RESUMO
We assessed the feasibility of transplanting a sheet of retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs) in a patient with neovascular age-related macular degeneration. The iPSCs were generated from skin fibroblasts obtained from two patients with advanced neovascular age-related macular degeneration and were differentiated into RPE cells. The RPE cells and the iPSCs from which they were derived were subject to extensive testing. A surgery that included the removal of the neovascular membrane and transplantation of the autologous iPSC-derived RPE cell sheet under the retina was performed in one of the patients. At 1 year after surgery, the transplanted sheet remained intact, best corrected visual acuity had not improved or worsened, and cystoid macular edema was present. (Funded by Highway Program for Realization of Regenerative Medicine and others; University Hospital Medical Information Network Clinical Trials Registry [UMIN-CTR] number, UMIN000011929 .).
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Degeneração Macular/terapia , Epitélio Pigmentado da Retina/citologia , Idoso , Técnicas de Cultura de Células , Diferenciação Celular , Estudos de Viabilidade , Feminino , Fibroblastos , Humanos , Masculino , Epitélio Pigmentado da Retina/transplante , Transplante AutólogoRESUMO
Injectable hydrogels are considered important to realize safe and effective minimally invasive therapy. Although animal-derived natural polymers are well studied, they typically lack injectability and fail to eliminate the potential risks of immunogenic reactions or unknown pathogen contamination. Despite extensive research activities to explore ideal injectable hydrogels, such state-of-the-art technology remains inaccessible to non-specialists. In this article, the design of a new injectable hydrogel platform that can be extemporaneously prepared from commercially available animal-component-free materials is described. The hydrogels can be prepared simply by mixing mutually reactive aqueous solutions without necessitating specialized knowledge or equipment. Their solidification time can be adjusted by choosing proper buffer conditions from immediate to an extended period of time, that is, few or several tens of minutes depending on the concentration of polymeric components, which not only provides injectability, but enables 3D encapsulation of cells. Mesenchymal stromal/stem cells can be encapsulated and cultured in the hydrogels at least for 2 weeks by traditional cell culture techniques, and retrieved by collagenase digestion with cell viability of approximately 80%. This hydrogel platform accelerates future cell-related research activities.
Assuntos
Técnicas de Cultura de Células , Colágeno Tipo I/metabolismo , Colagenases/metabolismo , Hidrogéis/metabolismo , Células-Tronco Mesenquimais/citologia , Animais , Sobrevivência Celular , Colágeno Tipo I/química , Colagenases/química , Humanos , Hidrogéis/química , Células-Tronco Mesenquimais/metabolismoRESUMO
BACKGROUND: The adrenocortical cells have been shown to produce various inflammatory cytokines such as TNFα and IL-6, which could modulate steroidogenesis. However, the role of inflammatory cytokines in aldosterone-producing adenomas (APAs) is not fully understood. In the present study, we examined the relationships between mRNA expression levels of the inflammation-related genes and somatic mutations in APA tissues. METHODS: We evaluated mRNA expression levels of TNFA, IL6, and NFKB1 in APA tissues obtained from 44 Japanese APA patients. RESULTS: We revealed that mRNA expression patterns of the inflammation-related genes depended on a KCNJ5 somatic mutation. In addition, we showed that mRNA expression levels of the inflammation-related genes correlated with those of the steroidogenic enzyme CYP11B1 in the patients with APAs. CONCLUSION: The present study documented for the first time the expression of inflammation-related genes in APAs and the correlation of their expression levels with the KCNJ5 mutation status and mRNA expression levels of steroidogenic enzymes, indicating the pathophysiological relevance of inflammation-related genes in APAs.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Adenoma Adrenocortical/genética , Adenoma Adrenocortical/metabolismo , Aldosterona/biossíntese , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Mutação , Adulto , Idoso , Citocinas/genética , Feminino , Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-6/genética , Masculino , Pessoa de Meia-Idade , Subunidade p50 de NF-kappa B/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Esteroide 11-beta-Hidroxilase/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
Changes in cell fluidity have been observed in various cellular tissues and are strongly linked to biological phenomena such as self-organization. Recent studies suggested variety of mechanisms and factors, which are still being investigated. This study aimed to investigate changes in cell fluidity in multi-layered cell sheets, by exploring the collective arrest of cell motion and its release in cultures of corneal epithelial cells. We constructed mathematical models to simulate the behaviors of individual cells, including cell differentiation and time-dependent changes in cell-cell connections, which are defined by stochastic or kinetic rules. Changes in cell fluidity and cell sheet structures were expressed by simulating autonomous cell behaviors and interactions in tissues using an agent-based model. A single-cell level spatiotemporal analysis of cell state transition between migratable and non-migratable states revealed that the release from collective arrest of cell motion was initially triggered by a decreased ability to form cell-cell connections in the suprabasal layers, and was propagated by chain migration. Notably, the disruption of cell-cell connections and stratification occurred in the region of migratable state cells. Hence, a modeling approach that considers time-dependent changes in cell properties and behavior, and spatiotemporal analysis at the single-cell level can effectively delineate emergent phenomena arising from the complex interplay of cells.
Assuntos
Células Epiteliais , Modelos Biológicos , Movimento CelularRESUMO
Background: Although intracardiac injection or intracoronary delivery of mesenchymal stem cells (MSCs) has been reported, there have been few studies on the intravenous injection of MSCs, particularly in Japan. MethodsâandâResults: Five patients with left ventricular ejection fraction (LVEF) ≤45% received 1.0×108 MSCs intravenously. The procedure did not induce significant changes in vital signs. One patient had an elevated body temperature after 1 day, but recovered spontaneously. Laboratory tests remained normal for 1 month after cell delivery. Computed tomography was performed after 1-2 years, and there was no evidence of malignancy. Conclusions: In this pilot study of patients with reduced LVEF, intravenous MSC delivery had no adverse effects.
RESUMO
This study aimed to examine the optimal cross-link density of recombinant peptide (RCP) particles, based on human collagen type I, for bone reconstruction in human alveolar cleft. Low- (group 1), medium- (group 2), and high- (group 3) cross-linked RCP particles were prepared by altering the duration of the heat-dependent dehydration reaction. Rat palatine fissures (n = 45), analogous to human congenital bone defects, were examined to evaluate the potential of bone formation by the three different RCP particles. Microcomputed tomography images were obtained to measure bone volume and bone mineral density at 4, 8, 12, and 16 weeks post grafting. Specimens were obtained for histological analysis at 16 weeks after grafting. Additionally, alkaline phosphatase and tartrate acid phosphatase staining were performed to visualize the presence of osteoblasts and osteoclasts. At 16 weeks, bone volume, bone mineral density, and new bone area measurements in group 2 were significantly higher than in any other group. In addition, the number of osteoblasts and osteoclasts on the new bone surface in group 2 was significantly higher than in any other group. Our results demonstrated that medium cross-linking was more suitable for bone formation-and could be useful in human alveolar cleft repairs as well.
RESUMO
INTRODUCTION: This study aimed to examine the bone-forming ability of medium-cross-linked recombinant collagen peptide (mRCP) particles developedbased on human collagen type I, contains an arginyl-glycyl-aspartic acid-rich motif, fabricated as bone filling material, compared to that of the autologous bone graft. METHODS: Calvarial bone defects were created in immunodeficient rats though a surgical procedure. The rats were divided into 2 groups: mRCP graft and tibia bone graft (bone graft). The bone formation potential of mRCP was evaluated by micro-computed tomography and hematoxylin-eosin staining at 1, 2, 3, and 4 weeks after surgery, and the data were analyzed and compared to those of the bone graft. RESULTS: The axial volume-rendered images demonstrated considerable bony bridging with the mRCP graft, but there was no significant difference in the bone volume and bone mineral density between the mRCP graft and bone graft at 4 weeks. The peripheral new bone density was significantly higher than the central new bone density and the bottom side score was significantly higher than the top side score at early stage in the regenerated bone within the bone defects. CONCLUSION: These results indicate that mRCP has a high potential of recruiting osteogenic cells, comparable to that of autologous bone chips.
RESUMO
Corneal limbal epithelial stem cell transplantation using cultivated human corneal epithelial cell sheets has been used successfully to treat limbal stem cell deficiencies. Here we report an investigation into the quality of cultivated human corneal epithelial cell sheets using time-lapse imaging of the cell culture process every 20 minutes over 14 days to ascertain the level of cell jamming, a phenomenon in which cells become smaller, more rounded and less actively expansive. In parallel, we also assessed the expression of p63, an important corneal epithelial stem cell marker. The occurrence of cell jamming was variable and transient, but was invariably associated with a thickening and stratification of the cell sheet. p63 was present in all expanding cell sheets in the first 9 days of culture, but it's presence did not always correlate with stratification of the cell sheet. Nor did p63 expression necessarily persist in stratified cell sheets. An assessment of cell jamming, therefore, can shed significant light on the quality and regenerative potential of cultivated human corneal epithelial cell sheets.
Assuntos
Doenças da Córnea/terapia , Epitélio Corneano/citologia , Proteínas de Membrana/metabolismo , Transplante de Células-Tronco , Células-Tronco/citologia , Células 3T3 , Animais , Engenharia Biomédica/métodos , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células/fisiologia , Células Epiteliais/citologia , Feminino , Humanos , Limbo da Córnea/citologia , Masculino , Camundongos , Pessoa de Meia-IdadeRESUMO
Epithelial cell rests of Malassez (ERM) are quiescent epithelial remnants of Hertwig's epithelial root sheath (HERS) that are involved in the formation of tooth roots. After completion of crown formation, HERS are converted from cervical loop cells, which have the potential to generate enamel for tooth crown formation. Cervical loop cells have the potential to differentiate into ameloblasts. Generally, no new ameloblasts can be generated from HERS, however this study demonstrated that subcultured ERM can differentiate into ameloblast-like cells and generate enamel-like tissues in combination with dental pulp cells at the crown formation stage. Porcine ERM were obtained from periodontal ligament tissue by explant culture and were subcultured with non-serum medium. Thereafter, subcultured ERM were expanded on 3T3-J2 feeder cell layers until the tenth passage. The in vitro mRNA expression pattern of the subcultured ERM after four passages was found to be different from that of enamel organ epithelial cells and oral gingival epithelial cells after the fourth passage using the same expansion technique. When subcultured ERM were combined with subcultured dental pulp cells, ERM expressed cytokeratin14 and amelogenin proteins in vitro. In addition, subcultured ERM combined with primary dental pulp cells seeded onto scaffolds showed enamel-like tissues at 8 weeks post-transplantation. Moreover, positive staining for amelogenin was observed in the enamel-like tissues, indicating the presence of well-developed ameloblasts in the implants. These results suggest that ERM can differentiate into ameloblast-like cells.
Assuntos
Ameloblastos/citologia , Diferenciação Celular , Células Epiteliais/citologia , Células 3T3 , Amelogenina/metabolismo , Animais , Proliferação de Células , Separação Celular , Transplante de Células , Células Cultivadas , Colágeno/metabolismo , Esmalte Dentário/citologia , Esmalte Dentário/metabolismo , Polpa Dentária/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Incisivo/citologia , Incisivo/metabolismo , Queratina-14/metabolismo , Camundongos , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suínos , Dente Decíduo/citologia , Dente Decíduo/metabolismoRESUMO
BACKGROUND: The aim of the present controlled clinical study was to compare the clinical response of human cultured periosteum (HCP) sheets in combination with platelet-rich plasma (PRP) and porous hydroxyapatite (HA) granules to a mixture of PRP and HA in the treatment of human infrabony periodontal defects. METHODS: Thirty interproximal infrabony osseous defects in 30 healthy, non-smoking subjects diagnosed with chronic periodontitis were included in this study. The subjects were randomly assigned to the test group (HCP sheets combined with PRP and HA) or the control group (PRP with HA). Clinical and radiographic measurements were made at baseline and the 12-month post-surgical evaluation. RESULTS: Compared to baseline, the 12-month results indicated that both treatment modalities resulted in statistically significant changes (P <0.01) in the gingival index, bleeding on probing, probing depth, clinical attachment level, and radiographic infrabony defect depth. Compared to the control group, the test group exhibited a statistically significantly more favorable change in clinical attachment gain (3.9 +/- 1.6 mm versus 2.7 +/- 1.3 mm; P <0.05), vertical relative attachment gain (83.5% +/- 31.7% versus 55.0% +/- 21.9%; P <0.05), and radiographic infrabony defect fill (4.9 +/- 1.2 mm versus 3.2 +/- 1.1 mm; P <0.01). CONCLUSIONS: Compared to PRP with HA, treatment with a combination of HCP sheets, PRP, and HA led to a significantly more favorable clinical improvement in infrabony periodontal defects. A factor likely contributing to these favorable clinical results is the presence of osteogenic cells in the HCP sheets, which provided greater regeneration potential.
Assuntos
Perda do Osso Alveolar/cirurgia , Regeneração Óssea/efeitos dos fármacos , Substitutos Ósseos/uso terapêutico , Durapatita/uso terapêutico , Periósteo , Plasma Rico em Plaquetas , Implantes Absorvíveis , Idoso , Perda do Osso Alveolar/complicações , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Índice Periodontal , Periodontite/complicações , Periodontite/cirurgia , Engenharia Tecidual/métodos , Resultado do TratamentoRESUMO
Recently, regenerative medicine has attracted attention worldwide and also in Japan. Several guidelines on regenerative medicine have been released from the Japanese regulatory agency. Within the guidelines, Japan Tissue Engineering Co., Ltd. has been developing tissue-engineered products of cultured epidermis, cartilage and cornea using autologous cells. In this paper, we describe the industrialization of regenerative medicine as well as regulations based on our experiences of the culture cartilage development.
Assuntos
Setor de Assistência à Saúde , Medicina Regenerativa , Engenharia Tecidual , Animais , Cartilagem , Ensaios Clínicos como Assunto , Setor de Assistência à Saúde/legislação & jurisprudência , Humanos , Japão , Controle de Qualidade , Medicina Regenerativa/legislação & jurisprudência , Segurança , Engenharia Tecidual/legislação & jurisprudênciaRESUMO
Cell therapies using adipose-derived stem cells (ADSCs) have been used to treat inflammatory bowel disease (IBD) in human and dog. We previously reported the CellSaic technique, which uses a recombinant scaffold to enhance the efficacy of cell therapy. To examine whether this technique can be applied to cell therapy for colitis, we evaluated the efficacy of CellSaic in colitis mouse models. Colitis mouse models were developed by administering dextran sulfate sodium (DSS) to C57BL/6 mice for 7 days. Then CellSaic comprising human/canine ADSCs (1.2 × 106 cells) or human/canine ADSCs only (1.2 × 106 cells) were administered to the mice. The body weights were measured, and the colon length measurements and histological evaluations were conducted at 7 days after administration. After in vitro culture of human ADSC (hADSC) CellSaic and hADSC spheroids in medium containing TNFα, the levels of the anti-inflammatory protein TSG-6 in each supernatant were measured. Furthermore, we conducted tumorigenicity and general toxicity tests of canine ADSC (cADSC) CellSaic in NOG mice for 8 weeks. In the colitis mouse models, the ADSC CellSaic group presented recovery of body weight and colon length compared with the ADSC-only group. Histological analysis showed that ADSC CellSaic decreased the number of inflammatory cells and repaired ulceration. In vitro, hADSC CellSaic secreted 3.1-fold more TSG-6 than the hADSCs. In addition, tumorigenicity and general toxicity of cADSC CellSaic were not observed. This study suggests that human and canine ADSC CellSaic has a therapeutic effect of colitis in human and dogs.
Assuntos
Colite/induzido quimicamente , Colite/terapia , Sulfato de Dextrana , Transplante de Células-Tronco , Tecido Adiposo/citologia , Animais , Materiais Biocompatíveis/química , Linhagem Celular , Colite/patologia , Colo/patologia , Modelos Animais de Doenças , Cães , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Alicerces Teciduais/químicaRESUMO
The pathophysiology of aldosterone-producing adenomas (APAs) has been investigated via genetic approaches and the pathogenic significance of a series of somatic mutations, including KCNJ5, has been uncovered. However, how the mutational status of an APA is associated with its molecular characteristics, including its transcriptome and methylome, has not been fully understood. This study was undertaken to explore the molecular characteristics of APAs, specifically focusing on APAs with KCNJ5 mutations as opposed to those without KCNJ5 mutations, by comparing their transcriptome and methylome status. Cortisol-producing adenomas (CPAs) were used as reference. We conducted transcriptome and methylome analyses of 29 APAs with KCNJ5 mutations, 8 APAs without KCNJ5 mutations and 5 CPAs. Genome-wide gene expression and CpG methylation profiles were obtained from RNA and DNA samples extracted from these 42 adrenal tumors. Cluster analysis of the transcriptome and methylome revealed molecular heterogeneity in APAs depending on their mutational status. DNA hypomethylation and gene expression changes in Wnt signaling and inflammatory response pathways were characteristic of APAs with KCNJ5 mutations. Comparisons between transcriptome data from our APAs and that from normal adrenal cortex obtained from the Gene Expression Omnibus suggested similarities between APAs with KCNJ5 mutations and zona glomerulosa. The present study, which is based on transcriptome and methylome analyses, indicates the molecular heterogeneity of APAs depends on their mutational status. Here, we report the unique characteristics of APAs with KCNJ5 mutations.
Assuntos
Adenoma/genética , Adenoma/metabolismo , Aldosterona/genética , Aldosterona/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Humanos , Pessoa de Meia-Idade , Mutação , Via de Sinalização Wnt/genéticaRESUMO
Non-alcoholic steatohepatitis (NASH) is characterized by steatosis with lobular inflammation and hepatocyte injury. Pirfenidone (PFD) is an orally bioavailable pyridone derivative that has been clinically used for the treatment of idiopathic pulmonary fibrosis. However, it remains unknown whether PFD improves liver fibrosis in a mouse model with human NASH-like phenotypes. In this study, we employed melanocortin 4 receptor-deficient (MC4R-KO) mice as a mouse model with human NASH-like phenotypes to elucidate the effect and action mechanisms of PFD on the development of NASH. PFD markedly attenuated liver fibrosis in western diet (WD)-fed MC4R-KO mice without affecting metabolic profiles or steatosis. PFD prevented liver injury and fibrosis associated with decreased apoptosis of liver cells in WD-fed MC4R-KO mice. Pretreatment of PFD inhibited the tumor necrosis factor-α (TNF-α)-induced liver injury and fibrogenic responses associated with decreased apoptosis of liver cells in wild-type mice. PFD also prevented TNF-α-induced hepatocyte apoptosis in vitro with reduced activation of caspase-8 and -3. This study provides evidence for the antifibrotic effect of PFD in a mouse model of human NASH. The data of this study highlight hepatocyte apoptosis as a potential therapeutic target, and suggest that PFD can be repositioned as an antifibrotic drug for human NASH.
Assuntos
Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Piridonas/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Células Cultivadas , Dieta Ocidental , Modelos Animais de Doenças , Comportamento Alimentar/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Inflamação/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Piridonas/farmacologia , Receptor Tipo 4 de Melanocortina/deficiência , Receptor Tipo 4 de Melanocortina/metabolismo , Fator de Necrose Tumoral alfa/efeitos adversos , Regulação para Cima/genéticaRESUMO
This study investigated the efficacy of the ureteral decellularized matrix (UDM) as a scaffold material for a tissue-engineered ureter, and the effect of bone marrow-derived mononuclear cells (BM-MNC) on the neovascularization of the scaffold. Canine ureters were treated with deoxycholic acid to remove all cells. Uroepithelial cells (UEC) were obtained from canine bladders, cultured, and then seeded onto the inner surface of the UDM before transplantation into the subcutaneous space of nude mice or the omentum of nude rats. The cultured UECs began showing vacuolar degeneration 3 days after transplantation and gradually disappeared thereafter. To facilitate neovascularization in the implant, BM-MNCs were seeded around the UDM before transplantation. This facilitated the survival of the UECs, which formed three to five cellular layers after 14 days. The mean microvessel density was significantly increased in tissues seeded with BM-MNCs. However, cell-tracking experiments revealed that the increased number of capillaries in the experimental group was not due to the direct differentiation of transplanted endothelial progenitor cells. Our results demonstrate that the UDM is a useful scaffold for a tissue-engineered ureter, especially when seeded with BM-MNCs to enhance angiogenesis.
Assuntos
Engenharia Tecidual/métodos , Ureter , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Cães , Células Epiteliais/citologia , Células Epiteliais/transplante , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/transplante , Camundongos , Camundongos Nus , Neovascularização Fisiológica , Ratos , Ratos Nus , Transplante Heterólogo , Ureter/irrigação sanguínea , Ureter/citologia , Urotélio/citologiaRESUMO
In the field of oral and maxillofacial surgery, tissue-engineering techniques have been found useful in regenerating lost tissues. Periodontal disease causes severe destruction of periodontal tissue, including the alveolar bone. In this study we attempted to regenerate canine periodontal tissue defects by grafting autologous cultured membrane derived from the periosteum. Under appropriate culture conditions, periosteal cells produce enough extracellular matrix to form sheets. Periosteum specimens were peeled from the mandibular body of adult hybrid dogs and were cultured until cells formed membrane. ALP activity was measured to determine an optimal time for grafting. The cultured periosteum (CP) was grafted and sutured on a mechanically made Class III furcation defect in the 4th mandibular premolars. After 3 months, the samples were harvested and observed radiologically and histologically. In cases of CP, the bone defects were regenerated and filled with newly formed hard tissue, whereas in the controls the defects remained. These results show that our novel treatment is effective in regenerating alveolar bone for the treatment of periodontal disease.
Assuntos
Periodonto , Periósteo , Regeneração , Engenharia Tecidual , Animais , Dente Pré-Molar/anormalidades , Dente Pré-Molar/citologia , Cães , Matriz Extracelular/fisiologia , Feminino , Mandíbula/anormalidades , Mandíbula/citologia , Doenças Periodontais/terapia , Periodonto/citologia , Periodonto/fisiologia , Periósteo/citologia , Periósteo/fisiologia , Regeneração/fisiologia , Técnicas de Cultura de Tecidos , Transplante AutólogoRESUMO
BACKGROUND: Cultured gingival substitute has been found to be a useful graft material for treatment of gingival recession. However, such substitutes include xenograft derivative materials that involve concomitant risk of viral contamination. To eliminate this risk, we designed new gingival substitutes made of recombinant human collagen types I and III sponges and cultured these substitutes in animal-free media (HFDM-1). METHODS: Gingival fibroblasts were seeded onto sponges of type I or III recombinant collagen. These sponges were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), HFDM-1 with 2% human serum (HS), or HFDM-1. Fibroblast proliferation in these samples was compared using the cell-counting kit assay. Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) released into the cultured media were examined by enzyme-linked immunosorbent assay. RESULTS: The fibroblasts proliferated significantly in all six combinations of collagen and medium types. The fibroblast growth rate after 9 days of culture was equal between HFDM-1 with 2% HS and DMEM with 10% FBS. The type III collagen sponge showed a higher fibroblast growth rate than the type I sponge. VEGF concentrations in HFDM-1 with 2% HS were higher than those in other media. The highest HGF levels were detected in DMEM with 10% FBS. CONCLUSIONS: The new cultured gingival substitute containing no animal-derived materials produced good cell proliferation and VEGF release. The results suggested that the substitute may provide a new tool for the treatment of gingival recession.
Assuntos
Órgãos Bioartificiais , Fibroblastos/transplante , Gengiva/citologia , Retração Gengival/cirurgia , Engenharia Tecidual/métodos , Animais , Bovinos , Técnicas de Cultura de Células , Proliferação de Células , Células Cultivadas , Colágeno Tipo I , Colágeno Tipo III , Meios de Cultivo Condicionados , Fibroblastos/metabolismo , Fator de Crescimento de Hepatócito/biossíntese , Humanos , Proteínas Recombinantes , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
OBJECTIVE: The pathophysiology of aldosterone-producing adenomas (APA) has been investigated intensively through genetic and genomic approaches. However, the role of epigenetics in APA is not fully understood. In the present study, we explored the relationship between gene expression and DNA methylation status in APA. METHODS: We conducted an integrated analysis of transcriptome and methylome data of paired APA-adjacent adrenal gland (AAG) samples from the same patient. The adrenal specimens were obtained from seven Japanese patients with APA who underwent adrenalectomy. Gene expression and genome-wide CpG methylation profiles were obtained from RNA and DNA samples that were extracted from those seven paired tissues. RESULTS: Methylome analysis showed global CpG hypomethylation in APA relative to AAG. The integration of gene expression and methylation status showed that 34 genes were up-regulated with CpG hypomethylation in APA. Of these, three genes (CYP11B2, MC2R, and HPX) may be related to aldosterone production, and five genes (PRRX1, RAB38, FAP, GCNT2, and ASB4) are potentially involved in tumorigenesis. CONCLUSION: The present study is the first methylome analysis to compare APA with AAG in the same patients. Our integrated analysis of transcriptome and methylome revealed DNA hypomethylation in APA and identified several up-regulated genes with DNA hypomethylation that may be involved in aldosterone production and tumorigenesis.
Assuntos
Adenoma/genética , Neoplasias das Glândulas Suprarrenais/genética , Metilação de DNA/genética , Hiperaldosteronismo/genética , Transcriptoma/genética , Adenoma/diagnóstico , Adenoma/cirurgia , Neoplasias das Glândulas Suprarrenais/diagnóstico , Neoplasias das Glândulas Suprarrenais/cirurgia , Adrenalectomia/tendências , Adulto , Idoso , Aldosterona/metabolismo , Feminino , Humanos , Hiperaldosteronismo/diagnóstico , Hiperaldosteronismo/cirurgia , Masculino , Pessoa de Meia-IdadeRESUMO
Using "real-time RT-PCR", we assessed the expression of three different hyaluronan synthase genes, HAS1, HAS2, and HAS3, by measuring their mRNA amounts in cultured human oral mucosal epithelial (COME) cells, oral mucosal fibroblasts, and dermal fibroblasts, and investigated the effects of interleukin-1beta (IL-1beta) and epidermal growth factor (EGF). When COME cells were treated with IL-1beta or EGF, early and marked increases and subsequent rapid decreases were observed for all HAS genes and, moreover, actual changes in hyaluronan synthesis subsequently occurred. The effects of IL-1beta stimulation were concentration-dependent and the maximal response to the EGF stimulation was observed at a low concentration (0.1 ng per mL). When two different types of fibroblasts were treated with IL-1beta or EGF, increased expression with different degrees and rates of three different HAS genes and subsequent increased synthesis of hyaluronan were also observed. In addition, HAS1 gene expression was not detectable in the mucosal fibroblasts, while weak HAS3 gene expression was detected in the dermal fibroblasts. Taken together, it is likely that the regulation of the expression of the three different HAS genes is different between oral mucosa and skin, which may be of significance for elucidating some of the differences between these tissues in wound healing.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Interleucina-1/farmacologia , Mucosa Bucal/enzimologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transferases/genética , Células Cultivadas , Células Epiteliais/enzimologia , Fibroblastos/enzimologia , Glucuronosiltransferase , Humanos , Hialuronan Sintases , CicatrizaçãoRESUMO
A new type of ceramic-polymer biomaterial having excellent apatite-forming ability in simulated body fluid was prepared by hot-pressing a mixture of poly(-L-lactic acid) (PLA) and calcium carbonate (vaterite). After PLA dissolved in methylene chloride was mixed with calcium carbonate consisting of vaterite, the mixture was dried completely and subsequently hot-pressed uniaxially under a pressure of 40 MPa at 180 degrees C. When 30 wt% vaterite was introduced, the modulus of elasticity was effectively improved by 3.5-6 GPa, which was about twice higher than the modulus of PLA. The composite showed no brittle fracture behavior and a comparably high bending strength of approximately 50 MPa. The composite containing 30 wt% vaterite formed a 5-15-microm-thick bonelike apatite layer on its surface after soaking in SBF at 37 degrees C even for 1-3d.