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1.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34199089

RESUMO

The meniscus possesses low self-healing properties. A perfect regenerative technique for this tissue has not yet been developed. This work aims to evaluate the role of hypoxia in meniscal development in vitro. Menisci from neonatal pigs (day 0) were harvested and cultured under two different atmospheric conditions: hypoxia (1% O2) and normoxia (21% O2) for up to 14 days. Samples were analysed at 0, 7 and 14 days by histochemical (Safranin-O staining), immunofluorescence and RT-PCR (in both methods for SOX-9, HIF-1α, collagen I and II), and biochemical (DNA, GAGs, DNA/GAGs ratio) techniques to record any possible differences in the maturation of meniscal cells. Safranin-O staining showed increments in matrix deposition and round-shape "fibro-chondrocytic" cells in hypoxia-cultured menisci compared with controls under normal atmospheric conditions. The same maturation shifting was observed by immunofluorescence and RT-PCR analysis: SOX-9 and collagen II increased from day zero up to 14 days under a hypoxic environment. An increment of DNA/GAGs ratio typical of mature meniscal tissue (characterized by fewer cells and more GAGs) was observed by biochemical analysis. This study shows that hypoxia can be considered as a booster to achieve meniscal cell maturation, and opens new opportunities in the field of meniscus tissue engineering.


Assuntos
Diferenciação Celular , Hipóxia/metabolismo , Menisco/citologia , Menisco/metabolismo , Animais , Biomarcadores , Células Cultivadas , Condrócitos/metabolismo , Expressão Gênica , Glicosaminoglicanos/metabolismo , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Suínos , Engenharia Tecidual/métodos
2.
Cells ; 9(3)2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32204578

RESUMO

Bone is an active tissue where bone mineralization and resorption occur simultaneously. In the case of fracture, there are numerous factors required to facilitate bone healing including precursor cells and blood vessels. To evaluate the interaction between bone marrow-derived mesenchymal stem cells (BMSC)-the precursor cells able to differentiate into bone-forming cells and human umbilical vein endothelial cells (HUVEC)-a cell source widely used for the study of blood vessels. We performed direct coculture of BMSC and HUVEC in normoxia and chemically induced hypoxia using Cobalt(II) chloride and Dimethyloxaloylglycine and in the condition where oxygen level was maintained at 1% as well. Cell proliferation was analyzed by crystal violet staining. Osteogenesis was examined by Alizarin Red and Collagen type I staining. Expression of angiogenic factor-vascular endothelial growth factor (VEGF) and endothelial marker-von Willebrand factor (VWF) were demonstrated by immunohistochemistry and enzyme-linked immunosorbent assay. The quantitative polymerase chain reaction was also used to evaluate gene expression. The results showed that coculture in normoxia could retain both osteogenic differentiation and endothelial markers while hypoxic condition limits cell proliferation and osteogenesis but favors the angiogenic function even after 1 of day treatment.


Assuntos
Diferenciação Celular , Células Endoteliais da Veia Umbilical Humana/citologia , Células-Tronco Mesenquimais/citologia , Neovascularização Fisiológica , Osteogênese , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Hipóxia Celular , Proliferação de Células , Técnicas de Cocultura , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Fator de von Willebrand/metabolismo
3.
In Vivo ; 33(6): 1851-1855, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31662512

RESUMO

AIM: To develop a method capable of identifying human corneal limbal stem cells (LSCs) and follow their proliferation and migration in the epithelium. MATERIALS AND METHODS: Ten fresh matched pairs of cadaveric normal human corneas were obtained from donors. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was used to target LSCs. The distribution of CFSE-positive cell clusters was analyzed by fluorescence microscopy by counterstaining with 4',6-diamidino-2-phenylindole (DAPI). Fluorescence was digitally recorded for seven days, and the rate of cell movement was determined. RESULTS: CFSE-labeled cells were tracked in corneas. Analysis of time sequences revealed that they moved centripetally. Daily average CFSE-labeled LSC movement was 0.073±0.01 cm (±SD). CONCLUSION: CFSE allowed us to identify LSCs and to track their centripetal migration from the limbal basal layer to the anterior ocular surface. This experimental system appears to be a valuable tool for further studies on corneal epithelial cell migration and proliferation.


Assuntos
Movimento Celular/fisiologia , Córnea/fisiologia , Epitélio Corneano/fisiologia , Fluoresceínas/metabolismo , Células-Tronco/fisiologia , Succinimidas/metabolismo , Técnicas de Cultura de Células/métodos , Proliferação de Células/fisiologia , Córnea/metabolismo , Epitélio Corneano/metabolismo , Humanos , Células-Tronco/metabolismo
4.
J Tissue Eng Regen Med ; 13(6): 1007-1018, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30811859

RESUMO

Due to their osteoconductive and inductive properties, a variety of calcium phosphate (CaP) scaffolds are commonly used in orthopaedics as graft material to heal bone defects. In this study, we have used two CaP scaffolds with different hydroxyapatite (HA) and ß-tricalcium phosphate (ß-TCP) ratios (MBCP®; 60/40 and MBCP+ ®; 20/80) to investigate their intrinsic capacity to favour human bone marrow stem cells (hBMSCs) osteogenic differentiation capacity. We report that MBCP+ ® showed in in vitro culture model a higher rate of calcium ion release in comparison with MBCP®. In two defined coculture systems, the hBMSC seeded onto MBCP+ ® presented an increased amount of VEGF secretion, resulting in an enhanced endothelial cell proliferation and capillary formation compared with hBMSC seeded onto MBCP®. When both ceramics combined with hBMSC were implanted in a nude mouse model, we observed a faster osteogenic differentiation and enhancement mature bone deposition sustained by the presence of a vast host vasculature within the MBCP+ ® ceramics. Bone formation was observed in samples highly positive to the activation of calcium sensing receptor protein (CaSr) on the surface of seeded hBMSC that also shown higher BMP-2 protein expression. With these data we provide valuable insights in the possible mechanisms of ossification and angiogenesis by hBMSC that we believe to be primed by calcium ions released from CaP scaffolds. Evidences could lead to an optimization of ceramic scaffolds to prime bone repair.


Assuntos
Fosfatos de Cálcio/farmacologia , Cerâmica/farmacologia , Células-Tronco Mesenquimais/citologia , Osteogênese/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Durapatita/química , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos Nus , Neovascularização Fisiológica/efeitos dos fármacos , Receptores de Detecção de Cálcio/metabolismo , Transdução de Sinais , Engenharia Tecidual
5.
J Biomed Mater Res B Appl Biomater ; 107(4): 951-964, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30176112

RESUMO

Highly porous small-diameter vascular grafts (SDVGs) prepared with elastomeric materials such as poly(ether urethane) (PEtU)-polydimethylsiloxane (PEtU-PDMS) are capable to biodegrade but may develop aneurismal dilatation. Through a compliance/patency assessment with ultrasound techniques, the current study investigated the functionality, in terms of patency and endothelialization, of a highly flexible and porous Nitinol mesh incorporated into PEtU-PDMS SDVGs in a sheep carotid model. Nitinol-PEtU-PDMS grafts with an internal diameter (ID) of 4 mm were manufactured by spray, phase-inversion technique. Compliance tests were performed by ultrasound (US) imaging using a high-resolution ultrasound diagnostic system. Ten adult sheep were implanted with 7 cm long grafts. The results of this study demonstrated an almost complete neointima luminal coverage in transmurally porous grafts reinforced with the Nitinol meshes after 6 months of implantation. Additionally, ultrasound has been used to quantitatively assess and monitor hemodynamic variables in an experimental model of synthetic vascular graft replacement. The use of reinforced PEtU-PDMS grafts may accelerate the endothelialization process of relatively long grafts, such as those needed for aortocoronary bypass. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 951-964, 2019.


Assuntos
Ligas , Implante de Prótese Vascular , Prótese Vascular , Artérias Carótidas , Ponte de Artéria Coronária , Elastômeros , Animais , Artérias Carótidas/diagnóstico por imagem , Artérias Carótidas/metabolismo , Artérias Carótidas/cirurgia , Porosidade , Ovinos
6.
Front Physiol ; 9: 181, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29593553

RESUMO

Bone remodeling process consists in a slow building phase and in faster resorption with the objective to maintain a functional skeleton locomotion to counteract the Earth gravity. Thus, during spaceflights, the skeleton does not act against gravity, with a rapid decrease of bone mass and density, favoring bone fracture. Several studies approached the problem by imaging the bone architecture and density of cosmonauts returned by the different spaceflights. However, the weaknesses of the previously reported studies was two-fold: on the one hand the research suffered the small statistical sample size of almost all human spaceflight studies, on the other the results were not fully reliable, mainly due to the fact that the observed bone structures were small compared with the spatial resolution of the available imaging devices. The recent advances in high-resolution X-ray tomography have stimulated the study of weight-bearing skeletal sites by novel approaches, mainly based on the use of the mouse and its various strains as an animal model, and sometimes taking advantage of the synchrotron radiation support to approach studies of 3D bone architecture and mineralization degree mapping at different hierarchical levels. Here we report the first, to our knowledge, systematic review of the recent advances in studying the skeletal bone architecture by high-resolution X-ray tomography after submission of mice models to microgravity constrains.

9.
Dis Model Mech ; 9(6): 685-96, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27125279

RESUMO

The ACVR1 gene encodes a type I receptor of bone morphogenetic proteins (BMPs). Activating mutations in ACVR1 are responsible for fibrodysplasia ossificans progressiva (FOP), a rare disease characterized by congenital toe malformation and progressive heterotopic endochondral ossification leading to severe and cumulative disability. Until now, no therapy has been available to prevent soft-tissue swelling (flare-ups) that trigger the ossification process. With the aim of finding a new therapeutic strategy for FOP, we developed a high-throughput screening (HTS) assay to identify inhibitors of ACVR1 gene expression among drugs already approved for the therapy of other diseases. The screening, based on an ACVR1 promoter assay, was followed by an in vitro and in vivo test to validate and characterize candidate molecules. Among compounds that modulate the ACVR1 promoter activity, we selected the one showing the highest inhibitory effect, dipyridamole, a drug that is currently used as a platelet anti-aggregant. The inhibitory effect was detectable on ACVR1 gene expression, on the whole Smad-dependent BMP signaling pathway, and on chondrogenic and osteogenic differentiation processes by in vitro cellular assays. Moreover, dipyridamole reduced the process of heterotopic bone formation in vivo Our drug repositioning strategy has led to the identification of dipyridamole as a possible therapeutic tool for the treatment of FOP. Furthermore, our study has also defined a pipeline of assays that will be useful for the evaluation of other pharmacological inhibitors of heterotopic ossification.


Assuntos
Receptores de Ativinas Tipo I/genética , Ensaios de Triagem em Larga Escala/métodos , Miosite Ossificante/tratamento farmacológico , Transcrição Genética , Animais , Biomarcadores/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Condrogênese/efeitos dos fármacos , Dipiridamol/farmacologia , Dipiridamol/uso terapêutico , Modelos Animais de Doenças , Camundongos , Miosite Ossificante/metabolismo , Miosite Ossificante/patologia , Ossificação Heterotópica/diagnóstico por imagem , Ossificação Heterotópica/patologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Transcrição Genética/efeitos dos fármacos
10.
J Mech Behav Biomed Mater ; 51: 1-12, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26188564

RESUMO

This manuscript reports the structural alterations occurring in mice skeleton as a consequence of the longest-term exposition (90 days) to simulated microgravity (hindlimb unloading) and hypergravity (2g) ever tested. Bone microstructural features were investigated by means of standard Cone Beam X-ray micro-CT, Synchrotron Radiation micro-CT and histology. Morphometric analysis confirmed deleterious bone architectural changes in lack of mechanical loading with a decrease of bone volume and density, while bone structure alterations caused by hypergravity were less evident. In the femurs from hypergravity-exposed mice, the head/neck cortical thickness increment was the main finding. In addition, in these mice the rate of larger trabeculae (60-75 µm) was significantly increased. Interestingly, the metaphyseal plate presented a significant adaptation to gravity changes. Mineralization of cartilage and bone deposition was increased in the 2g mice, whereas an enlargement of the growth plate cartilage was observed in the hindlimb unloaded group. Indeed, the presented data confirm and reinforce the detrimental effects on bone observed in real space microgravity and reveal region-specific effects on long bones. Finally these data could represent the starting point for further long-term experimentations that can deeply investigate the bone adaptation mechanisms to different mechanical force environments.


Assuntos
Fêmur , Hipergravidade/efeitos adversos , Simulação de Ausência de Peso/efeitos adversos , Animais , Fêmur/citologia , Fêmur/diagnóstico por imagem , Fêmur/fisiologia , Membro Posterior/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Tempo , Suporte de Carga , Microtomografia por Raio-X
11.
PLoS One ; 10(4): e0125110, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25897753

RESUMO

Demineralized bone matrix (DBM) is widely used for bone regeneration. Since DBM is prepared in powder form its handling properties are not optimal and limit the clinical use of this material. Various synthetic and biological carriers have been used to enhance the DBM handling. In this study we evaluated the effect of gamma irradiation on the physical-chemical properties of Pluronic and on bone morphogenetic proteins (BMPs) amount in DBM samples. In vivo studies were carried out to investigate the effect on bone regeneration of a gamma irradiated DBM-Pluronic F127 (DBM-PF127) composite implanted in the femur of rats. Gamma irradiation effects (25 kGy) on physical-chemical properties of Pluronic F127 were investigated by rheological and infrared analysis. The BMP-2/BMP-7 amount after DBM irradiation was evaluated by ELISA. Bone regeneration capacity of DBM-PF127 containing 40% (w/w) of DBM was investigated in transcortical holes created in the femoral diaphysis of Wistar rat. Bone porosity, repaired bone volume and tissue organization were evaluated at 15, 30 and 90 days by Micro-CT and histological analysis. The results showed that gamma irradiation did not induce significant modification on physical-chemical properties of Pluronic, while a decrease in BMP-2/BMP-7 amount was evidenced in sterilized DBM. Micro-CT and histological evaluation at day 15 post-implantation revealed an interconnected trabeculae network in medullar cavity and cellular infiltration and vascularization of DBM-PF127 residue. In contrast a large rate of not connected trabeculae was observed in Pluronic filled and unfilled defects. At 30 and 90 days the DBM-PF127 samples shown comparable results in term of density and thickness of the new formed tissue respect to unfilled defect. In conclusion a gamma irradiated DBM-PF127 composite, although it may have undergone a significant decrease in the concentration of BMPs, was able to maintains bone regeneration capability.


Assuntos
Materiais Biocompatíveis/química , Matriz Óssea/efeitos da radiação , Regeneração Óssea/fisiologia , Poloxâmero/química , Animais , Biomarcadores/metabolismo , Densidade Óssea , Matriz Óssea/química , Matriz Óssea/fisiologia , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 7/genética , Proteína Morfogenética Óssea 7/metabolismo , Fêmur/lesões , Fêmur/cirurgia , Raios gama , Expressão Gênica , Humanos , Masculino , Ratos , Ratos Wistar , Microtomografia por Raio-X
12.
J Craniomaxillofac Surg ; 42(5): e70-9, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23932544

RESUMO

The use of platelet rich plasma (PRP) in bone repair remains highly controversial. In this work, we evaluated the effect of lyophilized PRP on bone regeneration when associated with a silicon stabilized hydroxyapatite tricalcium phosphate scaffold in a rabbit calvarial defect (Skelite). Critical defects were created in the calvaria of twenty-four rabbits. The periosteum was removed and the defects were either left empty or filled with allogeneic PRP gel; Skelite particles; Skelite and PRP gel. Four animals were killed after 4 weeks, 10 animals after 8 and 10 after 16 weeks. Specimens were processed for X-ray microtomography (µCT) and for resin embedded histology. µCT analysis revealed significant osteoid-like matrix and new bone deposition in PRP + Skelite group at both 8 and 16 weeks in respect to Skelite alone. Histologically, PRP + Skelite defects were highly cellular with more abundant osteoid deposition and more regular collagen fibres. Moreover, in vitro migration assays confirmed the chemotactic effect of PRP to endothelial and osteoprogenitor cells. We conclude that the addition of PRP influenced the local tissue microenvironment by providing key cryptic factors for regeneration, thereby enhancing progenitor cell recruitment, collagen and bone matrix deposition, and by creating a bridging interface between the scaffold and bone.


Assuntos
Doenças Ósseas/cirurgia , Cerâmica/química , Hidroxiapatitas/química , Osteogênese/fisiologia , Plasma Rico em Plaquetas/fisiologia , Crânio/cirurgia , Tecidos Suporte/química , Animais , Matriz Óssea/patologia , Regeneração Óssea/fisiologia , Movimento Celular/fisiologia , Microambiente Celular/fisiologia , Colágeno , Células Endoteliais/patologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Masculino , Células-Tronco Mesenquimais/patologia , Osteoblastos/patologia , Inclusão em Plástico , Coelhos , Crânio/patologia , Fatores de Tempo , Engenharia Tecidual/métodos , Microtomografia por Raio-X/métodos
13.
J Cell Physiol ; 228(11): 2210-21, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23606520

RESUMO

Lipocalin-2 (LCN2) is a protein largely expressed in many tissues, associated with different biological phenomena such as cellular differentiation, inflammation and cancer acting as a survival/apoptotic signal. We found that LCN2 was expressed during osteoblast differentiation and we generated transgenic (Tg) mice over-expressing LCN2 in bone. Tg mice were smaller and presented bone microarchitectural changes in both endochondral and intramembranous bones. In particular, Tg bones displayed a thinner layer of cortical bone and a decreased trabecular number. Osteoblast bone matrix deposition was reduced and osteoblast differentiation was slowed-down. Differences were also observed in the growth plate of young transgenic mice where chondrocyte displayed a more immature phenotype and a lower proliferation rate. In bone marrow cell cultures from transgenic mice, the number of osteoclast progenitors was increased whereas in vivo it was increased the number of mature osteoclasts expressing tartrate-resistant acid phosphatase (TRAP). Finally, while osteoprotegerin (OPG) levels remained unchanged, the expression of the conventional receptor activator of nuclear factor-κB ligand (RANKL) and of the IL-6 was enhanced in Tg mice. In conclusion, we found that LCN2 plays a role in bone development and turnover having both a negative effect on bone formation, by affecting growth plate development and interfering with osteoblast differentiation, and a positive effect on bone resorption by enhancing osteoclast compartment.


Assuntos
Proteínas de Fase Aguda/metabolismo , Desenvolvimento Ósseo , Remodelação Óssea , Fêmur/metabolismo , Lipocalinas/metabolismo , Proteínas Oncogênicas/metabolismo , Fosfatase Ácida/metabolismo , Animais , Animais Recém-Nascidos , Tamanho Corporal , Reabsorção Óssea/diagnóstico por imagem , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Diferenciação Celular , Fêmur/diagnóstico por imagem , Fêmur/patologia , Lâmina de Crescimento/diagnóstico por imagem , Lâmina de Crescimento/metabolismo , Lâmina de Crescimento/patologia , Interleucina-6/metabolismo , Isoenzimas/metabolismo , Lipocalina-2 , Camundongos , Camundongos Transgênicos , Tamanho do Órgão , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteoclastos/patologia , Radiografia , Receptores de Superfície Celular/metabolismo , Reprodutibilidade dos Testes , Fosfatase Ácida Resistente a Tartarato , Transgenes/genética
14.
Tissue Eng Part A ; 19(1-2): 152-65, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22849574

RESUMO

The periosteum plays a pivotal role during bone development and repair contributing to bone vascularization and osteoprogenitor cells source. We propose a periosteal substitute engineered using a platelet-rich plasma (PRP) membrane incorporating autologous bone marrow-derived mesenchymal stem cells (PRP/BMSC gel membrane) to be wrapped around an osteoconductive scaffold for regeneration of compromised bone defects. The PRP/BMSC gel membrane was optimized using different compositions for optimal release of vascular endothelial growth factor (VEGF) and platelet derived growth factor-BB (PDGF-BB). Survival and proliferation of cells in the PRP gel membrane with time were confirmed in addition to their osteogenic capacity. Furthermore, to evaluate the possible effects of the PRP/BMSC gel membrane on surrounding progenitor cells in the injury area, we found that the PRP gel membrane products could significantly induce the migration of human endothelial cells in vitro, and increased the expression of bone morphogenetic protein 2 in cultured BMSC. These cells also secreted significant amounts of soluble proangiogenic factors, such as PDGF-BB, VEGF, and interleukin-8 (IL-8). Finally, the functionality of the PRP/BMSC gel membrane periosteal substitute for bone regeneration was tested in vivo both in an ectopic mouse model as well as in a rabbit segmental bone defect model providing evidence of its capacity to biomimic a periosteal response enhancing bone regeneration.


Assuntos
Regeneração Óssea/fisiologia , Substitutos Ósseos/uso terapêutico , Fraturas Ósseas/fisiopatologia , Fraturas Ósseas/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Neovascularização Fisiológica/fisiologia , Plasma Rico em Plaquetas , Animais , Materiais Biomiméticos/síntese química , Fraturas Ósseas/patologia , Humanos , Masculino , Camundongos , Osteogênese , Periósteo , Coelhos , Engenharia Tecidual/métodos , Resultado do Tratamento
15.
PLoS One ; 7(3): e33179, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22438896

RESUMO

Bone is a complex dynamic tissue undergoing a continuous remodeling process. Gravity is a physical force playing a role in the remodeling and contributing to the maintenance of bone integrity. This article reports an investigation on the alterations of the bone microarchitecture that occurred in wild type (Wt) and pleiotrophin-transgenic (PTN-Tg) mice exposed to a near-zero gravity on the International Space Station (ISS) during the Mice Drawer System (MDS) mission, to date, the longest mice permanence (91 days) in space. The transgenic mouse strain over-expressing pleiotrophin (PTN) in bone was selected because of the PTN positive effects on bone turnover. Wt and PTN-Tg control animals were maintained on Earth either in a MDS payload or in a standard vivarium cage. This study revealed a bone loss during spaceflight in the weight-bearing bones of both strains. For both Tg and Wt a decrease of the trabecular number as well as an increase of the mean trabecular separation was observed after flight, whereas trabecular thickness did not show any significant change. Non weight-bearing bones were not affected. The PTN-Tg mice exposed to normal gravity presented a poorer trabecular organization than Wt mice, but interestingly, the expression of the PTN transgene during the flight resulted in some protection against microgravity's negative effects. Moreover, osteocytes of the Wt mice, but not of Tg mice, acquired a round shape, thus showing for the first time osteocyte space-related morphological alterations in vivo. The analysis of specific bone formation and resorption marker expression suggested that the microgravity-induced bone loss was due to both an increased bone resorption and a decreased bone deposition. Apparently, the PTN transgene protection was the result of a higher osteoblast activity in the flight mice.


Assuntos
Remodelação Óssea/genética , Remodelação Óssea/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Citocinas/genética , Citocinas/fisiologia , Voo Espacial , Ausência de Peso/efeitos adversos , Animais , Osso e Ossos/citologia , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/fisiologia , Primers do DNA/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteócitos/citologia , Osteócitos/fisiologia , Compostos de Espiro , Suporte de Carga/fisiologia , Microtomografia por Raio-X
16.
J Biomater Appl ; 26(8): 1035-49, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-21363873

RESUMO

Bone augmentation procedures rely on osteogenic/osteoconductive properties of bone graft material (BGM). A further improvement is represented by use of autologous bone marrow stromal cells (BMSC), expanded in vitro and seeded on BGM before implantation in the bone defect. The effect of different BGMs on BMSC osteogenic differentiation was evaluated. BMSC were cultured in vitro in the presence of different BGM (natural, synthetic, and mixed origins). Cellular morphology was analyzed with scanning electron microscopy. The capability of BMSC to differentiate was determined in vitro by alkaline phosphatase gene expression and enzyme activity at different time points (7, 14, and 28 days) and in vivo by ectopic bone formation of implanted tissue constructs in an immunodeficient murine model. BGM supports the cell adhesion and osteogenic differentiation of BMSC developing a useful tool in the bone tissue engineering.


Assuntos
Células da Medula Óssea/citologia , Transplante Ósseo , Osso e Ossos/citologia , Células Estromais/citologia , Engenharia Tecidual , Adulto , Sequência de Bases , Adesão Celular , Primers do DNA , Humanos , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real
17.
Mol Biol Cell ; 21(19): 3487-96, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20719962

RESUMO

Sprouty (Spry) proteins are negative regulators of receptor tyrosine kinase signaling; however, their exact mechanism of action remains incompletely understood. We identified phosphatidylinositol-specific phospholipase C (PLC)-γ as a partner of the Spry1 and Spry2 proteins. Spry-PLCγ interaction was dependent on the Src homology 2 domain of PLCγ and a conserved N-terminal tyrosine residue in Spry1 and Spry2. Overexpression of Spry1 and Spry2 was associated with decreased PLCγ phosphorylation and decreased PLCγ activity as measured by production of inositol (1,4,5)-triphosphate (IP(3)) and diacylglycerol, whereas cells deficient for Spry1 or Spry1, -2, and -4 showed increased production of IP(3) at baseline and further increased in response to growth factor signals. Overexpression of Spry 1 or Spry2 or small-interfering RNA-mediated knockdown of PLCγ1 or PLCγ2 abrogated the activity of a calcium-dependent reporter gene, suggesting that Spry inhibited calcium-mediated signaling downstream of PLCγ. Furthermore, Spry overexpression in T-cells, which are highly dependent on PLCγ activity and calcium signaling, blocked T-cell receptor-mediated calcium release. Accordingly, cultured T-cells from Spry1 gene knockout mice showed increased proliferation in response to T-cell receptor stimulation. These data highlight an important action of Spry, which may allow these proteins to influence signaling through multiple receptors.


Assuntos
Proteínas de Membrana/metabolismo , Fosfolipase C gama/metabolismo , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Biomarcadores/metabolismo , Cálcio/metabolismo , Diglicerídeos/metabolismo , Ativação Enzimática , Imunoprecipitação , Inositol 1,4,5-Trifosfato/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Espaço Intracelular/metabolismo , Lectinas Tipo C/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células NIH 3T3 , Ligação Proteica , Proteínas Serina-Treonina Quinases , Linfócitos T/metabolismo , Transcrição Genética , Proteínas ras/metabolismo
18.
Brain Res ; 1152: 49-56, 2007 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-17459351

RESUMO

In vitro patch-clamp recordings were made to study the membrane properties and synaptic connectivity of fast-spiking interneurons in rat ventral striatum. Using a whole-cell configuration in acutely prepared slices, fast-spiking interneurons were recognized based on their firing properties and their morphological phenotype was confirmed by immunocytochemistry. Membrane properties of fast-spiking interneurons were distinguished from those of medium-sized spiny neurons by their more depolarized resting membrane potential, lower action potential amplitude and shorter half-width, short spike repolarization time and deep spike afterhyperpolarization. Firing patterns of interneurons could be subdivided in a bursting and non-bursting mode. Simultaneous dual whole-cell recordings revealed a high degree of connectivity of fast-spiking interneurons to medium-sized spiny neurons via unidirectional synapses. Burst firing in fast-spiking interneurons that were presynaptic to medium-sized spiny neurons resulted in barrages of postsynaptic potentials showing an initial amplitude increment, rapidly followed by a decrement. In conclusion, ventral striatal fast-spiking interneurons can be clearly distinguished from medium-sized spiny neurons by their membrane properties and their firing patterns can be subdivided in bursting and non-bursting modes. Their synaptic connectivity to medium-sized spiny neurons is unidirectional and characterized by frequency-dependent, dynamic changes in postsynaptic amplitude.


Assuntos
Potenciais de Ação , Gânglios da Base/fisiologia , Interneurônios/fisiologia , Transmissão Sináptica , Animais , Membrana Celular/fisiologia , Técnicas de Patch-Clamp , Ratos , Ratos Wistar
19.
J Neurophysiol ; 93(3): 1816-9, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15456801

RESUMO

One of the current hypotheses on dopamine in the physiology of motivation posits that this neurotransmitter regulates filtering and selection of inputs to the nucleus accumbens. The effects of dopamine (100 microM) and the D1-receptor agonist SKF 38393 (20-50 microM) on GABAergic synaptic transmission between pairs of principal cells of rat nucleus accumbens were studied by using simultaneous dual patch-clamp recordings in acutely prepared brain slices. Both compounds attenuated postsynaptic responses induced by presynaptic firing and this effect was reversed by the D1-receptor antagonist SCH 23390 (25 microM). This attenuating effect of dopamine D1-receptors may act to diminish competitive interactions between single projection neurons or ensembles in the nucleus accumbens.


Assuntos
Lateralidade Funcional/fisiologia , Lisina/análogos & derivados , Inibição Neural/fisiologia , Neurônios/fisiologia , Núcleo Accumbens/citologia , Receptores de Dopamina D1/fisiologia , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Animais , Benzazepinas/farmacologia , Dopamina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Lisina/metabolismo , Masculino , Inibição Neural/efeitos dos fármacos , Neurônios/classificação , Neurônios/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/antagonistas & inibidores , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia
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