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BACKGROUND AND AIMS: The future development of hepatocellular carcinoma (HCC) in patients after sustained virologic response (SVR) is an important issue. The purposes of this study were to investigate pathological alterations in organelle of the liver of SVR patients and to characterize organelle abnormalities that may be related to carcinogenesis after SVR. METHODS: The ultrastructure of liver biopsy specimens from patients with chronic hepatitis C (CHC) and SVR were compared to cell and mouse models and assessed semi-quantitatively using transmission electron microscopy. RESULTS: Hepatocytes in patients with CHC showed abnormalities in the nucleus, mitochondria, endoplasmic reticulum, lipid droplet, and pericellular fibrosis, comparable to those seen in hepatitis C virus (HCV)-infected mice and cells. DAA treatment significantly reduced organelle abnormalities such as the nucleus, mitochondria, and lipid droplet in the hepatocytes of patients and mice after SVR, and cured cells, but it did not change dilated/degranulated endoplasmic reticulum and pericellular fibrosis in patients and mice after SVR. Further, samples from patients with a post-SVR period of >1 year had significantly larger numbers of abnormalities in the mitochondria and endoplasmic reticulum than those of <1 year. A possible cause of organelle abnormalities in patients after SVR could be oxidative stress of the endoplasmic reticulum and mitochondria associated with abnormalities of the vascular system due to fibrosis. Interestingly, abnormal endoplasmic reticulum was associated with patients with HCC for >1 year after SVR. CONCLUSIONS: These results indicate that patients with SVR exhibit a persistent disease state and require long-term follow-up to detect early signs of carcinogenesis.
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Carcinoma Hepatocelular , Hepatite C Crônica , Hepatite C , Neoplasias Hepáticas , Animais , Camundongos , Carcinoma Hepatocelular/patologia , Antivirais/uso terapêutico , Neoplasias Hepáticas/patologia , Hepacivirus , Hepatite C/tratamento farmacológico , Hepatite C Crônica/complicações , Hepatite C Crônica/tratamento farmacológico , Resposta Viral Sustentada , Cirrose Hepática/complicações , Organelas/patologia , Carcinogênese/patologiaRESUMO
Keloids are fibroproliferative lesions caused by abnormal dermal wound healing. Keloidal collagen (KC) is a pathognomic feature of keloids, but the mechanism by which it forms is unknown. This study aimed to evaluate the histopathology of KC and thereby gain clues into how it forms. Methods: The cross-sectional study cohort consisted of a convenience series of patients with keloids who underwent surgical excision. Skin pieces (3 mm2) were collected from the keloid center and nearby control skin. Histopathology was conducted with light and electron microscopy and immunohistochemistry. KC composition was analyzed with protein shotgun analysis. Results: Microscopic analyses revealed the ubiquitous close association between KC and αSMA-positive spindle-shaped cells that closely resembled myofibroblasts. Neither KC nor the spindle-shaped cells were observed in the control tissues. Compared with control skin, the collagen fibers in the KC were overall thinner, their diameter varied more, and their spacing was irregular. These features were particularly pronounced in the collagens in the vicinity of the spindle-shaped cells. Protein shotgun analysis did not reveal a specific collagen in KC but showed abnormally high abundance of collagens I, III, VI, XII, and XIV. Conclusions: These findings suggest that KC may be produced directly by myofibroblasts rather than simply being denatured collagen fibers. Because collagens VI and XII associate with myofibroblast differentiation, and collagen XIV associates with local mechanical stress, these collagens may reflect, and perhaps contribute to, the keloid-specific local conditions that lead to the formation of KC.
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No standards for the assessment of axillary odor intensity and the effects of therapy for osmidrosis have been established. This study presents an objective method for assessing odor severity in patients with osmidrosis and investigates the volatile odorants and skin flora. Methods: The odor intensity was measured pre- and postoperatively using an industrial odor sensor in 79 patients with osmidrosis. Cultures of the axillary skin were obtained during skin flap surgery. Volatile odorants of the patients were assessed using an odor-sensor gas chromatograph mass spectrometer, and samples collected from clothing worn by the patients before and after surgery. The skin pH of the axilla was measured before and after surgery. The locations of odorants and bacteria in the skin were observed using electron microscopy. Results: The mean patient age was 28.8 years, and the male-to-female ratio was 4:3. The odor significantly decreased from 52.6 preoperatively to 20.5 postoperatively (P < 0.001). The bacterial flora on the skin included mostly Staphylococcus. Multiple causative substances (volatile proteins) were identified on gas chromatography. The mean preoperative axillary skin pH was 6.21, which was significantly different than that of patients without osmidrosis (5.92; P < 0.01). Conclusions: An odor sensor accurately assesses odor intensity in patients with osmidrosis. The neutralization of axillary pH may promote the production of odorants by creating the optimal pH for bacterial growth. Odor sensor and pH values can be used pre- and postoperatively as objective assessment measurements for patients with osmidrosis.
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Keloids are red' invasive scars that are driven by chronic inflammation in the reticular dermis. The role of blood vessels in keloid behavior remains poorly understood. In the present study with 32 keloid patients, we examined the hemodynamics of keloid tissue, the anatomy of the blood vessels feeding and draining the keloids, and the vascular histology of keloids. Methods: Ten patients with large anterior chest keloids underwent near-infrared spectroscopy, which measured regional saturation of oxygen and total hemoglobin index in the keloid and surrounding skin. Another 10 patients with large chest keloids and three healthy volunteers underwent multidetector-low computed tomography. The extirpated chest keloids of 12 patients were subjected to histology with optical, CD31 immunohistochemical, and electron microscopy. Results: All keloids had a low regional saturation of oxygen and a high total hemoglobin index, which is indicative of blood congestion. Multidetector-low computed tomography revealed dilation of the arteries and veins that were respectively feeding and draining the keloid leading edge. Hematoxylin-eosin staining and CD31 immunohistochemisty revealed considerable neovascularization in the keloid leading edge but not in the center. Electron microscopy showed that the lumens of many vessels in the keloid center appeared to be occluded or narrowed. Conclusions: Keloids seem to be congested because of increased neovascularization and arterial inflow at the leading edge and blocked outflow due to vascular destruction in the center. The surrounding veins seem to expand in response to this congested state. Methods that improve the blood circulation in keloids may be effective therapies.
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Some plus-stranded RNA viruses generate double-membrane vesicles (DMVs), one type of the membrane replication factories, as replication sites. Little is known about the lipid components involved in the biogenesis of these vesicles. Sphingomyelin (SM) is required for hepatitis C virus (HCV) replication, but the mechanism of SM involvement remains poorly understood. SM biosynthesis starts in the endoplasmic reticulum (ER) and gives rise to ceramide, which is transported from the ER to the Golgi by the action of ceramide transfer protein (CERT), where it can be converted to SM. In this study, inhibition of SM biosynthesis, either by using small-molecule inhibitors or by knockout (KO) of CERT, suppressed HCV replication in a genotype-independent manner. This reduction in HCV replication was rescued by exogenous SM or ectopic expression of the CERT protein, but not by ectopic expression of nonfunctional CERT mutants. Observing low numbers of DMVs in stable replicon cells treated with a SM biosynthesis inhibitor or in CERT-KO cells transfected with either HCV replicon or with constructs that drive HCV protein production in a replication-independent system indicated the significant importance of SM to DMVs. The degradation of SM of the in vitro-isolated DMVs affected their morphology and increased the vulnerability of HCV RNA and proteins to RNase and protease treatment, respectively. Poliovirus, known to induce DMVs, showed decreased replication in CERT-KO cells, while dengue virus, known to induce invaginated vesicles, did not. In conclusion, these findings indicated that SM is an essential constituent of DMVs generated by some plus-stranded RNA viruses.IMPORTANCE Previous reports assumed that sphingomyelin (SM) is essential for HCV replication, but the mechanism was unclear. In this study, we showed for the first time that SM and ceramide transfer protein (CERT), which is in the SM biosynthesis pathway, are essential for the biosynthesis of double-membrane vesicles (DMVs), the sites of viral replication. Low numbers of DMVs were observed in CERT-KO cells transfected with replicon RNA or with constructs that drive HCV protein production in a replication-independent system. HCV replication was rescued by ectopic expression of the CERT protein, but not by CERT mutants, that abolishes the binding of CERT to vesicle-associated membrane protein-associated protein (VAP) or phosphatidylinositol 4-phosphate (PI4P), indicating new roles for VAP and PI4P in HCV replication. The biosynthesis of DMVs has great importance to replication by a variety of plus-stranded RNA viruses. Understanding of this process is expected to facilitate the development of diagnosis and antivirus.
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Proteínas de Transporte/metabolismo , Hepacivirus/metabolismo , Esfingomielinas/metabolismo , Replicação Viral/fisiologia , Transporte Biológico , Proteínas de Transporte/genética , Linhagem Celular , Ceramidas , Retículo Endoplasmático/metabolismo , Técnicas de Inativação de Genes , Complexo de Golgi/metabolismo , Células HEK293 , Hepatite C/virologia , Humanos , Fosfatos de Fosfatidilinositol , RNA Viral/genéticaRESUMO
BACKGROUND: The authors developed a noncontact low-frequency ultrasound device that delivers high-intensity mechanical force based on phased-array technology. It may aid wound healing because it is likely to be associated with lower risks of infection and heat-induced pain compared with conventional ultrasound methods. The authors hypothesized that the microdeformation it induces accelerates wound epithelialization. Its effects on key wound-healing processes (angiogenesis, collagen accumulation, and angiogenesis-related gene transcription) were also examined. METHODS: Immediately after wounding, bilateral acute wounds in C57BL/6J mice were noncontact low-frequency ultrasound- and sham-stimulated for 1 hour/day for 3 consecutive days (10 Hz/90.6 Pa). Wound closure (epithelialization) was recorded every 2 days as the percentage change in wound area relative to baseline. Wound tissue was procured on days 2, 5, 7, and 14 (five to six per time point) and subjected to histopathology with hematoxylin and eosin and Masson trichrome staining, CD31 immunohistochemistry, and quantitative polymerase-chain reaction analysis. RESULTS: Compared to sham-treated wounds, ultrasound/phased-array-treated wounds exhibited significantly accelerated epithelialization (65 ± 27 percent versus 30 ± 33 percent closure), angiogenesis (4.6 ± 1.7 percent versus 2.2 ± 1.0 percent CD31 area), and collagen deposition (44 ± 14 percent versus 28 ± 13 percent collagen density) on days 5, 2, and 5, respectively (all p < 0.05). The expression of Notch ligand delta-like 1 protein (Dll1) and Notch1, which participate in angiogenesis, was transiently enhanced by treatment on days 2 and 5, respectively. CONCLUSIONS: The authors' noncontact low-frequency ultrasound phased-array device improved the wound-healing rate. It was associated with increased early neovascularization that was followed by high levels of collagen-matrix production and epithelialization. The device may expand the mechanotherapeutic proangiogenesis field, thereby helping stimulate a revolution in infected wound care.
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Pele/lesões , Terapia por Ultrassom/métodos , Cicatrização/fisiologia , Ferimentos e Lesões/terapia , Animais , Colágeno/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/fisiologia , Pele/metabolismo , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologiaRESUMO
In vitro chondrogenesis of mesenchymal stem cells (MSCs) mimics in vivo chondrogenesis of MSCs. However, the size of the cartilage pellets that can be attained in vitro is limited by current methods; therefore, some modifications are required to obtain larger pellets. Petaloid pieces of recombinant peptide (petaloid RCP) have the advantage of creating spaces between cells in culture. The RCP used here is based on the alpha-1 sequence of human collagen type I and contains 12 Arg-Gly-Asp motifs. We examined the effect and mechanisms of adding petaloid RCP on the in vitro chondrogenesis of human synovial MSCs by culturing 125k cells with or without 0.125 mg petaloid RCP in chondrogenic medium for 21 days. The cartilage pellets were sequentially analyzed by weight, sulfated glycosaminoglycan content, DNA retention, and histology. Petaloid RCP significantly increased the weight of the cartilage pellets: The petaloid RCP group weighed 7.7 ± 1.2 mg (n = 108), whereas the control group weighed 5.3 ± 1.6 mg. Sulfated glycosaminoglycan and DNA contents were significantly higher in the petaloid RCP group than in the control group. Light and transmission electron microscopy images showed that the petaloid RCP formed the framework of the pellet at day 1, the framework was broken by production of cartilage matrix by the synovial MSCs at day 7, and the cartilage pellet grew larger, with diffuse petaloid RCP remaining, at day 21. Therefore, petaloid RCP formed a framework for the pellet, maintained a higher cell number, and promoted in vitro cartilage formation of synovial MSCs. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. J Orthop Res 37:1350-1357, 2019.
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Condrogênese/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Peptídeos/farmacologia , Membrana Sinovial/citologia , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular , Feminino , Glicosaminoglicanos/metabolismo , Humanos , Proteínas Recombinantes/farmacologiaRESUMO
BACKGROUND: Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage and meniscus regeneration. Synovial tissue can be histologically classified into three regions; surface, stromal and perivascular region, but the localization of synovial MSCs has not been fully investigated. We identified markers specific for each region, and compared properties of MSCs derived from each region in the synovium. METHODS: The intensity of immunostaining with 19 antibodies was examined for surface, stromal, and perivascular regions of human synovium from six osteoarthritis patients. Specific markers were identified and synovial cells derived from each region were sorted. Proliferation, surface marker expression, chondrogenesis, calcification and adipogenesis potentials were compared in synovial MSCs derived from the three regions. RESULTS: We selected CD55+ CD271- for synovial cells in the surface region, CD55- CD271- in the stromal region, and CD55- CD271+ in the perivascular region. The ratio of the sorted cells to non-hematopoietic lineage cells was 5% in the surface region, 70% in the stromal region and 15% in the perivascular region. Synovial cells in the perivascular fraction had the greatest proliferation potential. After expansion, surface marker expression profiles and adipogenesis potentials were similar but chondrogenic and calcification potentials were higher in synovial MSCs derived from the perivascular region than in those derived from the surface and stromal regions. CONCLUSIONS: We identified specific markers to isolate synovial cells from the surface, stromal, and perivascular regions of the synovium. Synovial MSCs in the perivascular region had the highest proliferative and chondrogenic potentials among the three regions.
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Biomarcadores/metabolismo , Células-Tronco Mesenquimais/metabolismo , Microscopia Eletrônica de Transmissão/métodos , Membrana Sinovial/metabolismo , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular , Citometria de Fluxo , Humanos , Pessoa de Meia-IdadeRESUMO
Osseointegrated implants have been recognized as being very reliable and having long-term predictability. However, host defense mechanisms against infection have been known to be impaired around a dental implant because of the lack of a periodontal ligament (PDL). The purpose of our experimental design was to produce cementum and PDL on the implant surface adopting cell sheet technology. To this aim we used PDL-derived cells, which contain multipotential stem cells, as the cell source and we cultured them on an implant material constituted of commercially pure titanium treated with acid etching, blasting, and a calcium phosphate (CaP) coating to improve cell attachment. Implants with adhered human PDL cell sheets were transplanted into bone defects in athymic rat femurs as a xenogeneic model. Implants with adhered canine PDL-derived cell sheets were transplanted into canine mandibular bone as an autologous model. We confirmed that PDL-derived cells cultured with osteoinductive medium had the ability to induce cementum formation. The attachment of PDL cells onto the titanium surface with three surface treatments was accelerated, compared with that onto the smooth titanium surface, at 40 min after starting incubation. Results in the rat model showed that cementum-like and PDL-like tissue was partly observed on the titanium surface with three surface treatments in combination with adherent PDL-derived cell sheets. On the other hand, osseointegration was observed on almost all areas of the smooth titanium surface that had PDL-derived cell sheets, but did not have the three surface treatments. In the canine model, histological observation indicated that formation of cementum-like and PDL-like tissue was induced on the titanium surface with surface treatments and that the PDL-like tissue was perpendicularly oriented between the titanium surface with cementum-like tissue and the bone. Results demonstrate that a periodontal-like structure was formed around a titanium implant, which is similar to the environment existing around a natural tooth. The clinical application of dental implants combined with a cell sheet technique may be feasible as an alternative implant therapy. Furthermore, application of this methodology may play an innovative role in the periodontal, prosthetic, and orthodontic fields in dentistry.
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Prótese Ancorada no Osso , Interface Osso-Implante , Implantação Dentária Endóssea , Implantes Dentários , Ligamento Periodontal , Titânio , Animais , Cães , Xenoenxertos , Masculino , Ligamento Periodontal/citologia , Ligamento Periodontal/transplante , Ratos , Ratos Nus , Propriedades de SuperfícieRESUMO
We previously reported transcriptional repression-induced atypical cell death of neuron (TRIAD), a new type of necrosis that is mainly regulated by Hippo pathway signaling and distinct from necroptosis regulated by RIP1/3 pathway. Here, we examined the ultrastructural and biochemical features of neuronal cell death in the brains of human HD patients in parallel with the similar analyses using mutant Htt-knock-in (Htt-KI) mice. LATS1 kinase, the critical regulator and marker of TRIAD, is actually activated in cortical neurons of postmortem human HD and of Htt-KI mouse brains, while apoptosis promoter kinase Plk1 was inactivated in human HD brains. Expression levels of YAP/YAPdeltaC were decreased in cortical neurons of human HD brains. Ultra-structural analyses revealed extreme enlargement of endoplasmic reticulum (ER), which characterizes TRIAD, in cortical neurons of human HD and those of Htt-KI mice. These biochemical and morphological results support that TRIAD occurs in human and mouse neurons under the HD pathology.
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Doença de Huntington/metabolismo , Doença de Huntington/patologia , Necrose/metabolismo , Necrose/patologia , Neurônios/metabolismo , Neurônios/ultraestrutura , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose/fisiologia , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Proteínas de Ciclo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Técnicas de Introdução de Genes , Humanos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição , Proteínas de Sinalização YAP , Quinase 1 Polo-LikeRESUMO
The trigger for bone remodeling is bone resorption by osteoclasts. Osteoclast differentiation only occurs on the old bone, which needs to be repaired under physiological conditions. However, uncontrolled bone resorption is often observed in pro-inflammatory bone diseases, such as rheumatoid arthritis. Mature osteoclasts are multinuclear cells that differentiate from monocyte/macrophage lineage cells by cell fusion. Although Osteoclast precursors should migrate across osteoblast layer to reach bone matrix before maturation, the underlying mechanisms have not yet been elucidated in detail. We herein found that osteoclast precursors utilize two routes to migrate across osteoblast layer by confocal- and electro-microscopic observations. The osteoclast supporting activity of osteoblasts inversely correlated with osteoblast density and was positively related to the number of osteoclast precursors under the osteoblast layer. Osteoclast differentiation was induced by IL-1ß, but not by PGE2 in high-density osteoblasts. Osteoblasts and osteoclast precursors expressed CX3CL1 and CX3CR1, respectively, and the expression of CX3CL1 increased in response to interleukin-1ß. An anti-CX3CL1-neutralizing antibody inhibited the migration of osteoclast precursors and osteoclast differentiation. These results strongly suggest the involvement of CX3CL1 in the migration of osteoclast precursors and osteoclastogenesis, and will contribute to the development of new therapies for bone diseases. J. Cell. Physiol. 232: 1739-1745, 2017. © 2016 Wiley Periodicals, Inc.
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Movimento Celular/efeitos dos fármacos , Quimiocina CX3CL1/metabolismo , Interleucina-1beta/farmacologia , Osteoblastos/citologia , Osteoclastos/citologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Contagem de Células , Dinoprostona/farmacologia , Células HEK293 , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Camundongos , Testes de Neutralização , Osteoblastos/efeitos dos fármacos , Osteoblastos/ultraestrutura , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Imagem com Lapso de TempoRESUMO
The main pathogenesis of intervertebral disc (IVD) herniation involves disruption of the annulus fibrosus (AF) caused by ageing or excessive mechanical stress and the resulting prolapse of the nucleus pulposus. Owing to the avascular nature of the IVD and lack of understanding the mechanisms that maintain the IVD, current therapies do not lead to tissue regeneration. Here we show that homeobox protein Mohawk (Mkx) is a key transcription factor that regulates AF development, maintenance and regeneration. Mkx is mainly expressed in the outer AF (OAF) of humans and mice. In Mkx(-/-) mice, the OAF displays a deficiency of multiple tendon/ligament-related genes, a smaller OAF collagen fibril diameter and a more rapid progression of IVD degeneration compared with the wild type. Mesenchymal stem cells overexpressing Mkx promote functional AF regeneration in a mouse AF defect model, with abundant collagen fibril formation. Our results indicate a therapeutic strategy for AF regeneration.
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Anel Fibroso/fisiologia , Proteínas de Homeodomínio/metabolismo , Disco Intervertebral/fisiologia , Regeneração , Adulto , Animais , Anel Fibroso/metabolismo , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Disco Intervertebral/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND AND OBJECTIVE: Mid-infrared erbium: yttrium-aluminum-garnet (Er:YAG) and erbium, chromium: yttrium-scandium-gallium-garnet (Er,Cr:YSGG) lasers (2.94- and 2.78-µm, respectively) are utilized for effective dental hard tissue treatment because of their high absorption in water, hydroxide ion, or both. Recently, a mid-infrared tunable, nanosecond pulsed, all-solid-state chromium-doped: cadmium-selenide (Cr:CdSe) laser system was developed, which enables laser oscillation in the broad spectral range around 2.9 µm. The purpose of this study was to evaluate the ablation of dental hard tissue by the nanosecond pulsed Cr:CdSe laser at a wavelength range of 2.76-3.00 µm. STUDY DESIGN/MATERIALS AND METHODS: Enamel, dentin, and cementum tissue were irradiated at a spot or line at a fluence of 0-11.20 J/cm2 /pulse (energy output: 0-2.00 mJ/pulse) with a repetition rate of 10 Hz and beam diameter of â¼150 µm on the target (pulse width â¼250 ns). After irradiation, morphological changes, ablation threshold, depth, and efficiency, and thickness of the structurally and thermally affected layer of irradiated surfaces were analyzed using stereomicroscopy, scanning electron microscopy (SEM), and light microscopy of non-decalcified histological sections. RESULTS: The nanosecond pulsed irradiation without water spray effectively ablated dental hard tissue with no visible thermal damage such as carbonization. The SEM analysis revealed characteristic micro-irregularities without major melting and cracks in the lased tissue. The ablation threshold of dentin was the lowest at 2.76 µm and the highest at 3.00 µm. The histological analysis revealed minimal thermal and structural changes â¼20 µm wide on the irradiated dentin surfaces with no significant differences between wavelengths. The efficiency of dentin ablation gradually increased from 3.00 to 2.76 µm, at which point the highest ablation efficiency was observed. CONCLUSION: The nanosecond pulsed Cr:CdSe laser demonstrated an effective ablation ability of hard dental tissues, which was remarkably wavelength-dependent on dentin at the spectral range of 2.76-3.00 µm. These results demonstrate the potential feasibility of the use of pulsed Cr:CdSe laser as a novel laser system for dental treatment. Lasers Surg. Med. 48:965-977, 2016. © 2016 Wiley Periodicals, Inc.
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Cemento Dentário/cirurgia , Esmalte Dentário/cirurgia , Dentina/cirurgia , Lasers de Estado Sólido/uso terapêutico , Cemento Dentário/diagnóstico por imagem , Cemento Dentário/patologia , Esmalte Dentário/diagnóstico por imagem , Esmalte Dentário/patologia , Dentina/diagnóstico por imagem , Dentina/patologia , Humanos , Técnicas In Vitro , Microscopia Eletrônica de VarreduraRESUMO
OBJECTIVE: Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs) are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1) whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2) whether aggregates of human MSCs promoted lubricin expression, and (3) whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats. METHODS: For in vitro analysis, human bone marrow (BM) MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages. RESULTS: In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4), which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in the superficial cartilage. CONCLUSION: Cartilage derived from MSCs expressed lubricin protein both in vitro and in vivo. Aggregation promoted lubricin expression of MSCs in vitro and transplantation of aggregates of MSCs regenerated cartilage including the superficial zone in a rat osteochondral defect model. Our results indicate that aggregated MSCs could be clinically relevant for therapeutic approaches to articular cartilage regeneration with an appropriate superficial zone in the future.
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Células da Medula Óssea/metabolismo , Cartilagem/metabolismo , Regulação da Expressão Gênica , Glicoproteínas/biossíntese , Células-Tronco Mesenquimais/metabolismo , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Células Cultivadas , Condrogênese , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Articulação do Joelho/fisiopatologia , Transplante de Células-Tronco Mesenquimais/métodos , Microscopia Eletrônica de Transmissão , Proteoglicanas/biossíntese , Proteoglicanas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Transgênicos , Regeneração , Microtomografia por Raio-XRESUMO
Though the Er:YAG laser (ErL) has been used in periodontal therapy, the irradiated tip damage has not been studied in detail. In this study, the change in the energy output, surface morphology, and temperature of quartz tips was evaluated following contact irradiation. Soft tissue, calculus on extracted human teeth, and porcine bone were irradiated by ErL for 60 min at 14.2 or 28.3 J/cm(2)/pulse and 20 Hz with or without water spray. The energy output ratio declined the most in the calculus group, followed by the bone and soft tissue groups with and/or without water spray. Carbon contamination was detected in all groups, and contamination by P, Ca, and/or other inorganic elements was observed in the calculus and bone groups. The rate of energy output reduction and the degree of surface alteration/contamination is variously influenced by the targeting tissue, temperature elevation of the tip and water spray.
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Processo Alveolar/efeitos da radiação , Cálculos Dentários/radioterapia , Instrumentos Odontológicos , Transferência de Energia/efeitos da radiação , Gengiva/efeitos da radiação , Terapia a Laser/instrumentação , Lasers de Estado Sólido , Quartzo/química , Quartzo/efeitos da radiação , Animais , Contaminação de Equipamentos , Humanos , Técnicas In Vitro , Microscopia Eletrônica de Varredura , Espectrometria por Raios X , Propriedades de Superfície/efeitos da radiação , Suínos , Temperatura , ÁguaRESUMO
PURPOSE: To examine whether the T1 rho value reflects histological changes in menisci we analyzed the relationship between T1 rho value and histological findings in intact and radially incised menisci of pigs. MATERIALS AND METHODS: Seven microminipigs were used for this experiment. A radial incision was created and repaired in the medial meniscus, which was evaluated 4 weeks after surgery. Sagittal T1 rho mapping images were taken by 3.0T magnetic resonance imaging (MRI). The region of interest was set by dividing the meniscus into six zones (from zone 1 to zone 6). For histological evaluation of intact menisci, characteristics of each zone were determined. In incised menisci, a histological score was used to evaluate pathological change. RESULTS: In intact lateral menisci, the zone where histological findings indicated fibrocartilage showed a lower T1 rho value (34.2 ± 2.3 msec) than hyaline-like cartilage (38.2 ± 2.5 msec) or fibrous tissue (37.2 ± 2.0 msec). In incised medial menisci, T1 rho values increased (about 50-90 msec) in the zone where histological findings indicated that synovial ingrowth, scar tissue formation, and degenerative changes had occurred. There were correlations between T1 rho values and histological scores in all zones (r = 0.62-0.92, P = 0.001-0.026). CONCLUSION: Zonal variations of the T1 rho value were observed in intact menisci due to varying structure in each zone. T1 rho values were correlated with histological changes such as collagen fiber organization and safranin-o stainability in incised menisci. This study supports T1 rho mapping as useful for evaluating ultrastructural composition in menisci.
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Imageamento por Ressonância Magnética , Meniscos Tibiais/anatomia & histologia , Animais , Processamento de Imagem Assistida por Computador , Suínos , Porco MiniaturaRESUMO
Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage and meniscus regeneration. Use of primary MSCs is the preferable because these cells are safer than cells passaged several times in terms of probability of chromosome abnormalities. The effect of hypoxia on the proliferation of MSCs is controversial and remains unknown in primary synovial MSCs. Primary synovial MSCs were cultured at normoxia or hypoxia, and colony number, cell number, surface epitopes, mitochondria activity, TEM finding, and chondrogenic potential were analyzed. To investigate the effect of hypoxia on attachment of synovial MSCs, cells were cultured at hypoxia for the first 3 days, then cultured at normoxia. To investigate the effect of hypoxia on proliferation, cells were also cultured at hypoxia for the last 11 days. Hypoxia increased colony number and cell number per dish in primary synovial MSCs. Hypoxia did not affect cell number per colony, surface epitopes, mitochondria activity, TEM finding or chondrogenic potential. Hypoxia for the first 3 days did not alter colony number per dish or cell number per dish, while hypoxia for the last 11 days increased. Hypoxia enhanced proliferation through increase of colony formation rate with chondrogenic potential in primary synovial MSCs.
Assuntos
Condrogênese/fisiologia , Células-Tronco Mesenquimais/citologia , Membrana Sinovial/citologia , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular/fisiologia , Hipóxia Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Citocinas , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco , Membrana Sinovial/metabolismoRESUMO
BACKGROUND: The brain vascular system arises from the perineural vascular plexus (PNVP) which sprouts radially into the neuroepithelium and subsequently branches off laterally to form a secondary plexus in the subventricular zone (SVZ), the subventricular vascular plexus (SVP). The process of SVP formation remains to be fully elucidated. We investigated the role of Foxc1 in early stage vascular formation in the ventral telencephalon. RESULTS: The Foxc1 loss of function mutant mouse, Foxc1(ch/ch) , showed enlarged telencephalon and hemorrhaging in the ventral telencephalon by embryonic day 11.0. The mutant demonstrated blood vessel dilation and aggregation of endothelial cells in the SVZ after the invasion of endothelial cells through the radial path, which lead to failure of SVP formation. During this early stage of vascular development, Foxc1 was expressed in endothelial cells and pericytes, as well as in cranial mesenchyme surrounding the neural tube. Correspondingly, abnormal deposition pattern of basement membrane proteins around the vessels and increased strong Vegfr2 staining dots were found in the aggregation sites. CONCLUSIONS: These observations reveal an essential role for Foxc1 in the early stage of vascular formation in the telencephalon.
Assuntos
Circulação Cerebrovascular/fisiologia , Embrião de Mamíferos , Fatores de Transcrição Forkhead/metabolismo , Telencéfalo , Animais , Embrião de Mamíferos/irrigação sanguínea , Embrião de Mamíferos/embriologia , Fatores de Transcrição Forkhead/genética , Camundongos , Camundongos Mutantes , Telencéfalo/irrigação sanguínea , Telencéfalo/embriologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVE: The aim of this study is to elucidate the structure of the resin-dentin interface formed by photochemical dentin treatment using an argon fluoride (ArF) excimer laser. BACKGROUND DATA: The ArF excimer laser processes material by photochemical reaction without generating heat, while also providing surface conditioning that enhances material adhesion. In the case of bonding between resin and dentin, we demonstrated in a previous study that laser etching using an ArF excimer laser produced bonding strength comparable to that of the traditional bonding process; however, conditions of the bonding interface have not been fully investigated. METHODS: A dentin surface was irradiated in air with an ArF excimer laser followed by bonding treatment. Cross sections were observed under light microscope, transmission electron microscope (TEM), and scanning electron microscope, then analyzed using an energy dispersive X-ray spectroscope (EDS): EDS line profiles of the elements C, O, Si, Cl, P, and Ca at the resin-dentin interface were obtained. RESULTS: The density of C in resin decreased as it approached the interface, reaching its lowest level within the dentin at a depth of 2 µm from the resin-dentin interface on EDS. There was no hybrid layer observed at the interface on TEM. Therefore, it was suggested that the resin monomer infiltrated into the microspaces produced on the dentin surface by laser abrasion. CONCLUSIONS: The monomer infiltration without hybrid layer is thought to be the adhesion mechanism after laser etching. Therefore, the photochemical processes at the bonding interface achieved using the ArF excimer laser has great potential to be developed into a new bonding system in dentistry.