP-2 expression. The amount of extracellularly deposited LTBP-2 was decreased by about 70% as a result of stretching, compared with the control. LTBP-2 interacted with fibulin-5 on the fibers, and stretching decreased the amount of the LTBP-2 interacted with fibulin-5 by about 60%. Oxytalan fiber coalescence did not occur when LTBP-2 was suppressed by about 95%, whereas it occurred when LTBP-2 was suppressed by about 40%, fibulin-5 being colocalized with oxytalan fibers. These results suggest that LTBP-2, in response to tension stress, may negatively control the function of fibulin-5, thereby modulating the mechanism of oxytalan fiber coalescence.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Proteínas de Ligação a TGF-beta Latente/biossíntese , Estresse Fisiológico , Células Cultivadas , Proteínas da Matriz Extracelular/biossíntese , Feminino , Humanos , MasculinoRESUMO
Fragments of Hertwig's epithelial root sheath persist in the periodontal ligament (PDL) in small clusters known as epithelial rests of Malassez (ERM). It is generally agreed that ERM are maintained as a quiescent and exclusively dental epithelial cluster in PDL. However, we speculate that homeostasis and cellular turnover underlies cluster maintenance. We also hypothesize that the fate of ERM clusters - diminishing or remaining - might be regulated via the presence or absence of epithelial stem cells therein. Histological analysis of aging mouse molar PDL showed that ERM clusters gradually increase in size with increasing age. Immunocytochemistry and cell culture revealed that ERM clusters contained Ki67-positive cells and were able to expand when brought in culture. The TdT-mediated biotin-dUTP nick-end labeling (TUNEL) procedure also detected signs of apoptosis. Finally, we identified putative epithelial stem cells in the clusters by 5-bromo-2'-deoxyuridine (BrdU) pulse-chase experiments and immunohistochemistry, using the stem-cell marker leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5). The results suggest that ERM clusters are maintained in the PDL, via cellular turnover, throughout life.
Assuntos
Apoptose/genética , Células Epiteliais/citologia , Dente Molar/citologia , Ligamento Periodontal/citologia , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/citologia , Raiz Dentária/citologia , Animais , Bromodesoxiuridina/metabolismo , Técnicas de Cultura de Células , Regulação da Expressão Gênica , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dente Molar/crescimento & desenvolvimento , Receptores Acoplados a Proteínas G/genética , Células-Tronco/metabolismo , Raiz Dentária/crescimento & desenvolvimentoRESUMO
The ciliary zonules, also known as the zonules of Zinn, help to control the thickness of the lens during focusing. The ciliary zonules are composed of oxytalan fibers, which are synthesized by human nonpigmented ciliary epithelial cells (HNPCEC). The ciliary zonules are exposed to ultraviolet (UV), especially UV-A and UV-B, throughout life. We previously demonstrated that UV-B, but not UV-A, degrades fibrillin-1- and fibrillin-2-positive oxytalan fibers. However, the mechanism by which UV-B degrades oxytalan fibers remains unknown. In this study, we investigate the involvement of matrix metalloproteinase-2 (MMP-2) in the UV-B-induced degradation of fibrillin-1- and fibrillin-2-positive oxytalan fibers in cultured HNPCECs. Enzyme-linked immunosorbent assay revealed that UV-B irradiation at levels of 100 and 150 mJ/cm2 significantly increased the level of active MMP-2. Notably, MMP-2 inhibitors completely suppressed the degradation of fibrillin-1- and fibrillin-2-positive oxytalan fibers. In addition, we show that UV-B activates MMP-2 via stress-responsive kinase p38. Taken together, the results suggest that UV-B activates a production of active type of MMP-2 via the p38 pathway, and subsequently, an active-type MMP-2 degrades the fibrillin-1- and fibrillin-2-positive oxytalan fibers in cultured HNPCECs.
RESUMO
The periodontal ligaments (PDLs) are soft connective tissue between the cementum covering the tooth root surface and alveolar bone. PDLs are composed of collagen and elastic system fibers, blood vessels, nerves, and various types of cells. Elastic system fibers are generally formed by elastin and microfibrils, but PDLs are mainly composed of the latter. Compared with the well-known function of collagen fibers to support teeth, little is known about the role of elastic system fibers in PDLs. To clarify their role, we examined PDLs of mice under-expressing fibrillin-1 (mgR mice), which is one of the major microfibrillar proteins. The PDLs of homozygous mgR mice showed one-quarter of the elastic system fibers of wild-type (WT) mice. A close association between the elastic system fibers and the capillaries was noted in WT, homozygous and heterozygous mgR mice. Interestingly, capillaries in PDLs of homozygous mice were dilated or enlarged compared with those of WT mice. A comparable level of type I collagen, which is the major collagen in PDLs, was expressed in PDL-cells of mice with three genotypes. However, multi-oriented collagen fiber bundles with a thinner appearance were noted in homozygous mice, whereas well-organized collagen fiber bundles were seen in WT mice. Moreover, there was a marked decrease in periostin expression, which is known to regulate the fibrillogenesis and crosslinking of collagen. These observations suggest that the microfibrillar protein, fibrillin-1, is indispensable for normal tissue architecture and gene expression of PDLs.
Assuntos
Capilares/patologia , Doenças do Colágeno/complicações , Dilatação Patológica/complicações , Tecido Elástico/patologia , Proteínas dos Microfilamentos/genética , Ligamento Periodontal/metabolismo , Animais , Colágeno/metabolismo , Doenças do Colágeno/genética , Doenças do Colágeno/metabolismo , Dilatação Patológica/genética , Dilatação Patológica/metabolismo , Tecido Elástico/metabolismo , Tecido Elástico/ultraestrutura , Fibrilina-1 , Fibrilinas , Regulação da Expressão Gênica , Heterozigoto , Homozigoto , Hibridização In Situ , Masculino , Camundongos , Camundongos Transgênicos , Microfibrilas/metabolismo , Microfibrilas/patologia , Microfibrilas/ultraestrutura , Proteínas dos Microfilamentos/metabolismo , Ligamento Periodontal/patologia , Ligamento Periodontal/ultraestrutura , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismoRESUMO
The upper and lower tooth-bearing jaws of the filefish (Stephanolepis cirrhifer) were scanned using a micro-CT system in order to address the existing gaps between the traditional pictures of the morphology and histology. 2D tomograms, reconstructed 3D models and virtual dissection were employed to examine and evaluate the in situ geometry of tooth implantation and the mode of tooth attachment both separately and collectively. No distinct sockets comparable to those in mammals were evident, but shallow depressions were observed in the premaxillary and the dentary. The opening of the tooth pulp cavity was not simply oriented towards the apparent tooth base in a direction opposite to the tooth apex. The opening was distorted basoposteriorly or basoanteriorly depending on the position of the tooth, and the edge of the pulp cavity opening was barely ankylosed; i.e. the sites of pleurodont ankylosis along the basoposterior or basoanterior edge of the opening appeared to closely match the contour of the shallow depression in the bone. These 3D findings appear to be very informative when considering the phylogeny of tooth attachment, suggesting that micro-CT would be a useful modality concurrent with or in advance of histological investigations.
Assuntos
Anquilose Dental , Dente/diagnóstico por imagem , Animais , Dentição , Arcada Osseodentária , Ligamento PeriodontalRESUMO
The morphogenesis of long bones is a multistep process that generates a variety of genetically defined forms. The tarsometatarsal (TMT) long bone morphology in birds develops through lateral fusion of three initially independent periosteal bone cylinders (BCs). Previous studies have clarified the histological details and chronology of the changes occurring during development. The present study investigated the temporospatial distribution of osteogenic and osteoclastic cells in the embryonic chicken using histochemistry for alkaline phosphatase and tartrate-resistant acid phosphatase, with particular reference to the radial growth of BCs and their subsequent fusion process. Osteogenic cells were localized preferentially in the periosteum of radially growing BCs, leaving open cancellous spaces in the BC wall. Osteoclasts observed later than embryonic day 10 were localized preferentially in the endosteal surface, and therefore the radial growth of BCs resulting from osteoblast activity was accompanied by endosteal resorption by osteoclasts, with progressive enlargement of the bone marrow spaces. During BC fusion, trabecular bridges were formed by periosteal osteogenic cells, with removal of the bone septum by endosteal osteoclasts. These findings suggest that fusion of BCs in the embryonic chicken is mediated by cellular events constituting ordinary long bone development, and not through a defined mechanism specific for fusion.
Assuntos
Galinhas , Osteoclastos , Animais , Osso e Ossos , Embrião de Galinha , Osteoblastos , OsteogêneseRESUMO
OBJECTIVE: In orthodontic tooth movement, some cytokines released from periodontal ligament fibroblasts and alveolar bone osteoblasts on the pressure side can alter the normal processes of bone remodelling, resulting in physiological bone resorption. We examined the effect of compressive force and interleukin (IL)-1 type I receptor antagonist (IL-1ra) on the expression of inflammatory cytokines that promote osteoclast formation, as well as on their receptors, in osteoblastic Saos-2 cells. DESIGN: The cells were cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum with or without continuous compressive force (0.5-3.0 g/cm(2)) and/or IL-1ra for up to 24h. The gene expression levels of the cytokines and their receptors were estimated by determining mRNA levels using real-time PCR; the protein levels were determined using ELISA or immunohistochemical staining. RESULTS: The expression of IL-1beta, IL-1 receptor, IL-6, IL-6 receptor, IL-8 receptor, IL-11 and tumor necrosis factor-alpha (TNFalpha) increased depending on the strength and duration of the compressive force, whereas the expression of IL-8, IL-11 receptor and TNFalpha receptor did not change with the application of compressive force. The expression of cytokines and their receptors produced by 3.0 g/cm(2) of compressive force decreased with the simultaneous addition of IL-1ra and the decrease was remarkable in IL-8 receptor, IL-11 and TNFalpha. CONCLUSION: These results indicate that mechanical stress induces the production of inflammatory cytokines and their receptors in osteoblasts and the phenomenon is enhanced by the autocrine action of IL-1beta, which is increased in amount by mechanical stress.
Assuntos
Citocinas/metabolismo , Osteoblastos/metabolismo , Receptores de Citocinas/metabolismo , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/fisiologia , Força Compressiva , Citocinas/genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Humanos , Imuno-Histoquímica , Inflamação/metabolismo , Osteoblastos/fisiologia , Receptores de Citocinas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse MecânicoRESUMO
Fibrillin is a primary component of elastin-associated microfibrils. Since microfibrils are distributed rather ubiquitously in embryonic tissues, attention has focused on the types of cells responsible for producing fibrillin. To clarify this issue, we employed monensin-induced perturbation of secretory activity in embryonic primary cultures, as this would allow examination of both the secreted protein and the formation of extracellular fibrils in the same culture. Micromasses of avian limb bud mesoderm, its ectodermal covering and several explants from other sources were cultured in the presence and absence of monensin, and evaluated immunohistochemically using antibodies against fibrillin and cell lineage markers. The results indicated that monensin perturbation induced intracellular accumulation of fibrillin and prevented the formation of microfibrils. It was shown specifically that not only mesodermally derived fibrogenic cells and myogenic cells of skeletal and smooth muscle cell lineage, but also epithelial-type cells such as endothelial and ectodermal cells, are producers of fibrillin. This dual cellular origin of fibrillin at the ectomesenchymal interface is considered significant for understanding the formation and remodeling of microfibrils originating from the basal lamina.
Assuntos
Ectoderma/citologia , Ionóforos/farmacologia , Mesoderma/citologia , Microfibrilas , Proteínas dos Microfilamentos/metabolismo , Monensin/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Linhagem da Célula , Embrião de Galinha , Ectoderma/efeitos dos fármacos , Ectoderma/metabolismo , Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Fibrilinas , Histocitoquímica , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Células Musculares/metabolismo , Miocárdio/citologia , Transporte Proteico/efeitos dos fármacosRESUMO
The ciliary zonules link the lens to the ciliary body in the eye, controlling the thickness of the lens for focusing through their characteristic elasticity. The ciliary zonules are composed of oxytalan fibers. Physiological or pathological damage to the ciliary zonules, including exposure to ultraviolet (UV)-A and UV-B components, can lead to lens dislocation. However, no studies have shown whether UV affects the ciliary zonule. Here, we assessed the effects of UV light on human nonpigmented ciliary epithelial cells (HNPCECs). HNPCECs were cultured for 4 weeks, and expression of fibrillin-1 and fibrillin-2 was confirmed. In control cultures (0 mJ/cm2), some fibrillin-1-positive fibers were merged with fibrillin-2. After UV-A irradiation, the appearance of both fibrillin-1- and fibrillin-2-positive fibers was unchanged. However, after UV-B irradiation, fibrillin-1-positive fibers became thin at an irradiation level of 100 mJ/cm2, and the fiber structure became amorphous at 150 mJ/cm2. Fibrillin-2-positive fibers lost their continuity and disappeared after being exposed to 150 mJ/cm2 UV-B. UV-B irradiation did not affect cell viability, possibly because of the sensitivity of fibrillin-1 and fibrillin-2 to UV-B. Thus, dislocation of the lens with age may be attributable to cumulative exposure to UV-B.
RESUMO
Bone marrow-derived multipotent stromal cells (BMSCs) have potent antiinflammatory effects. This study aimed to investigate the antiinflammatory potential of BMSCs using a mouse model of ligature-induced periodontitis. BMSCs were isolated from the femurs and tibiae of mice. Periodontitis was induced by placing a ligature around the right maxillary second molar. After 3 days, the mice were administered BMSC in the gingiva of the mesial interdental papilla around the ligatured molar. The ligatured and non-ligatured mice that were not administered BMSC served as controls. Differences in inflammatory infiltration and bone resorption around the roots of the second molar were assessed and were subsequently quantified using microcomputed tomography (micro-CT), histological analysis, and tartrate-resistant acid phosphatase (TRAP) staining. Micro-CT revealed that alveolar bone loss around the ligatured molars increased in a time-dependent manner; however, the effect was significantly less in BMSC-treated mice compared with ligatured control mice. Tissue histopathology revealed that BMSC administration mitigated inflammatory infiltration in ligatured BMSC mice. In addition, the number of TRAP-positive osteoclasts was markedly elevated in ligatured control mice compared with those in BMSC-treated mice. These findings indicate that local BMSC administration can mitigate inflammation and alveolar bone resorption, suggesting that administering BMSC leads to new therapeutics for periodontitis.
Assuntos
Células-Tronco Mesenquimais , Periodontite/terapia , Perda do Osso Alveolar , Animais , Reabsorção Óssea , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Periodontite/etiologiaRESUMO
The formation of cross-linkages between and within collagen is catalyzed by lysyl oxidase, which can be inhibited by ß-aminopropionitrile (BAPN), a lathyrogen from sweet pea (Lathyrus odoratus) seeds. The quality and integrity of the collagenous template of skeletal elements depend on an appropriate concentration of collagen cross-links. In this study, chick embryos treated in ovo with BAPN on embryonic days (ED) 4-9 were found to develop multiple skeletal deformities. The most readily discernible and highly reproducible deformity was evident in the tibiotarsus, on which we focused to explore the chronology of the malformation process. Several lines of observation indicated that the bending deformity observable at ED10 in the tibiotarsus was inducible by BAPN administered on ED4-8; in other words, administration of BAPN on ED8 was sufficient to induce the deformity by ED10, whereas administration on ED9 was ineffective. Ultrastructurally, osteoclasts appeared to show enhanced activity in the medullary surface of the bone collar after BAPN administration. In addition, bone hyperplasia associated with the bending deformity was suggested to be correlated with higher osteoblast activity on the concave (or flexor) side of the tibiotarsal skeleton. These findings indicate that the bending deformity due to reduced mechanical integrity of the collagenous template is also associated with aberrant bone remodeling. (J Oral Sci 58, 255-263, 2016).
Assuntos
Aminopropionitrilo/toxicidade , Osso e Ossos/efeitos dos fármacos , Animais , Embrião de Galinha , Relação Dose-Resposta a Droga , Reprodutibilidade dos TestesRESUMO
Dedifferentiated fat (DFAT) cells derived from mature adipocytes have mesenchymal stem cells' (MSCs) characteristics. Generally, mature adipocytes are 60-110 µm in diameter; however, association between adipocyte size and dedifferentiation efficiency is still unknown. This study, therefore, investigated the dedifferentiation efficiency of adipocytes based on cell diameter. Buccal fat pad was harvested from five human donors and dissociated by collagenase digestion. After exclusion of unwanted stromal cells by centrifugation, floating adipocytes were collected and their size distribution was analyzed. The floating adipocytes were then separated into two groups depending on cell size using 40- and 100-µm nylon mesh filters: cell diameters less than 40 µm (small adipocytes: S-adipocytes) and cell diameters of 40-100 µm (large adipocytes: L-adipocytes). Finally, we evaluated the efficiency of adipocyte dedifferentiation and then characterized the resultant DFAT cells. The S-adipocytes showed a higher capacity to dedifferentiate into DFAT cells (S-DFAT cells) compared to the L-adipocytes (L-DFAT cells). The S-DFAT cells also showed a relatively higher proportion of CD146-positive cells than L-DFAT cells, and exhibited more osteogenic differentiation ability based on the alkaline phosphatase activity and amount of calcium deposition. These results suggested that the S- and L-DFAT cells had distinct characteristics, and that the higher dedifferentiation potential of S-adipocytes compared to L-adipocytes gives the former group an advantage in yielding DFAT cells.
Assuntos
Tecido Adiposo/citologia , Diferenciação Celular , Boca/citologia , Osteogênese , Adipócitos/citologia , Adipogenia , Adulto , Desdiferenciação Celular , Proliferação de Células , Separação Celular , Forma Celular , Tamanho Celular , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Feminino , Fibroblastos/citologia , Citometria de Fluxo , Fluorescência , Perfilação da Expressão Gênica , Células-Tronco Embrionárias Humanas/citologia , Humanos , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Adulto JovemRESUMO
Lipid-free fibroblast-like cells, known as dedifferentiated fat (DFAT) cells, can be generated from mature adipocytes with a large single lipid droplet. DFAT cells can re-establish their active proliferation ability and can transdifferentiate into various cell types under appropriate culture conditions. The first objective of this study was to compare the multilineage differentiation potential of DFAT cells with that of adipose-derived stem cells (ASCs) on mesenchymal stem cells. We obtained DFAT cells and ASCs from inbred rats and found that rat DFAT cells possess higher osteogenic differentiation potential than rat ASCs. On the other hand, DFAT cells show similar adipogenic differentiation, and chondrogenic differentiation potential in comparison with ASCs. The second objective of this study was to assess the regenerative potential of DFAT cells combined with novel solid scaffolds composed of PLGA (Poly d, l-lactic-co-glycolic acid) on periodontal tissue, and to compare this with the regenerative potential of ASCs combined with PLGA scaffolds. Cultured DFAT cells and ASCs were seeded onto PLGA scaffolds (DFAT/PLGA and ASCs/PLGA) and transplanted into periodontal fenestration defects in rat mandible. Micro computed tomography analysis revealed a significantly higher amount of bone regeneration in the DFAT/PLGA group compared with that of ASCs/PLGA and PLGA-alone groups at 2, 3, and 5 weeks after transplantation. Similarly, histomorphometric analysis showed that DFAT/PLGA groups had significantly greater width of cementum, periodontal ligament and alveolar bone than ASCs/PLGA and PLGA-alone groups. In addition, transplanted fluorescent-labeled DFAT cells were observed in the periodontal ligament beside the newly formed bone and cementum. These findings suggest that DFAT cells have a greater potential for enhancing periodontal tissue regeneration than ASCs. Therefore, DFAT cells are a promising cell source for periodontium regeneration.
RESUMO
The existence of progenitor/mesenchymal stem cells (MSCs) was demonstrated previously in human primary/deciduous teeth. In this study, we examined dental pulp cells from root portion (root cells) of primary teeth without discernible root resorption and compared them with pulp cells from the crown portion (crown cells). Root cells and crown cells were characterized and compared to each other based on progenitor/MSC characteristics and on their generation efficiency of induced pluripotent stem (iPS) cells. Root cells and crown cells included cells manifesting typical progenitor/MSC properties such as osteogenic and adipogenic differentiation potential and clonogenicity. Interestingly, root cells showed a higher expression level of embryonic stem cell marker, KLF4, than crown cells. Moreover, the number of colony-forming unit-fibroblast and cell proliferation rate were higher for root cells than crown cells, and the efficiency of generating iPS cells from root cells was approximately four times higher than that from crown cells. Taken together, these results suggest that root cells from primary teeth show the MSC-like properties and thus could be a potent alternative source for iPS cell generation and the subsequent transplantation therapy.
Assuntos
Polpa Dentária/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Mesenquimais/citologia , Coroa do Dente/citologia , Raiz Dentária/citologia , Dente Decíduo/citologia , Adipócitos/citologia , Adipócitos/metabolismo , Biomarcadores/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proliferação de Células , Reprogramação Celular/genética , Polpa Dentária/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Cultura Primária de Células , Coroa do Dente/metabolismo , Raiz Dentária/metabolismo , Dente Decíduo/metabolismoRESUMO
Cell sheet technology has been used to deliver cells in single-sheet form with an intact extracellular matrix for soft tissue repair and regeneration. Here, we hypothesized that titanium-reinforced cell sheets could be constructed for bone tissue engineering and regeneration. Fifty-µm-thick titanium plates containing apertures were prepared and roughened by acid etching, some of which were photofunctionalized with 12 min of UV light treatment. Cell sheets were prepared by culturing rat calvarial periosteum-derived cells on temperature-responsive culture dishes and attached to titanium plates. Titanium-reinforced osteogenic cell sheet construction was conditional on various technical and material factors: cell sheets needed to be double-sided and sandwich the titanium plate, and the titanium plates needed to be micro thin and contain apertures to allow close apposition of the two cell sheets. Critically, titanium plates needed to be UV-photofunctionalized to ensure adherence and retention of cell sheets. Single-sided cell sheets or double-sided cell sheets on as-made titanium contracted and deformed within 4 days of incubation. Titanium-reinforced cell sheets on photofunctionalized titanium were structurally stable at least up to 14 days, developed the expected osteogenic phenotypes (ALP production and mineralization), and maintained structural integrity without functional degradation. Successful construction of titanium-reinforced osteogenic cell sheets was associated with increased cell attachment, retention, and expression of vinculin, an adhesion protein by photofunctionalization. This study identified the technical and material requirements for constructing titanium-reinforced osteogenic cell sheets. Future in vivo studies are warranted to test these titanium-reinforced cell sheets as stably transplantable, mechanically durable, and shape controllable osteogenic devices.
Assuntos
Materiais Biocompatíveis , Regeneração Óssea , Osteogênese , Titânio , Animais , Células Cultivadas , Teste de Materiais , Microscopia Eletrônica de Varredura , Periósteo/citologia , Ratos , Propriedades de Superfície , Engenharia Tecidual/métodosRESUMO
In this study, a unique fiber system in the subectodermal mesenchyme of the chick limb bud was visualized immunohistochemically with the use of a novel monoclonal antibody termed "FB1." This antibody stained a subset of extracellular fibers in the embryonic mesenchyme. Among the fibers visualized, those running perpendicularly to the limb bud ectoderm became progressively prominent in their thickness and length, and organized into a parallel array in the subectodermal region. This fiber system was distinct from that of major collagens, fibronectin, or tenascin. A molecule immunoprecipitated with FB1 comigrated with JB3 antigen, or chicken fibrillin-2. The fibers visualized immunohistochemically by FB1 and JB3 were indistinguishable from each other, and ultrastructurally appeared to be bundles composed of tubular-like microfibrils that originated directly from the ectodermal basal lamina. They lacked the amorphous deposits that are characteristic of elastin. A similar array of subectodermal fibers was also found in the developing axilla and some truncal regions, again well before the development of a definitive dermis. These findings suggest that a parallel array of subectodermal FB1-positive fibers constitutes a precocious fiber system in the presumptive dermis prior to the substantial formation of collagenous fibers. These fibers could be developmentally linked to oxytalan fibers, which are known to be present in the papillary dermis in mature cutaneous tissue.