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1.
Neuropharmacology ; 221: 109277, 2022 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-36223864

RESUMO

Ischemic stroke is characterized by the loss of cerebral blood flow, which frequently leads to neurological deficits. Tissue plasminogen activator is the only therapeutic agent approved to treat ischemic stroke but increases the risk of intracranial hemorrhage and mortality. The fibrinogen-depleting agent lumbrokinase has been used to improve myocardial perfusion in symptomatic stable angina and to prevent secondary ischemic stroke. Lumbrokinase is highly fibrin-specific and only active in the presence of fibrin. Therefore, lumbrokinase has a low risk of hemorrhage due to excessive fibrinolysis. In this study, we aimed to clarify the neuroprotection of lumbrokinase in mice subjected to permanent middle cerebral artery occlusion. Lumbrokinase significantly attenuated infarct volume and improved neurological dysfunction. Lumbrokinase dramatically decreased the expressions of the endoplasmic reticulum (ER) transmembrane receptor protein inositol-requiring enzyme-1 (IRE1) and its downstream transcription factor, XBP-1, caspase-12, and NF-κB activity, thereby significantly inhibiting apoptosis and autophagy and decreasing the NLRP3 inflammasome. Our evidence indicates that post-stroke treatment with lumbrokinase protects against ischemic stroke, thereby regulating ER stress through the collective inhibitory effect of the IRE1 signaling pathways to decrease apoptosis, autophagy, and inflammatory responses. We suggest that lumbrokinase is potential as an adjuvant treatment for ischemic stroke.


Assuntos
Isquemia Encefálica , AVC Isquêmico , Animais , Camundongos , Ativador de Plasminogênio Tecidual/uso terapêutico , Estresse do Retículo Endoplasmático , Infarto da Artéria Cerebral Média/tratamento farmacológico , Proteínas Serina-Treonina Quinases , Apoptose , Proteínas de Membrana/metabolismo , Fibrina/farmacologia , Fibrina/uso terapêutico , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo
2.
Acta Chir Belg ; 122(3): 164-168, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33635752

RESUMO

PURPOSE: There is no clear consensus as to which topical hemostatic agent is best used during cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy. The aim of this study was to evaluate the effect of hyperthermic chemotherapy on the biomechanical properties of organic topical hemostatic agents and histologically fibrin formation rates. METHODS: Four topical hemostatic agents (Spongostan™, Surgicel®, Fibrillar™, Arista®) were evaluated. All agents were mixed with 3 ml blood in sterile tubes separately to form clot formation. The resulting clot formations were incubated with 36 °C and 42 °C with saline or cisplatin for 1 h. Strength and flexibility of hemostatic samples were evaluated under weight of 0 g, 50 g, 100 g, 200 g and 300 g. All samples were stained with hemotoxylin-eosin and compared histologically for fibrin clot formation under light microscope. RESULTS: There were no statistically significant differences according to strength and flexibility of topical hemostatic agents on hyperthermic chemotherapy. Histopathologically, the highest fibrin formation was observed in Surgicel®, followed by Fibrillar™. The least fibrin formation was detected in Arista®. CONCLUSIONS: This study demonstrated that exposure to hyperthermic chemotherapy did not significantly affect the biomechanical properties of organic topical hemostatic agents and the fibrin clot formation.


Assuntos
Hemostáticos , Hipertermia Induzida , Cisplatino , Terapia Combinada , Procedimentos Cirúrgicos de Citorredução , Fibrina/farmacologia , Hemostasia , Hemostáticos/uso terapêutico , Humanos
3.
Biomolecules ; 10(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32121647

RESUMO

Bone defects cause aesthetic and functional changes that affect the social, economic and especially the emotional life of human beings. This complication stimulates the scientific community to investigate strategies aimed at improving bone reconstruction processes using complementary therapies. Photobiomodulation therapy (PBMT) and the use of new biomaterials, including heterologous fibrin biopolymer (HFB), are included in this challenge. The objective of the present study was to evaluate the influence of photobiomodulation therapy on bone tibial reconstruction of rats with biomaterial consisting of lyophilized bovine bone matrix (BM) associated or not with heterologous fibrin biopolymer. Thirty male rats were randomly separated into three groups of 10 animals. In all animals, after the anesthetic procedure, a noncritical tibial defect of 2 mm was performed. The groups received the following treatments: Group 1: BM + PBMT, Group 2: BM + HFB and Group 3: BM + HFB + PBMT. The animals from Groups 1 and 3 were submitted to PBMT in the immediate postoperative period and every 48 h until the day of euthanasia that occurred at 14 and 42 days. Analyses by computed microtomography (µCT) and histomorphometry showed statistical difference in the percentage of bone formation between Groups 3 (BM + HB + PBMT) and 2 (BM + HFB) (26.4% ± 1.03% and 20.0% ± 1.87%, respectively) at 14 days and at 42 days (38.2% ± 1.59% and 31.6% ± 1.33%, respectively), and at 42 days there was presence of bone with mature characteristics and organized connective tissue. The µCT demonstrated BM particles filling the defect and the deposition of new bone in the superficial region, especially in the ruptured cortical. It was concluded that the association of PBMT with HFB and BM has the potential to assist in the process of reconstructing bone defects in the tibia of rats.


Assuntos
Materiais Biocompatíveis , Matriz Óssea , Regeneração Óssea , Fibrina , Terapia com Luz de Baixa Intensidade , Tíbia , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Matriz Óssea/química , Matriz Óssea/transplante , Bovinos , Fibrina/química , Fibrina/farmacologia , Masculino , Ratos , Ratos Wistar , Tíbia/lesões , Tíbia/fisiologia
4.
J Vis Exp ; (122)2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28448053

RESUMO

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Engenharia Tecidual/métodos , Animais , Automação , Dimetilpolisiloxanos , Avaliação Pré-Clínica de Medicamentos/instrumentação , Canal de Potássio ERG1/antagonistas & inibidores , Fibrina/farmacologia , Coração/efeitos dos fármacos , Humanos , Camundongos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Ratos
5.
Minerva Stomatol ; 65(6): 385-392, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27711028

RESUMO

Research into regenerative dentistry has contributed momentum to the field of molecular biology. Periapical surgery aims at removing periapical pathology to achieve complete wound healing and regeneration of bone and periodontal tissue. Regenerative endodontic procedures are widely being added to the current armamentarium of pulp therapy procedures. The regenerative potential of platelets has been deliberated. Platelet-rich fibrin (PRF) is a wonderful tissue-engineering product and has recently gained much popularity due its promising results in wound healing bone induction. The features of this product are an attribute of platelets which, after cellular interactions, release growth factors and have shown application in diverse disciplines of dentistry. This paper is intended to shed light onto the various prospects of PRF and to provide clinical insight into regenerative endodontic therapy.


Assuntos
Terapia Biológica/métodos , Plaquetas , Endodontia/métodos , Fibrina/uso terapêutico , Regeneração Tecidual Guiada/métodos , Medicina Regenerativa/métodos , Plaquetas/química , Fibrina/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Células-Tronco Mesenquimais/efeitos dos fármacos , Neovascularização Fisiológica , Periodontite Periapical/terapia , Cicatrização
6.
Tsitologiia ; 58(10): 785-91, 2016.
Artigo em Inglês, Russo | MEDLINE | ID: mdl-30198704

RESUMO

In recent years, engineering of blood vessels, which can provide the effective transport of nutrients and various metabolites, is one of the major challenges in tissue reconstruction. Many researches are carried out to develop cell-seeded bioconstructs based on natural polymers, particularly on PEGylated fibrin. Therefore, the aim of this study was to reveal the optimal component ratio for modified fibrin hydrogels in order to provide favorable conditions for vascular development of endothelial and mesenchymal stem cell co-culture. It has been found out that the PEGylated fibrin hydrogels can support 3D cell growth in HUVECs and hASCs co-culture. The microporous filamentous hydrogel prepared from PEGylated 5 : 1 fibrinogen and using the 1 : 0.2 protein to thrombin ratio had the most favorable microenvironment for cell distribution, growth and development in the studied co-culture that resulted in high levels of expression of proteins required for angiogenesis.


Assuntos
Fibrina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Hidrogéis/farmacologia , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Bovinos , Avaliação Pré-Clínica de Medicamentos , Fibrina/química , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Hidrogéis/química , Células-Tronco Mesenquimais/citologia
7.
Eur Cell Mater ; 27: 112-23; discussion 123, 2014 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-24488855

RESUMO

Hydrogels pose interesting features for cartilage regeneration strategies, such as the option for injectability and in situ gelation resulting in optimal filling of defects. We aimed to study different hydrogels for their capability to support chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs). hBMSCs were encapsulated in alginate, alginate with hyaluronic acid (alginate/HA), fibrin or thermoresponsive HA grafted with poly(N-isopropyl acrylamide) side-chains (HA-pNIPAM). Glycosaminoglycan production and cartilage-related gene expression were significantly higher in hBMSC-alginate and hBMSC-fibrin constructs than in the other constructs. Supplementation of alginate with HA was not beneficial. hBMSC-alginate, hBMSC-fibrin and hBMSC-HA-pNIPAM constructs were placed in simulated defects in osteochondral biopsies and cultured in vitro for 28 d. Biopsies containing hBMSC-alginate and hBMSC-fibrin were implanted subcutaneously in nude mice for 12 weeks. hBMSC-alginate constructs had significantly higher cartilage-related gene expression after 28 d of culture as well as significantly more safranin-O positive repair tissue after 12 weeks in vivo than hBMSC-fibrin constructs. Although initial experiments with hBMSC-hydrogel constructs suggested comparable results of hBMSC-alginate, hBMSC-fibrin and hBMSC-HA-pNIPAM constructs, culture in the osteochondral biopsy model in vitro as well as in vivo revealed differences, suggests that chondrogenesis of hBMSCs in an osteochondral environment is hydrogel-dependent.


Assuntos
Condrócitos/citologia , Condrogênese , Hidrogéis/farmacologia , Células-Tronco Mesenquimais/citologia , Resinas Acrílicas/farmacologia , Adulto , Alginatos/farmacologia , Animais , Cartilagem/metabolismo , Cartilagem/fisiologia , Bovinos , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Fibrina/farmacologia , Ácido Glucurônico/farmacologia , Regeneração Tecidual Guiada , Ácidos Hexurônicos/farmacologia , Humanos , Ácido Hialurônico/farmacologia , Hidrogéis/química , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Nus , Osteocondrose/cirurgia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regeneração , Alicerces Teciduais/química
8.
Can J Vet Res ; 77(3): 231-6, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24101802

RESUMO

The objective of this study was to describe the results of thromboelastography platelet mapping (TEG-PM) carried out using 2 techniques in 20 healthy dogs. Maximum amplitudes (MA) generated by thrombin (MAthrombin), fibrin (MAfibrin), adenosine diphosphate (ADP) receptor activity (MAADP), and thromboxane A2 (TxA2) receptor activity (stimulated by arachidonic acid, MAAA) were recorded. Thromboelastography platelet mapping was carried out according to the manufacturer's guidelines (2-analyzer technique) and using a variation of this method employing only 1 analyzer (1-analyzer technique) on 2 separate blood samples obtained from each dog. Mean [± standard deviation (SD)] MA values for the 1-analyzer/2-analyzer techniques were: MAthrombin = 51.9 mm (± 7.1)/52.5 mm (± 8.0); MAfibrin = 20.7 mm (± 21.8)/23.0 mm (± 26.1); MAADP = 44.5 mm (± 15.6)/45.6 mm (± 17.0); and MAAA = 45.7 mm (± 11.6)/45.0 mm (± 15.4). Mean (± SD) percentage aggregation due to ADP receptor activity was 70.4% (± 32.8)/67.6% (± 33.7). Mean percentage aggregation due to TxA2 receptor activity was 77.3% (± 31.6)/78.1% (± 50.2). Results of TEG-PM were not significantly different for the 1-analyzer and 2-analyzer methods. High correlation was found between the 2 methods for MAfibrin [concordance correlation coefficient (r) = 0.930]; moderate correlation was found for MAthrombin (r = 0.70) and MAADP (r = 0.57); correlation between the 2 methods for MAAA was lower (r = 0.32). Thromboelastography platelet mapping (TEG-PM) should be further investigated to determine if it is a suitable method for measuring platelet dysfunction in dogs with thrombopathy.


Cette étude visait à décrire les résultats de la cartographie de la thromboélastographie des plaquettes (TEG-PM) effectuée à l'aide de deux techniques chez 20 chiens en santé. L'amplitude maximale (MA) générée par la thrombine (MAthrombine), la fibrine (MAfibrine), l'activité du récepteur de l'adénosine diphosphate (ADP) (MAADP), l'activité du récepteur de la thromboxane A2 (TxA2) (stimulée par l'acide arachidonique, MAAA) ont été mesurées. La TEG-PM a été effectuée selon les recommandations du manufacturier (technique à 2 analyseurs) ainsi qu'une variation de cette méthode en utilisant seulement un analyseur (technique à 1 analyseur) sur deux échantillons sanguins séparés obtenus de chaque chien. Les valeurs moyennes [± écart-type (SD)] de MA pour les techniques à 1 analyseur/2 analyseurs étaient: MAthrombine = 51,9 mm (± 7,1)/52,5 mm (± 8,0); MAfibrine = 20,7 mm (± 21,8)/23,0 mm (± 26,1); MAADP = 44,5 mm (± 15,6)/45,6 mm (± 17,0); et MAAA = 45,7 mm (± 11,6)/45,0 mm (± 15,4). La moyenne (± SD) du pourcentage d'agrégation due à l'activité du récepteur ADP était de 70,4 % (± 32,8)/67,6 % (± 33,7). La moyenne du pourcentage d'agrégation due à l'activité du récepteur TxA2 était de 77,3 % (± 31,6)/78,1 % (± 50,2). Il n'y avait pas de différence significative dans les résultats de TEG-PM entre les méthodes à 1 analyseur ou à 2 analyseurs. Une corrélation élevée a été trouvée entre les deux méthodes pour la MAfibrine [coefficient de concordance de corrélation (r) = 0,930]; une corrélation modérée a été trouvée pour MAthrombine (r = 0,70) et MAADP (r = 0,57); la corrélation entre les deux méthodes pour MAAA était plus faible (r = 0,32). Des études supplémentaires devraient être effectuées pour déterminer si la TEG-PM est une méthode qui convient pour mesurer le dysfonctionnement des plaquettes chez les chiens avec thrombopathie.(Traduit par Docteur Serge Messier).


Assuntos
Plaquetas/fisiologia , Cães/fisiologia , Agregação Plaquetária/fisiologia , Tromboelastografia/veterinária , Difosfato de Adenosina/farmacologia , Animais , Feminino , Fibrina/farmacologia , Masculino , Estatísticas não Paramétricas , Tromboelastografia/métodos , Trombina/farmacologia , Tromboxano A2/farmacologia
9.
J Biomed Mater Res A ; 101(6): 1531-8, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23554144

RESUMO

Modular tissue engineering applies biomaterials-based approaches to create discrete cell-seeded microenvironments, which can be further assembled into larger constructs for the repair of injured tissues. In the current study, we embedded human bone marrow-derived mesenchymal stem cells (MSC) and human adipose-derived stem cells (ASC) in collagen/fibrin (COL/FIB) and collagen/fibrin/hydroxyapatite (COL/FIB/HA) microbeads, and evaluated their suitability for bone tissue engineering applications. Microbeads were fabricated using a water-in-oil emulsification process, resulting in an average microbead diameter of approximately 130 ± 25 µm. Microbeads supported both cell viability and cell spreading of MSC and ASC over 7 days in culture. The embedded cells also began to remodel and compact the microbead matrix as demonstrated by confocal reflectance microscopy imaging. After two weeks of culture in media containing osteogenic supplements, both MSC and ASC deposited calcium mineral in COL/FIB microbeads, but not in COL/FIB/HA microbeads. There were no significant differences between MSC and ASC in any of the assays examined, suggesting that either cell type may be an appropriate cell source for orthopedic applications. This study has implications in the creation of defined microenvironments for bone repair, and in developing a modular approach for delivery of pre-differentiated cells.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Cerâmica/farmacologia , Colágeno/farmacologia , Congressos como Assunto , Fibrina/farmacologia , Células-Tronco Mesenquimais/citologia , Osteogênese/efeitos dos fármacos , Tecido Adiposo/citologia , Análise de Variância , Animais , Materiais Biocompatíveis/farmacologia , Células da Medula Óssea/citologia , Boston , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Microscopia Confocal , Microesferas , Tamanho da Partícula
10.
Hum Reprod ; 28(8): 2187-200, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23608357

RESUMO

STUDY QUESTION: Does fibrin introduced into the extracellular matrix affect the growth and maturation of individual primate follicles during encapsulated three-dimensional (3D) culture? SUMMARY ANSWER: While not altering follicle survival, fibrin-alginate (FIBRIN) improves macaque primary, but not secondary, follicle development during encapsulated 3D culture in terms of growth, steroidogenesis, anti-Müllerian hormone (AMH)/vascular endothelial growth factor (VEGF) production and oocyte maturation. WHAT IS KNOWN ALREADY: Efforts to grow non-human primate ovarian follicles from the secondary to the antral stage during encapsulated 3D culture have been successful. However, the growth and maturation of primary follicles in vitro has not been reported in primates, especially in chemically defined conditions. STUDY DESIGN, SIZE, DURATION: In vitro follicle maturation was investigated using the rhesus macaque (Macaca mulatta). Ovaries (n = 7 pairs) were obtained during the early follicular phase of the menstrual cycle (cycle day 1-4). Primary (80-120 µm diameter) and secondary (125-225 µm diameter) follicles were isolated mechanically, randomly assigned to experimental groups, encapsulated into alginate (0.25% w/v) or FIBRIN (25 mg/ml fibrinogen-0.25% alginate) and cultured for 13 and 5 weeks, respectively. MATERIALS, SETTING, METHODS: Individual follicles were cultured in alpha minimum essential medium supplemented with FSH. Follicle survival and growth were assessed by microscopy. Follicles that reached the antral stage were treated with recombinant hCG. Metaphase II (MII) oocytes were inseminated via ICSI. Follicle morphology was evaluated by hematoxylin and eosin (H&E) staining. Immunohistochemistry was performed for cytochrome P450 family 17 subfamily A polypeptide 1 (CYP17A1) and 19 subfamily A polypeptide 1 (CYP19A1). Culture medium was analyzed for estradiol (E2) and progesterone by chemiluminescence, androstenedione (A4) by radioimmunoassay, as well as anti-Müllerian hormone (AMH) and vascular endothelial growth factor (VEGF) by enzyme-linked immunosorbent assay. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 105 primary and 133 secondary follicles were collected. The presence of fibrin in the alginate matrix had no effect on either primary or secondary follicle survival. Growing primary and secondary follicles formed an antrum at Weeks 9 and 3, respectively. The percentage of growing follicles was higher (P < 0.05) for primary follicles cultured in FIBRIN than alginate at Week 13. The diameters were larger for the growing secondary follicles cultured in alginate than FIBRIN at Week 5 (P < 0.05). H&E staining revealed the typical morphology for small antral follicles. CPY17A1 immunostaining was detected in theca cells, while CYP19A1 was observed in granulosa cells. E2 increased (P < 0.05) during antrum formation in growing follicles at Week 9 for primary and Week 3 for secondary follicles. AMH levels in medium from growing primary follicles increased (P < 0.05) after Week 4 with peak levels at Weeks 9-11. AMH increased (P < 0.05) in growing secondary follicles at Weeks 3-5. VEGF levels in medium were elevated (P < 0.05) in growing primary follicles at Week 9. VEGF increased (P < 0.05) in medium from growing secondary follicles at Weeks 3-5. E2, AMH and VEGF production was higher (P < 0.05) in primary follicle culture with FIBRIN than alginate alone. One primary follicle cultured in FIBRIN (1 of 5 follicles harvested) and a secondary follicle cultured in alginate alone (1 of 15 follicles harvested) yielded an MII oocyte. The fertilized oocyte from primary follicle culture arrested without cell division after fertilization, while the oocyte from secondary follicle culture cleaved and reached the morula stage. LIMITATIONS, REASONS FOR CAUTION: The study reports on in vitro development and function of individual macaque follicles, that is limited to the interval from the primary and secondary stage to the small antral stage. The findings await translation to human ovarian follicles. WIDER IMPLICATIONS OF THE FINDINGS: The 3D model for primate follicle development offers a unique opportunity to investigate the growth and regulation of primate primary, as well as secondary follicles, and their enclosed oocytes, as they grow to the antral stage by monitoring and manipulating factors or signaling pathways in vitro. Since primate primary follicles, in addition to secondary follicles, can be cultured to the antral stage to provide mature oocytes, they represent an additional source of pre-antral follicles for in vitro follicle maturation with the potential to provide gametes for assisted reproductive technology as an option for fertility preservation in women, including patients with cancer. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by The Oncofertility Consortium (NIH U54 RR024347-HD058294, PL1-EB008542), NIH U54-HD18185 (Eunice Kennedy Shriver Specialized Cooperative Centers Program in Reproduction and Infertility Research), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), Oregon National Primate Research Center 8P51OD011092. There are no conflicts of interest.


Assuntos
Fibrina/farmacologia , Macaca/fisiologia , Folículo Ovariano/efeitos dos fármacos , Animais , Hormônio Antimülleriano/metabolismo , Técnicas de Cultura de Células/veterinária , Gonadotropina Coriônica/farmacologia , Feminino , Fertilização , Hormônios Esteroides Gonadais/metabolismo , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
11.
J Biomed Mater Res A ; 101(2): 428-37, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22865465

RESUMO

In this study, we created self-assembled smooth muscle cell (SMC) tissue rings (comprised entirely of cells and cell-derived matrix; CDM) and compared their structure and material properties with tissue rings created from SMC-seeded fibrin or collagen gels. All tissue rings were cultured statically for 7 days in supplemented growth medium (with ε-amino caproic acid, ascorbic acid, and insulin-transferrin-selenium), prior to uniaxial tensile testing and histology. Self-assembled CDM rings exhibited ultimate tensile strength and stiffness values that were two-fold higher than fibrin gel and collagen gel rings. Tensile testing of CDM, fibrin gel and collagen gel rings treated with deionized water to lyse cells showed little to no change in mechanical properties relative to untreated ring samples, indicating that the ECM dominates the measured ring mechanics. In addition, CDM rings cultured in supplemented growth medium were significantly stronger than CDM rings cultured in standard, unsupplemented growth medium. These results illustrate the potential utility of self-assembled cell rings as model CDM constructs for tissue engineering and biomechanical analysis of ECM material properties.


Assuntos
Colágeno/farmacologia , Fibrina/farmacologia , Géis/química , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/fisiologia , Resistência à Tração/efeitos dos fármacos , Engenharia Tecidual/métodos , Animais , Meios de Cultura/farmacologia , Matriz Extracelular , Miócitos de Músculo Liso/efeitos dos fármacos , Ratos , Ratos Wistar , Sefarose
12.
Int J Med Sci ; 9(10): 872-80, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23155361

RESUMO

INTRODUCTION: Maxillary bone losses often require additional regenerative procedures: as a supplement to the procedures of tissue regeneration, a platelet concentrate called PRF (Platelet Rich Fibrin) was tested for the first time in France by Dr. Choukroun. Aim of the present study is to investigate, clinically and histologically, the potential use of PRF, associated with deproteinized bovine bone (Bio-Oss), as grafting materials in pre-implantology sinus grafting of severe maxillary atrophy, in comparison with a control group, in which only deproteinized bovine bone (Bio-Oss) was used as reconstructive material. MATERIALS AND METHODS: 60 patients were recruited using the cluster-sampling method; inclusion criteria were maxillary atrophy with residual ridge < 5mm. The major atrophies in selected patients involved sinus-lift, with a second-look reopening for the implant insertion phase. The used grafting materials were: a) Bio-Oss and b) amorphous and membranous PRF together with Bio-Oss. We performed all operations by means of piezosurgery in order to reduce trauma and to optimize the design of the operculum on the cortical bone. The reopening of the surgical area was scheduled at 3 different times. RESULTS: 72 sinus lifts were performed with subsequent implants insertions.We want to underline how the histological results proved that the samples collected after 106 days (Early protocol) with the adding of PRF were constituted by lamellar bone tissue with an interposed stroma that appeared relaxed and richly vascularized. CONCLUSIONS: The use of PRF and piezosurgery reduced the healing time, compared to the 150 days described in literature, favoring optimal bone regeneration. At 106 days, it is already possible to achieve good primary stability of endosseous implants, though lacking of functional loading.


Assuntos
Atrofia , Fibrina , Reconstrução Mandibular , Maxila , Plasma Rico em Plaquetas , Adulto , Animais , Regeneração Óssea , Bovinos , Implantação Dentária Endóssea , Feminino , Fibrina/química , Fibrina/farmacologia , França , Humanos , Masculino , Maxila/crescimento & desenvolvimento , Maxila/cirurgia , Seio Maxilar/patologia , Seio Maxilar/cirurgia , Pessoa de Meia-Idade , Minerais/uso terapêutico , Piezocirurgia , Plasma Rico em Plaquetas/química
14.
Tissue Eng Part A ; 18(15-16): 1596-607, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22439983

RESUMO

The ability to study the gross morphological changes occurring during tissue formation is vital to producing tissue-engineered structures of clinically relevant dimensions in vitro. Here, we have used nondestructive methods of digital imaging and optical coherence tomography to monitor the early-stage formation and subsequent maturation of fibrin-based tissue-engineered ligament constructs. In addition, the effect of supplementation with essential promoters of collagen synthesis, ascorbic acid (AA) and proline (P), has been assessed. Contraction of the cell-seeded fibrin gel occurs unevenly within the first 5 days of culture around two fixed anchor points before forming a longitudinal ligament-like construct. AA+P supplementation accelerates gel contraction in the maturation phase of development, producing ligament-like constructs with a higher collagen content and distinct morphology to that of unsupplemented constructs. These studies highlight the importance of being able to control the methods of tissue formation and maturation in vitro to enable the production of tissue-engineered constructs with suitable replacement tissue characteristics for repair of clinical soft-tissue injuries.


Assuntos
Fibrina/farmacologia , Ligamentos/efeitos dos fármacos , Ligamentos/fisiologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Ácido Ascórbico/farmacologia , Galinhas , Colágeno/metabolismo , Géis , Processamento de Imagem Assistida por Computador , Ligamentos/citologia , Prolina/farmacologia , Tomografia de Coerência Óptica
15.
Acta Biomater ; 7(2): 691-9, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20837168

RESUMO

Modifying the relative concentrations of fibrinogen and thrombin can control the physical properties of fibrin gels, while the viability of associated cells has been linked to the gel's final network structure. It was hypothesized that increasing the gel ionic strength during fabrication through supplementation with sodium chloride (NaCl) would provide an improved approach for tailoring the physical properties of fibrin gels and maintaining the viability and osteogenic potential of entrapped cells. Fibrin gels were formed by mixing fibrinogen, thrombin and calcium chloride with varying masses of NaCl (0-4.40% w/v), and the osteogenic potential of entrapped human mesenchymal stem cells (MSC) was examined over 14 days. Physical properties including gelation time, compressive modulus and fiber diameter were dependent upon NaCl content, with gels containing 2.60% NaCl possessing compressive moduli threefold higher than gels without NaCl. Alkaline phosphatase activity was highest for MSC entrapped in gels containing 2.15-2.60% NaCl after 14 days, and all gels exhibited increased calcium incorporation over the culture period. These data confirm that varying the salt concentration of the pre-gel solution can modulate the material properties of fibrin constructs without additional fibrinogen or thrombin, thereby offering a new approach for generating improved cell transplantation vehicles for use in bone tissue regeneration.


Assuntos
Fibrina/farmacologia , Géis/farmacologia , Teste de Materiais , Osteogênese/efeitos dos fármacos , Fenômenos Físicos , Cloreto de Sódio/farmacologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Microscopia Eletrônica de Varredura , Concentração Osmolar , Fatores de Tempo
16.
Clin Transplant ; 23(6): 968-74, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19712088

RESUMO

Calciphylaxis, also known as calcific uremic arteriolopathy (CUA), is a rare complication in patients with end-stage renal disease as well as in patients after renal transplantation. It should be suspected in patients with typical painful violaceous skin lesions on the extremities or on the trunk. Active multidisciplinary management approach, with intensive local wound care, is vital in these patients. Controlling parathyroid hormone, hyperbaric oxygenation, sodium thiosulphate, bisphosphonates, cinacalcet and skin grafting could be effective. In our report, we describe a case of CUA in a 43-year-old patient two years after kidney transplantation. Despite intensive standard treatment, his wounds progressed; therefore, we decided to use iloprost, in combination with hyperbaric oxygenation. The clean wounds were then covered with cultivated autologous skin cells to enhance wound epithelialization. Seven months after finishing iloprost and hyperbaric oxygen treatment and the first application of skin substitute, the wounds healed completely and remained healed during the four-yr follow-up period. We conclude that in patients with severe CUA-induced wounds, the combined treatment with iloprost, hyperbaric oxygen and autologous cultured fibrin-based skin substitutes can be effective. A combination of different treatment modalities is vital in patients with CUA.


Assuntos
Calciofilaxia/terapia , Fibrina/farmacologia , Oxigenoterapia Hiperbárica/métodos , Iloprosta/uso terapêutico , Transplante de Rim/efeitos adversos , Pele Artificial , Pele/citologia , Adulto , Calciofilaxia/etiologia , Transplante de Células/métodos , Células Cultivadas , Humanos , Masculino , Índice de Gravidade de Doença , Vasodilatadores/uso terapêutico
17.
Angiogenesis ; 12(3): 275-85, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19449108

RESUMO

The fibrinous exudate of a wound or tumor stroma facilitates angiogenesis. We studied the involvement of RGD-binding integrins during tube formation in human plasma-derived fibrin clots and human purified fibrin matrices. Capillary-like tube formation by human microvascular endothelial cells in a 3D plasma-derived fibrinous matrix was induced by FGF-2 and TNF-alpha and depended largely on cell-bound u-PA and plasmin activities. While tube formation was minimally affected by the addition of either the alphavbeta3-integrin inhibiting mAb LM609 or the alpha5-integrin inhibiting mAb IIA1, the general RGD-antagonist echistatin completely inhibited this process. Remarkably, when alphavbeta3- and alpha5beta1-integrins were inhibited simultaneously, tube formation was reduced by 78%. It was accompanied by a 44% reduction of u-PA antigen accumulation and 41% less production of fibrin degradation products. alphavbeta5-integrin-blocking antibodies further enhanced the inhibition by mAb LM609 and mAb IIA1 to 94%, but had no effect by themselves. alphav-specific cRGD only inhibited angiogenesis when alpha5beta1-integrin was simultaneously blocked. Endostatin mimicked the effect of alpha5beta1-integrin and inhibited tube formation only in the presence of LM609 or cRGD (73 and 80%, respectively). Comparable results were obtained when purified fibrin matrices were used instead of the plasma-derived fibrinous matrices. These data show that blocking of tube formation in a fibrinous exudate requires the simultaneous inhibition of alphavbeta3- and alpha5beta1-integrins. This may bear impact on attempts to influence angiogenesis in a fibrinous environment.


Assuntos
Anticorpos Monoclonais/farmacologia , Capilares/efeitos dos fármacos , Fibrina/farmacologia , Integrina alfa5beta1/antagonistas & inibidores , Integrina alfaVbeta3/antagonistas & inibidores , Neovascularização Fisiológica/efeitos dos fármacos , Inibidores da Angiogênese/farmacologia , Anticorpos Monoclonais/administração & dosagem , Capilares/crescimento & desenvolvimento , Capilares/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Combinação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Endostatinas/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Matriz Extracelular/química , Fibrina/química , Humanos , Integrina alfa5beta1/imunologia , Integrina alfa5beta1/fisiologia , Integrina alfaVbeta3/imunologia , Integrina alfaVbeta3/fisiologia , Oligopeptídeos/metabolismo , Ligação Proteica , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
18.
J Am Coll Surg ; 203(6): 817-26, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17116549

RESUMO

BACKGROUND: Earlier observations implicate arterial thrombosis causing endothelial dysfunction by decreasing nitric oxide (NO) levels. NO levels are restored by regional L-arginine supplementation in animal models. The purpose of this study was to investigate the roles of thrombus components in NO generation. STUDY DESIGN: Human umbilical vein endothelial cells were harvested and cultured. The thrombus components thrombin, thrombin receptor agonist peptide (TRAP), and fibrin were added to a media of confluent human umbilical vein endothelial cells. Endothelial nitric oxide synthase (eNOS) activity was assayed by measuring conversion of L-arginine to L-citrulline. Endothelial NOS mRNA levels were quantitated using real-time polymerase chain reaction. Cellular membrane transport of L-arginine through the y+ channel was assayed with (14)C-labeled L-arginine. Arginase activity was determined as the conversion of (14)C L-arginine to (14)C urea and trapped as Na(2)(14)CO(3) for scintillation counting. Arginase protein amounts were assessed using Western blotting. RESULTS: Endothelial cells exposed to thrombin for 4 hours led to increased arginase activity. Thrombin (10 U/mL) caused a 1.6-fold increase compared with that in controls (320+/-29 microM urea/min versus 194+/-10 microM urea/min, p=0.03), and thrombin (30 U/mL) increased arginase activity 2.1-fold (398+/-27 microM urea/min, p < 0.001, versus controls); thrombin at 1 U/mL and fibrin had no effect. TRAP (50 microM) had an effect similar to that of thrombin 10 U/mL (316+/-21 microM urea/min, p < 0.01, versus controls). Protein amounts of arginase corresponded with activity levels. Neither eNOS nor inducible nitric oxide synthase (iNOS) activities were affected by exposure to thrombin and TRAP for 4 hours. Similarly, quantification of eNOS, iNOS, and endothelin-1 mRNA did not change, although CL-100, a known thrombin-inducible gene, was upregulated. Finally, transport of L-arginine into endothelial cells was unaffected by thrombin, TRAP, and fibrin exposure. CONCLUSIONS: Endothelial cells exposed to thrombin have increased arginase enzymatic activity, and the remainder of NO generation capability is unaffected. L-arginine supplementation or arginase blockade may counteract endothelial dysfunction in the setting of acute arterial thrombosis.


Assuntos
Arginase/metabolismo , Endotélio Vascular/metabolismo , Trombina/farmacologia , Trombose/metabolismo , Arginase/genética , Arginina/metabolismo , Transporte Biológico , Células Cultivadas , Endotélio Vascular/fisiopatologia , Fibrina/farmacologia , Expressão Gênica , Humanos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Receptores de Trombina/metabolismo , Trombose/fisiopatologia , Veias Umbilicais
19.
Ital J Anat Embryol ; 109(4): 199-208, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15717455

RESUMO

Transected peripheral nerve can be protected with different supplementations. One of them is implantation of dead-ended connective tissue chambers filled with fibrin and growth-promoting substances. The aim of this study was to find whether nerve growth factor (NGF) applied by means of such method exerts neuroprotective effect upon transected sciatic nerves. Study was performed on the adult male Wistar C rats. Connective tissue chambers grew around the silicone tubes implanted under their skin. Chambers were then filled with fibrin (control group) or fibrin with NGF solution (NGF group). Right sciatic nerve was cut, its distal stump was removed and its proximal stump was introduced into the chamber. Following 4 weeks DiI was applied to the free end of implant. The labeled motoneurons in the slices obtained from L3-L4 spinal cord segments and the number of myelinated nerve fibers present in the middle part of the chambers were counted. Acetylcholinesterase-positive fiber endings inside the chambers were also visualized. Our data showed that the number of motor neurons and their diameters as well as the number of myelinated fibers were higher in the NGF group when compared to the control group, but these differences were not significant. In both groups parallelly arranged acetylcholinesterase-positive nerve fibers were present. The obtained results show that NGF has no influence on regeneration of the motor component of the rat sciatic nerves in adult animals.


Assuntos
Implantes Experimentais , Fator de Crescimento Neural/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Neuropatia Ciática/terapia , Acetilcolina/metabolismo , Acetilcolinesterase/metabolismo , Animais , Carbocianinas , Tecido Conjuntivo/fisiologia , Tecido Conjuntivo/transplante , Cultura em Câmaras de Difusão/instrumentação , Cultura em Câmaras de Difusão/métodos , Modelos Animais de Doenças , Fibrina/farmacologia , Masculino , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/fisiologia , Fibras Nervosas Mielinizadas/fisiologia , Fibras Nervosas Mielinizadas/ultraestrutura , Fator de Crescimento Neural/uso terapêutico , Regeneração Nervosa/fisiologia , Ratos , Ratos Wistar , Nervo Isquiático/lesões , Nervo Isquiático/cirurgia , Neuropatia Ciática/metabolismo , Neuropatia Ciática/patologia , Silicones , Medula Espinal/citologia , Medula Espinal/fisiologia
20.
Thromb Res ; 57(6): 957-66, 1990 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-2116685

RESUMO

Based on the results of previous investigations that pollen Typhae, a Traditional Chinese Medicine, Had antiatherogenic effects, several components were isolated successively from the drug and their effects on porcine aortic endothelial cell (EC) and smooth muscle cell (SMC) cultures as well as on platelet aggregation were examined. 12 components isolated from Pollen Typhae have been identified on their chemical structures and biological effects. 4 of them showed different evident antiatherogenic effects. 1) Isorhamnetin-3-O-rhamnosyl-glucoside could stimulate EC to produce tPA and PGI2; 2) Quercetin-3-O-neohesperidose could protect EC from injury by fibrin, as well as raise tPA activity; 3) beta-Sitosterol palmitate could inhibit SMC proliferation and 4) beta-Sitosterol glucoside showed an inhibitory effect on platelet aggregation. These results would provide some information for the search of new drugs in the treatment and prevention of atherosclerosis.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Endotélio Vascular/efeitos dos fármacos , Epoprostenol/biossíntese , Flavonóis , Músculo Liso Vascular/efeitos dos fármacos , Pólen/análise , Ativador de Plasminogênio Tecidual/biossíntese , Animais , Divisão Celular/efeitos dos fármacos , Dissacarídeos/farmacologia , Fibrina/farmacologia , Hesperidina/análogos & derivados , Hesperidina/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária , Quercetina/análogos & derivados , Quercetina/farmacologia , Sitosteroides/farmacologia , Suínos
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