RESUMO
OBJECTIVES: This study investigated the miRNA expression profile in Notch-activated human dental stem pulp stem cells (DPSCs) and validated the functions of miRNAs in modulating the odonto/osteogenic properties of DPSCs. METHODS: DPSCs were treated with indirect immobilized Jagged1. The miRNA expression profile was examined using NanoString analysis. Bioinformatic analysis was performed, and miRNA expression was validated. Odonto/osteogenic differentiation was examined using alkaline phosphatase staining, Alizarin Red S staining, as well as odonto/osteogenic-related gene and protein expression. RESULTS: Fourteen miRNAs were differentially expressed in Jagged1-treated DPSCs. Pathway analysis revealed that altered miRNAs were associated with TGF-ß, Hippo, ErbB signalling pathways, FoxO and Ras signalling. Target prediction analysis demonstrated that 7604 genes were predicted to be targets for these altered miRNAs. Enrichment analysis revealed relationships to various DNA bindings. Among differentially expressed miRNA, miR-296-3p and miR-450b-5p were upregulated under Jagged1-treated conditions. Overexpression of miR-296-3p and miR-450b-5p enhanced mineralization and upregulation of odonto/osteogenic-related genes, whereas inhibition of these miRNAs revealed opposing results. The miR-296-3p and miR-450b-5p inhibitors attenuated the effects of Jagged1-induced mineralization in DPSCs. CONCLUSIONS: Jagged-1 promotes mineralization in DPSCs that are partially regulated by miRNA. The novel understanding of these miRNAs could lead to innovative controlled mechanisms that can be applied to modulate biology-targeted dental materials.
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Capsaicin (trans-8-methyl-N-vanilyl-6-nonenamide) is a unique alkaloid isolated from hot chili peppers of the capsicum family. Capsaicin is an agonist of transient receptor potential vanilloid subtype 1 (TRPV1), which is expressed in nociceptive sensory neurons and a range of secretory epithelia, including salivary glands. Capsaicin has analgesic and anti-inflammatory properties in sensory neurons. Recently, increasing evidence has indicated that capsaicin also affects saliva secretion and inflammation in salivary glands. Applying capsaicin increases salivary secretion in human and animal models. Capsaicin appears to increase salivation mainly by modulating the paracellular pathway in salivary glands. Capsaicin activates TRPV1, which modulates the permeability of tight junctions (TJ) by regulating the expression and function of putative intercellular adhesion molecules in an ERK (extracelluar signal-regulated kinase) -dependent manner. Capsaicin also improved dysfunction in transplanted salivary glands. Aside from the secretory effects of capsaicin, it has anti-inflammatory effects in salivary glands. The anti-inflammatory effect of capsaicin is, however, not mediated by TRPV1, but by inhibition of the NF-κB pathway. In conclusion, capsaicin might be a potential drug for alleviating dry mouth symptoms and inflammation of salivary glands.
Assuntos
Capsaicina/farmacologia , Glândulas Salivares/efeitos dos fármacos , Glândulas Salivares/fisiopatologia , Fármacos do Sistema Sensorial/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Capsaicina/uso terapêutico , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Receptores Muscarínicos/metabolismo , Glândulas Salivares/metabolismo , Salivação/efeitos dos fármacos , Fármacos do Sistema Sensorial/uso terapêutico , Canais de Cátion TRPV/metabolismoRESUMO
G protein-coupled receptors (GPCRs) play important roles in various pathogeneses and physiological regulations. Owing to their functional diversity, GPCRs are considered one of the primary pharmaceutical targets. However, drugs targeting GPCRs have not been developed yet to regenerate hard tissues such as teeth and bones. Mesenchymal stromal cells (MSCs) have high proliferation and multi-lineage differentiation potential, which are essential for hard tissue regeneration. Here, we present a strategy for targeting class A GPCRs for hard tissue regeneration by promoting the differentiation of endogenous MSCs into osteogenic and odontogenic progenitor cells. Through in vitro screening targeted at class A GPCRs, we identified six target receptors (LPAR1, F2R, F2RL1, F2RL2, S1PR1, and ADORA2A) and candidate drugs with potent biomineralization effects. Through a combination of profiling whole transcriptome and accessible chromatin regions, we identified that p53 acts as a key transcriptional activator of genes that modulate the biomineralization process. Moreover, the therapeutic potential of class A GPCR-targeting drugs was demonstrated in tooth pulpotomy and calvarial defect models. The selected drugs revealed potent regenerative effects in both tooth and bone defects, represented by newly formed highly mineralized regions. Consequently, this study provides translational evidence for a new regenerative strategy for damaged hard tissue.
Assuntos
Células-Tronco Mesenquimais , Osteogênese , Células-Tronco , Diferenciação Celular , Receptores Acoplados a Proteínas G , Regeneração ÓsseaRESUMO
Dental pulp-derived stromal cells (DPSCs) are a crucial cell population for maintaining the tissue integrity of the pulp-dentin complex. The oxytocin receptor (OXTR), a member of the G protein-coupled receptor (GPCR) superfamily, plays versatile roles in diverse biological contexts. However, the role of OXTR in dental pulp has not yet been fully understood. Here, we demonstrate the biological functions and significance of OXTR in DPSCs through a multidisciplinary approach. Microarray data of 494 GPCR genes revealed high OXTR expression in human DPSCs (hDPSCs). Blocking OXTR activity increased the expression of osteogenic and odontogenic marker genes, promoting hDPSC differentiation. Additionally, we found that OXTR is involved in extracellular matrix (ECM) remodeling through the regulation of the gene expression related to ECM homeostasis. We further demonstrated that these genetic changes are mediated by trascriptional activity of Yes-associated protein (YAP). Based on the results, a preclinical experiment was performed using an animal model, demonstrating that the application of an OXTR inhibitor to damaged pulp induced significant hard tissue formation. These results provide new insight into the oxytocin-OXTR system in the regenerative process of pulp-dentin complex.
Assuntos
Receptores de Ocitocina , Células-Tronco , Animais , Humanos , Receptores de Ocitocina/genética , Receptores de Ocitocina/metabolismo , Proteínas/metabolismo , Matriz Extracelular , Diferenciação Celular/fisiologia , Dentina/fisiologia , Polpa Dentária , Células Cultivadas , Proliferação de CélulasRESUMO
Oral biofilms or dental plaques are one of the major etiological factors for diverse oral diseases. We aimed to evaluate the effect of a multichannel oral irrigator (MCOI) on periodontal health in 29 participants randomly divided into two groups: the MCOI group and the control group. To evaluate the effect of the MCOI on periodontal health, the modified Quigley Hein Plaque Index (PI), Mühlemann-Son Sulcus Bleeding Index (SBI), bleeding on probing (BOP), and swelling were evaluated and compared before and after MCOI use for 3 days. Although PI and SBI showed statistically significant increases in the control group, the MCOI group showed no significant changes in either parameter. Moreover, the percentage of BOP was significantly lower in the MCOI group. Saliva samples were analyzed by 16s rRNA amplicon sequencing to investigate changes in the oral microbiome. Sequencing results showed that Porphyromonas spp. were significantly increased in the control group, whereas no significant change was detected in the MCOI group. Using the MCOI, enriched populations and functional pathways were detected in pioneer species comprising non-mutans streptococci. These findings provide evidence of the effectiveness of the MCOI in maintaining periodontal health and a healthy microbial ecology in the oral cavity.
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Microbiota , Doenças Periodontais , Humanos , Administração Oral , Índice de Placa Dentária , RNA Ribossômico 16S/genéticaRESUMO
Hybrid ionomer cements (HICs) are aesthetic polyelectrolyte cements that have been modified with a resin. The setting of HICs occurs by both monomer polymerization and an acid-base reaction. In addition, HICs contain a resin, which is substituted for water. Thus, the competition between the setting reactions and reduced water content inherently limits polysalt formation and, consequently the bioactive interactions. In this study, we explored the effects of polybetaine zwitterionic derivatives (mZMs) on the augmentation of the bioactive response of HICs. The polybetaines were homogenized into an HIC in different proportions (α, ß, and γ) at 3% w/v. Following basic characterization, the bioactive response of human dental pulp stem cells (hDPSCs) was evaluated. The augmented release of the principal constituent ions (strontium, silica, and fluoride) from the HIC was observed with the addition of the mZMs. Modification with α-mZM elicited the most favorable bioactive response, namely, increased ion elution, in vitro calcium phosphate precipitation, and excellent biofouling resistance, which deterred the growth of the bridging species of Veillonella. Moreover, α-mZM resulted in a significant increase in the hDPSC response, as confirmed by a significant increase (p < 0.05) in alizarin red staining. The results of mRNA expression tests, performed using periodically refreshed media, showed increased and early peak expression levels for RUNX2, OCN, and OPN in the case of α-mZM. Based on the results of the in vitro experiments, it can be concluded that modification of HICs with polybetaine α-mZM can augment the overall biological response.
Assuntos
Fluoretos , Cimentos de Ionômeros de Vidro , Humanos , Cimentos Ósseos , Teste de MateriaisRESUMO
Guided bone regeneration aided by the application of occlusive membranes is a promising therapy for diverse inflammatory periodontal diseases. Symbiosis, homeostasis between the host microbiome and cells, occurs in the oral environment under normal, but not pathologic, conditions. Here, we develop a symbiotically integrating occlusive membrane by mimicking the tooth enamel growth or multiple nucleation biomineralization processes. We perform human saliva and in vivo canine experiments to confirm that the symbiotically integrating occlusive membrane induces a symbiotic healing environment. Moreover, we show that the membrane exhibits tractability and enzymatic stability, maintaining the healing space during the entire guided bone regeneration therapy period. We apply the symbiotically integrating occlusive membrane to treat inflammatory-challenged cases in vivo, namely, the open and closed healing of canine premolars with severe periodontitis. We find that the membrane promotes symbiosis, prevents negative inflammatory responses, and improves cellular integration. Finally, we show that guided bone regeneration therapy with the symbiotically integrating occlusive membrane achieves fast healing of gingival soft tissue and alveolar bone.
Assuntos
Perda do Osso Alveolar , Periodontite , Humanos , Regeneração Tecidual Guiada Periodontal , Cicatrização/fisiologia , Gengiva , Membranas Artificiais , Regeneração Óssea/fisiologiaRESUMO
Acidic extracellular pH promotes osteoporotic bone loss by osteoclast activation. However, the change of osteoclastic cell behavior in acidosis-stimulated bone resorption process is unknown. We found that lowering extracellular pH induced an increase in the survival, adhesion, and migration of mature osteoclasts with a full actin ring, leading to enhanced pit formation on dentine slices. Acidosis upregulated osteopontin, which is an Arg-Gly-Asp (RGD) motif-containing matrix protein secreted from osteoclasts and acts as a common modulator for their survival, adhesion, and migration. A synthetic RGD peptide treatment blocked acidosis-induced osteoclast adhesion and migration, likely by competing with the RGD motif-containing extracellular matrix proteins for cell surface integrin binding. We finally observed that acidosis was associated with activation of osteoclast survival/adhesion/migration-related Pyk2, Cbl-b, and Src signals. Collectively, the findings indicate that extracellular acidosis stimulates bone resorption by extending osteoclast survival and facilitating osteoclast adhesion and migration.
Assuntos
Acidose/fisiopatologia , Reabsorção Óssea/fisiopatologia , Movimento Celular , Osteoclastos/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Reabsorção Óssea/metabolismo , Adesão Celular , Sobrevivência Celular , Células Cultivadas , Proteínas da Matriz Extracelular/metabolismo , Quinase 2 de Adesão Focal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Quinases da Família src/metabolismoRESUMO
Lymphatic vessels in the diaphragm are essential for draining peritoneal fluid, but little is known about their pathological changes during inflammation. Here we characterized diaphragmatic lymphatic vessels in a peritonitis model generated by daily i.p. administration of lipopolysaccharide (LPS) in mice. Intraperitoneal LPS increased lymphatic density, branching, sprouts, connections, and network formation in the diaphragm in time- and dose-dependent manners. These changes were reversible on discontinuation of LPS administration. The LPS-induced lymphatic density and remodeling occur mainly through proliferation of lymphatic endothelial cells. CD11b+ macrophages were massively accumulated and closely associated with the lymphatic vessels changed by i.p. LPS. Both RT-PCR assays and experiments with vascular endothelial growth factor-C/D blockade and macrophage-depletion indicated that the CD11b+ macrophage-derived lymphangiogenic factors vascular endothelial growth factor-C/D could be major mediators of LPS-induced lymphangiogenesis and lymphatic remodeling through paracrine activity. Functional assays with India ink and fluorescein isothiocyanate-microspheres indicated that impaired peritoneal fluid drainage in diaphragm of LPS-induced peritonitis mice was due to inflammatory fibrosis and massive attachment of CD11b+ macrophages on the peritoneal side of the diaphragmatic lymphatic vessels. These findings reveal that CD11b+ macrophages play an important role in i.p. LPS-induced aberrant lymphangiogenesis and lymphatic dysfunction in the diaphragm.
Assuntos
Antígeno CD11b/metabolismo , Diafragma/fisiopatologia , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/farmacologia , Linfangiogênese/efeitos dos fármacos , Vasos Linfáticos/fisiopatologia , Macrófagos/imunologia , Animais , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/patologia , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ácido Clodrônico/farmacologia , Diafragma/efeitos dos fármacos , Diafragma/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Injeções Intraperitoneais , Ligantes , Lipossomos , Linfonodos/efeitos dos fármacos , Linfonodos/patologia , Vasos Linfáticos/efeitos dos fármacos , Vasos Linfáticos/patologia , Macrófagos/efeitos dos fármacos , Camundongos , Tamanho do Órgão/efeitos dos fármacos , Peritonite/induzido quimicamente , Peritonite/fisiopatologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Dental pulp is composed of nerves, blood vessels, and various types of cells and surrounded by a thick and hard enamel-dentin matrix. Due to its importance in the maintenance of tooth vitality, there have been intensive efforts to analyze the complex cellular-level organization of the dental pulp in teeth. Although conventional histologic analysis has provided microscopic images of the dental pulp, 3-dimensional (3D) cellular-level visualization of the whole dental pulp in an intact tooth has remained a technically challenging task. This is mainly due to the inevitable disruption and loss of microscopic structural features during the process of mechanical sectioning required for the preparation of the tooth sample for histological observation. To accomplish 3D microscopic observation of thick intact tissue, various optical clearing techniques have been developed mostly for soft tissue, and their application for hard tissues such as bone and teeth has only recently started to be investigated. In this work, we established a simple and rapid optical clearing technique for intact mouse teeth without the time-consuming process of decalcification. We achieved 3D cellular-level visualization of the microvasculature and various immune cell distributions in the whole dental pulp of mouse teeth under normal and pathologic conditions. This technique could be used to enable diverse research methods on tooth development and regeneration by providing 3D visualization of various pulpal cells in intact mouse teeth.
Assuntos
Polpa Dentária , Dente , Animais , Camundongos , Camundongos Endogâmicos C57BL , OdontogêneseRESUMO
Mineral trioxide aggregate (MTA) has been introduced as a choice material for regenerative dentistry. To date, the diverse biological activities of MTA, including its anti-inflammatory effects, have been extensively discussed. However, there is limited insight into the link between MTA and immune cell migration. In this study, we report the role of MTA in enhancing both chemotactic and chemokinetic immune cell migration through distinct signaling pathways. By using versatile live imaging techniques, we demonstrated that MTA-mediated CaSR activation induced diverse downstream pathways to govern cell migratory capacity. In this context, Cdc42 generates cytoskeleton-driven cellular protrusions to steer directional cell migration (chemotaxis) whereas Ca2+-calmodulin dependent myosin light chain kinase induces cell contractility that plays an important role in speeding up the average migration speed (chemokinesis). Our findings illuminate an unrecognized role for MTA and the related CaSR signaling network in immune cell migration, providing evidence that can drive development of novel approaches to immunological therapy.
Assuntos
Compostos de Alumínio/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Compostos de Cálcio/farmacologia , Quimiotaxia/efeitos dos fármacos , Óxidos/farmacologia , Receptores de Detecção de Cálcio/metabolismo , Silicatos/farmacologia , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Combinação de Medicamentos , Células HL-60 , Humanos , Células Jurkat , Masculino , Camundongos , Cultura Primária de Células , Células THP-1 , Células U937 , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Polycaprolactone (PCL) cylindrical scaffolds with gradually increasing pore size along the longitudinal direction were fabricated by a novel centrifugation method to investigate pore size effect on cell and tissue interactions. The scaffold was fabricated by the centrifugation of a cylindrical mold containing fibril-like PCL and the following fibril bonding by heat treatment. The scaffold showed gradually increasing pore size (from approximately 88 to approximately 405 microm) and porosity (from approximately 80% to approximately 94%) along the cylindrical axis by applying the centrifugal speed, 3000 rpm. The scaffold sections were examined for their in vitro cell interactions using different kinds of cells (chondrocytes, osteoblasts, and fibroblasts) and in vivo tissue interactions using a rabbit model (skull bone defects) in terms of scaffold pore sizes. It was observed that different kinds of cells and bone tissue were shown to have different pore size ranges in the scaffold for effective cell growth and tissue regeneration. The scaffold section with 380-405 microm pore size showed better cell growth for chondrocytes and osteoblasts, while the scaffold section with 186-200 microm pore size was better for fibroblasts growth. Also the scaffold section with 290-310 microm pore size showed faster new bone formation than those of other pore sizes. The pore size gradient scaffolds fabricated by the centrifugation method can be a good tool for the systematic studies of the interactions between cells or tissues and scaffolds with different pore size.
Assuntos
Substitutos Ósseos/uso terapêutico , Condrócitos/fisiologia , Osteoblastos/fisiologia , Poliésteres/química , Fraturas Cranianas/patologia , Fraturas Cranianas/cirurgia , Engenharia Tecidual/métodos , Células 3T3 , Animais , Materiais Biocompatíveis/química , Substitutos Ósseos/química , Técnicas de Cultura de Células/métodos , Linhagem Celular , Centrifugação , Condrócitos/citologia , Humanos , Teste de Materiais , Camundongos , Osteoblastos/citologia , Porosidade , Coelhos , Propriedades de SuperfícieRESUMO
OBJECTIVE: To test the antiadherent and antibacterial properties of surface modification of orthodontic wires with photocatalytic titanium oxide (TiO(2)). MATERIALS AND METHODS: TiO(2) was coated on the surface of the orthodontic wires by a sol-gel thin film dip-coating method. Bacterial adhesion to the wires was evaluated by the weight change of the wires. The antibacterial activity of the surface-modified orthodontic wires was demonstrated by the dilution agar plate method for Streptococcus mutans and spectrophotometry for Porphyromonas gingivalis. RESULTS: The orthodontic wires coated with the photocatalytic TiO(2) showed an antiadherent effect against S. mutans compared with the uncoated wires. The bacterial mass that bound to the TiO(2)-coated orthodontic wires remained unchanged, whereas that of the uncoated wires increased by 4.97%. Furthermore, the TiO(2)-coated orthodontic wires had a bactericidal effect on S. mutans and P. gingivalis, which cause dental caries and periodontitis, respectively. The antiadherent and antibacterial mechanisms of TiO(2) to break down the cell wall of those bacteria were revealed by scanning electron microscopy. CONCLUSION: The surface modification of orthodontic wires with photocatalytic TiO(2) can be used to prevent the development of dental plaque during orthodontic treatment.
Assuntos
Antibacterianos/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/farmacologia , Fios Ortodônticos , Titânio/farmacologia , Porphyromonas gingivalis/efeitos dos fármacos , Porphyromonas gingivalis/fisiologia , Streptococcus mutans/efeitos dos fármacos , Streptococcus mutans/fisiologia , Propriedades de SuperfícieRESUMO
Mineral trioxide aggregate (MTA) is a calcium silicate-based bioactive material that has been extensively used in dentistry. MTA has been highlighted in its diverse biological functions and excellent clinical outcomes. However, limited insight into the intracellular signaling pathways has been provided to explain the biological activities of MTA. Here, we firstly elucidate that the extracellular calcium-sensing receptor (CaSR) is a major signaling mediator of MTA-induced biological reactions through versatile live imaging techniques of human dental pulp cells (hDPCs). We found that MTA activates diverse CaSR downstream pathways; notably, CaSR activation essentially requires dual modulation of extracellular Ca2+ and pH via MTA. Among the CaSR downstream pathways, Ca2+ mobilization from intracellular stores by the phospholipase C pathway plays an important role in osteogenic differentiation of hDPCs by regulating transcriptional activity. Our findings shed light on the signal transduction mechanism of MTA, thus providing a crucial molecular basis for the use of MTA in regenerative dental therapy.
Assuntos
Compostos de Alumínio/farmacologia , Compostos de Cálcio/farmacologia , Óxidos/farmacologia , Receptores de Detecção de Cálcio/metabolismo , Silicatos/farmacologia , Adolescente , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Combinação de Medicamentos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Espaço Intracelular/metabolismo , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Adulto JovemRESUMO
This study examined the influences of titanium (Ti) discs with similar surface roughnesses (R(a) values), but with different topographies and chemical compositions, on the adhesion, spreading, and the alkaline phosphatase (ALP) activity of osteoblast-like cells and normal human fibroblasts. The presence of adhesion molecules on the Ti surfaces and their effects on cell activity were also investigated. Two types of Ti discs were prepared. One kind was a mechanically polished Ti disc, and the other type was a disc obtained by the heating of hydroxyapatite (HA) dip-coated Ti. Scanning electron microscopy, optical interferometry, and scanning Auger electron spectroscopy were used to examine the surface morphology, roughness, and chemical composition, respectively, of the superficial Ti layer. The two types of Ti discs had different topographies and chemical compositions, but had similar R(a) values. The cells on both surface types had similar behaviors and ALP activities. A biological evaluation of the surface-modified Ti discs showed that the type I collagen coating was functionally active in terms of cell spreading in both types of Ti discs. In the mechanically polished Ti discs, fibronectin was functionally active in the normal human fibroblasts, but not in the osteoblast-like cells. Cell adhesion was slightly better on the heat-treated HA dip-coated Ti discs, but not on the mechanically polished Ti discs. Type I collagen and fibronectin mediated the adhesion and spreading of osteoblast-like cells through alpha2beta1 integrin and alpha5beta1 integrin, respectively. These results suggest that type I collagen might be a good candidate for the biochemical modification of Ti surfaces, particularly those surfaces obtained by heating of HA dip-coated Ti.
Assuntos
Proliferação de Células , Materiais Revestidos Biocompatíveis , Durapatita , Fibroblastos/fisiologia , Osteoblastos/fisiologia , Titânio , Adesão Celular , Células Cultivadas , Colágeno Tipo I/biossíntese , Durapatita/química , Fibroblastos/citologia , Humanos , Integrina alfa2beta1/biossíntese , Integrina alfa5beta1/biossíntese , Teste de Materiais/métodos , Osteoblastos/citologia , Titânio/químicaRESUMO
hBMSCs are multipotent cells that are useful for tissue regeneration to treat degenerative diseases and others for their differentiation ability into chondrocytes, osteoblasts, adipocytes, hepatocytes and neuronal cells. In this study, biodegradable elastic hydrogels consisting of hydrophilic poly(ethylene glycol) (PEG) and hydrophobic poly(ε-caprolactone) (PCL) scaffolds were evaluated for tissue engineering because of its biocompatibility and the ability to control the release of bioactive peptides. The primary cultured cells from human bone marrow are confirmed as hBMSC by immunohistochemical analysis. Mesenchymal stem cell markers (collagen type I, fibronectin, CD54, integrin1ß, and Hu protein) were shown to be positive, while hematopoietic stem cell markers (CD14 and CD45) were shown to be negative. Three different hydrogel scaffolds with different block compositions (PEG:PCL=6:14 and 14:6 by weight) were fabricated using the salt leaching method. The hBMSCs were expanded, seeded on the scaffolds, and cultured up to 8 days under static conditions in Iscove's Modified Dulbecco's Media (IMDM). The growth of MSCs cultured on the hydrogel with PEG/PCL= 6/14 was faster than that of the others. In addition, the morphology of MSCs seemed to be normal and no cytotoxicity was found. The coating of the vascular endothelial growth factor (VEGF) containing scaffold with Matrigel slowed down the release of VEGF in vitro and promoted the angiogenesis when transplanted into BALB/c nude mice. These results suggest that hBMSCs can be supported by a biode gradable hydrogel scaffold for effective cell growth, and enhance the angiogenesis by Matrigel coating.
Assuntos
Colágeno/metabolismo , Laminina/metabolismo , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica , Poliésteres/metabolismo , Polietilenoglicóis/metabolismo , Proteoglicanas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Medula Óssea/metabolismo , Transplante de Células , Células Cultivadas , Colágeno/toxicidade , Combinação de Medicamentos , Humanos , Hidrogéis/metabolismo , Hidrogéis/toxicidade , Laminina/toxicidade , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos BALB C , Poliésteres/toxicidade , Polietilenoglicóis/toxicidade , Proteoglicanas/toxicidadeRESUMO
All-trans retinoic acid (ATRA) inhibits matrix metalloproteinase (MMP)-2 and MMP-9 in synovial fibroblasts, skin fibroblasts, bronchoalveolar lavage cells and cancer cells, but activates MMP-9 in neuroblast and leukemia cells. Very little is known regarding whether ATRA can activate or inhibit MMPs in human dental pulp cells (HDPCs). The purpose of this study was to determine the effects of ATRA on the production and secretion of MMP-2 and -9 in HDPCs. The productions and messenger RNA (mRNA) expressions of MMP-2 and -9 were accessed by gelatin zymography and real-time polymerase chain reaction (PCR), respectively. ATRA was found to decrease MMP-2 level in a dose-dependent manner. Significant reduction in MMP-2 mRNA expression was also observed in HDPCs treated with 25 µmolâ L(-1) ATRA. However, HDPCs treated with ATRA had no effect on the pattern of MMP-9 produced or secreted in either cell extracts or conditioned medium fractions. Taken together, ATRA had an inhibitory effect on MMP-2 expression in HDPCs, which suggests that ATRA could be a candidate as a medicament which could control the inflammation of pulp tissue in vital pulp therapy and regenerative endodontics.
Assuntos
Polpa Dentária/enzimologia , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Tretinoína/farmacologia , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados , Polpa Dentária/citologia , Polpa Dentária/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , RNA Mensageiro/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Gênica/efeitos dos fármacos , Tretinoína/administração & dosagemRESUMO
Recently, we developed a novel method to fabricate a nerve guide conduit (NGC) with asymmetrical pore structure and hydrophilicity using poly(lactic-co-glycolic acid) (PLGA) and Pluronic F127 by a modified immersion precipitation method. From the animal study using a rat model (sciatic nerve defect of rat), we recognized that the unique PLGA/Pluronic F127 tube provided good environments for nerve regeneration. In this study, we applied low-intensity pulsed ultrasound as a simple and noninvasive stimulus at the PLGA/F127 NGC-implanted site transcutaneously in rats to investigate the feasibility of ultrasound for the enhanced nerve regeneration through the tube. The nerve regeneration behaviors within the ultrasound-stimulated PLGA/Pluronic F127 NGCs were compared with the NGCs without the ultrasound treatment as well as normal nerve by histological and immunohistochemical observations. It was observed that the PLGA/Pluronic F127 tube-implanted group applied with the ultrasound had more rapid nerve regeneration behavior (approximately 0.71 mm/day) than the tube-implanted group without the ultrasound treatment (approximately 0.48 mm/day). The ultrasound-treated tube group also showed greater neural tissue area as well as larger axon diameter and thicker myelin sheath than the tube group without the ultrasound treatment, indicating better nerve regeneration. The better nerve regeneration behavior in the our NGC/ultrasound system may be caused by the synergistic effect of the asymmetrically porous PLGA/Pluronic F127 tube with unique properties (selective permeability, hydrophilicity, and structural stability, which can provide good environment for nerve regeneration) and physical stimulus (stimulation of the Schwann cells and activation of the neurotrophic factors).
Assuntos
Regeneração Tecidual Guiada/métodos , Regeneração Nervosa/efeitos da radiação , Nervos Periféricos/fisiologia , Terapia por Ultrassom/métodos , Animais , Regeneração Tecidual Guiada/normas , Ácido Láctico , Fatores de Crescimento Neural/metabolismo , Nervos Periféricos/efeitos da radiação , Poloxâmero , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Porosidade , Ratos , Células de Schwann/efeitos da radiação , UltrassomRESUMO
A novel guided bone regeneration (GBR) membrane was fabricated by an immersion precipitation of poly (glycolic-co-lactic acid) (PLGA)/Pluronic F127 solution impregnated in an electrospun polycaprolactone (PCL)/Tween 80 nanofiber mesh. The prepared PCL/Tween 80 nanofiber mesh-embedded PLGA/Pluronic F127 membrane (hydrophilized PCL/PLGA hybrid membrane) had nano-size pores on the top side (which can prevent from fibrous connective tissue infiltration but allow permeation of oxygen and nutrients) and micro-size pores on the bottom side (which can improve adhesiveness with bone). From the comparisons of mechanical properties (tensile and suture pullout strengths), model nutrient (FITC-labeled bovine serum albumin) permeability, and bone regeneration behavior using a rat model (skull bone defect) of the hybrid membrane with those of PLGA/Pluronic F127 membrane (asymmetrically porous, hydrophilized PLGA membrane), PCL/Tween 80 nanofiber mesh (electrospun, hydrophilized PCL nanofiber mesh), and a commercialized GBR membrane, Bio-Gide (collagen type I/III membrane), it was observed that the PCL/PLGA hybrid membrane seems to be highly desirable as a GBR membrane for the selective permeability caused by its unique morphology and osteoconductivity provided by several tens micro-size pores of the bottom side as well as the excellent mechanical strengths by the hybridization of porous PLGA membrane and PCL nanofiber mesh.
Assuntos
Materiais Biocompatíveis/química , Regeneração Óssea , Ácido Láctico/química , Membranas Artificiais , Poliésteres/química , Ácido Poliglicólico/química , Animais , Osso e Ossos/lesões , Osso e Ossos/ultraestrutura , Teste de Materiais , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos , Ratos Sprague-DawleyRESUMO
Injectable polycaprolactone (PCL) porous beads were fabricated for use as cell carriers by a novel isolated particle-melting method (for nonporous beads) and the following melt-molding particulate-leaching method (for porous beads). The prepared beads showed highly porous and uniform pore structures with almost the same surface and interior porosities (porosity, over 90%). The PCL porous beads (bead size, 400-550 microm) with different pore sizes (25-50 and 50-100 microm) were compared for their in vitro cell (human chondrocyte) growth behavior with the nonporous beads. The porous beads showed higher cell seeding density and growth than the nonporous beads. The pore size effect between the porous beads was not significant up to 7 days, but after that time the beads with pore sizes of 50-100 microm showed significantly higher cell growth than those of 25-50 microm. To evaluate the tissue compatibility of the PCL porous beads, the beads were dispersed, uniformly, in cold Pluronic F127 solution and injected into hairless mice, subcutaneously, in the gel state of Pluronic F127 at room temperature, leading to the homogeneous bead delivery. The histological findings confirmed that the PCL porous beads in Pluronic F127 gel are biocompatible: surrounding tissues gradually infiltrated into the porous beads for up to 4 weeks with little inflammatory response. The PCL porous beads with highly porous and uniform pore structures fabricated in this study can be widely applicable as cell carriers.