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Coping with a zero-waste, more sustainable economy represents the biggest challenge for food market nowadays. We have previously demonstrated that by applying smart multidisciplinary waste management strategies to purple sea urchin (Paracentrotus lividus) food waste, it is possible to obtain both a high biocompatible collagen to produce novel skin substitutes and potent antioxidant pigments, namely polyhydroxynapthoquinones (PHNQs). Herein, we have analyzed the biological activities of the PHNQs extract, composed of Spinochrome A and B, on human skin fibroblast cells to explore their future applicability in the treatment of non-healing skin wounds with the objective of overcoming the excessive oxidative stress that hinders wound tissue regeneration. Our results clearly demonstrate that the antioxidant activity of PHNQs is not restricted to their ability to scavenge reactive oxygen species; rather, it can be traced back to an upregulating effect on the expression of superoxide dismutase 1, one of the major components of the endogenous antioxidant enzymes defense system. In addition, the PHNQs extract, in combination with Antimycin A, displayed a synergistic pro-apoptotic effect, envisaging its possible employment against chemoresistance in cancer treatments. Overall, this study highlights the validity of a zero-waste approach in the seafood chain to obtain high-value products, which, in turn, may be exploited for different biomedical applications.
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Epithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal properties. EMT has been closely associated with cancer cell aggressiveness. The aim of this study was to evaluate the mRNA and protein expression of EMT-associated markers in mammary tumors of humans (HBC), dogs (CMT), and cats (FMT). Real-time qPCR for SNAIL, TWIST, and ZEB, and immunohistochemistry for E-cadherin, vimentin, CD44, estrogen receptor (ER), progesterone receptor (PR), ERBB2, Ki-67, cytokeratin (CK) 8/18, CK5/6, and CK14 were performed. Overall, SNAIL, TWIST, and ZEB mRNA was lower in tumors than in healthy tissues. Vimentin was higher in triple-negative HBC (TNBC) and FMTs than in ER+ HBC and CMTs (p < 0.001). Membranous E-cadherin was higher in ER+ than in TNBCs (p < 0.001), whereas cytoplasmic E-cadherin was higher in TNBCs when compared with ER+ HBC (p < 0.001). A negative correlation between membranous and cytoplasmic E-cadherin was found in all three species. Ki-67 was higher in FMTs than in CMTs (p < 0.001), whereas CD44 was higher in CMTs than in FMTs (p < 0.001). These results confirmed a potential role of some markers as indicators of EMT, and suggested similarities between ER+ HBC and CMTs, and between TNBC and FMTs.
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Sarcoids are the most common equine skin tumours Although they do not metastasize, they can be locally aggressive and cause significant clinical symptoms in affected horses. Despite being common, very little is known about the host immune response and the biological mechanisms underlying persistence and recurrence of equine sarcoids. The latter reflects the need for further research in this field. This in-vitro study used sarcoid explants from horses with naturally occurring sarcoids (n = 12) to evaluate the induction of a humoral immune response directed against equine sarcoid-derived bovine papilloma-virus (BPV)- 1 infected fibroblasts using a flow cytometric crossmatch assay. The presence of antibodies against exogenous bovine serum albumin (BSA) and fibroblast-like mesenchymal stromal cells (MSCs) was also evaluated by ELISA and flow cytometry, respectively. The viral load in the sarcoid explants, the corresponding cultured sarcoid fibroblasts, and matched peripheral blood mononuclear cells (PBMCs) from affected horses were determined by quantitative BPV-1/- 2 PCR analysis. Antibodies against autologous sarcoid cells were present in six out of twelve sarcoid-affected horses. Serum from all horses showed cross reactivity with allogeneic sarcoid cells, while only a part reacted with BSA or MSCs. Screening of host PBMCs demonstrated the absence of BPV E1 nucleic acids. Statistical analysis revealed a significantly higher mean viral load in the parental sarcoid tissue compared to the low passage fibroblasts (P < 0.001). These results support the hypothesis that sarcoid-affected horses may develop antibodies recognizing tumour-specific antigens. In contrast to sarcoid explants, equine PBMCs do not seem to contain complete BPV genomes. These results provide a basis for future investigations on the clinical relevance of these antibodies.
Assuntos
Doenças dos Cavalos , Sarcoidose , Dermatopatias , Neoplasias Cutâneas , Animais , Cavalos , Leucócitos Mononucleares , Neoplasias Cutâneas/veterinária , Dermatopatias/veterinária , Sarcoidose/veterinária , Fibroblastos , DNA ViralRESUMO
OBJECTIVE: The use of mesenchymal stem cells (MSCs) for the treatment of equine joint disease is widely investigated because of their regenerative and immunomodulatory potential. Allogeneic MSCs provide a promising alternative to autologous MSCs, since the former are immediately available and enable a thorough donor screening. However, questions have been raised concerning the immunogenic potential of allogeneic MSCs, especially after repeated administration. METHODS: Current retrospective study assessed the cellular and humoral immunogenicity of ten jumping and dressage horses with naturally occurring degenerative joint disease which were treated 3 times intra-articularly with a 1 mL stem cell suspension containing 1.4-2.5 million chondrogenic induced equine allogeneic peripheral blood-derived MSCs (ciMSCs) combined with 1 mL equine allogeneic plasma. Stem cells from 2 donor horses were used. Horses were clinically evaluated for joint effusion, presence of pain to palpation and skin surface temperature at the local injection site, joint range of motion, occurrence of adverse events and the presence of ectopic tissue. The cellular immune response was analyzed using a modified mixed lymphocyte reaction and the humoral immune response was investigated using a flow cytometric crossmatch assay by which the presence of alloantibodies against the ciMSCs was evaluated. Presence of anti-bovine serum albumin antibodies was detected via ELISA. RESULTS: Clinical evaluation of the horses revealed no serious adverse effects or suspected adverse drug reactions and no ectopic tissue formation at the local injection site or in other areas of the body. Generally, repeated administration led to a decrease of horses with joint effusion of the affected joint. Pain to palpation, skin surface temperature and joint range of motion did not increase or even decreased after treatment administration. Allogeneic ciMSCs did not induce a cellular immune response and no alloantibodies were detected in the recipients' serum, regardless the presence of BSA antibodies in 70 % of the horses. CONCLUSION: Repeated intra-articular injections with allogeneic equine ciMSCs did not elicit clinically relevant adverse events. Furthermore, current study indicates the absence of a cellular or a humoral immune response following repeated intra-articular injections.
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Transplante de Células-Tronco Hematopoéticas , Cavalos , Células-Tronco Mesenquimais , Animais , Transplante de Células-Tronco Hematopoéticas/veterinária , Imunidade Celular , Imunidade Humoral , Injeções Intra-Articulares , Estudos RetrospectivosRESUMO
Skin wound healing may sometimes lead to open sores that persist for long periods and expensive hospitalization is needed. Among different kinds of therapeutic innovative approaches, mesenchymal stem cells (MSCs) and low-temperature atmospheric pressure cold plasma (ionized gas) have been recently tested to improve this regenerative process. To optimize wound healing the present study intended to combine, for the first time, these two novel approaches in a large size animal wound healing model with the aim of assessing the putative dual beneficial effects. Based on clinical, histopathological, and molecular results a synergistic action in a second intention healing wound in sheep has been observed. Experimental wounds treated with cold plasma and MSCs showed a slower but more effective healing compared to the single treatment, as observed in previous studies. The combined treatment improved the correct development of skin appendages and structural proteins of the dermis showing the potential of the dual combination as a safe and effective tool for skin regeneration in the veterinary clinical field.
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Células-Tronco Mesenquimais/fisiologia , Gases em Plasma/farmacologia , Regeneração , Carneiro Doméstico , Fenômenos Fisiológicos da Pele , Cicatrização , Animais , Modelos Animais de DoençasRESUMO
Mammary cancer is a common neoplasm in women, dogs, and cats that still represents a therapeutic challenge. Wnt/ß-catenin and Hippo pathways are involved in tumor progression, cell differentiation, and metastasis. The aim of this study was to evaluate mRNA and protein expression of molecules involved in these pathways in human (HBC), canine (CMT), and feline mammary tumors (FMT). Real-time quantitative polymerase chain reaction (qPCR) for ß-catenin, CCND1, YAP, TAZ, CTGF, and ANKRD1, western blotting for YAP, TAZ, and ß-catenin, and immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR), ERBB2, ß-catenin, and YAP/TAZ were performed on mammary tumor tissues. The protein expression of active ß-catenin was higher in tumors than in healthy tissues in all 3 species. The mRNA expression of the downstream gene CCND1 was increased in HBC ER+ and CMTs compared to healthy tissues. Membranous and cytoplasmic protein expression of ß-catenin were strongly negatively correlated in all 3 species. Tumors showed an increased protein expression of YAP/TAZ when compared to healthy tissues. Notably, YAP/TAZ expression was higher in triple negative breast cancers when compared to HBC ER+ and in FMTs when compared to CMTs. The mRNA expression of ß-catenin, YAP, TAZ, CTGF, and ANKRD1 was not different between tumors and healthy mammary gland in the 3 species. This study demonstrates deregulation of Wnt/ß-catenin and Hippo pathways in mammary tumors, which was more evident at the protein rather than the mRNA level. Wnt/ß-catenin and Hippo pathways seem to be involved in mammary carcinogenesis and therefore represent interesting therapeutic targets that should be further investigated.
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Neoplasias da Mama , Doenças do Gato , Doenças do Cão , Neoplasias Mamárias Animais , Animais , Neoplasias da Mama/veterinária , Gatos , Transformação Celular Neoplásica , Cães , Feminino , Via de Sinalização Hippo , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , beta CateninaRESUMO
In ungulates the stability of the fetlock joint is dependent on several muscles, which are exposed to high stress and strain. Among those muscles, the proximal sesamoidean ligament or PSL (also known as the suspensory ligament or Ruini's elasto-tendinous organ) is organized at birth in layers of muscle fibres alternated with abundant tendinous tissue that, during the postnatal development, becomes the predominant tissue. In this study we analysed the PSL of the sheep at the age of 1, 30 and 180â¯days and determined the expression of several genes which either (a) are markers of muscle fibre growth and maturation, or (b) play a role as signal molecules. We observed an accelerated maturation, as indicated by the transition of MyHC isoform expression towards the slow isoforms and a reduced regenerative potential indicated by the low Pax7 expression and the altered Wnt signalling. We also found a specific myogenic expression pattern of MyoD, Myf5 and Myogenin in the developing PSL and high mRNA levels of specific fibrogenic factors, as TGF-ß1, that, undoubtedly, stimulate the growth of connective tissue. Our observations confirmed, at molecular level, the peculiarity of the fast involution observed in PSL a muscle that undergoes a very specific active differentiation process during early development, which implies myofibres involution and their replacement with connective tissue.
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Ligamentos/crescimento & desenvolvimento , Desenvolvimento Muscular/genética , Miosinas/genética , Carneiro Doméstico/genética , Fatores Etários , Animais , Diferenciação Celular , Fatores de Regulação Miogênica , Miosinas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ossos Sesamoides , Carneiro Doméstico/crescimento & desenvolvimentoRESUMO
BACKGROUND: Conversely to common coated iron oxide nanoparticles, novel naked surface active maghemite nanoparticles (SAMNs) can covalently bind DNA. Plasmid (pDNA) harboring the coding gene for GFP was directly chemisorbed onto SAMNs, leading to a novel DNA nanovector (SAMN@pDNA). The spontaneous internalization of SAMN@pDNA into cells was compared with an extensively studied fluorescent SAMN derivative (SAMN@RITC). Moreover, the transfection efficiency of SAMN@pDNA was evaluated and explained by computational model. METHODS: SAMN@pDNA was prepared and characterized by spectroscopic and computational methods, and molecular dynamic simulation. The size and hydrodynamic properties of SAMN@pDNA and SAMN@RITC were studied by electron transmission microscopy, light scattering and zeta-potential. The two nanomaterials were tested by confocal scanning microscopy on equine peripheral blood-derived mesenchymal stem cells (ePB-MSCs) and GFP expression by SAMN@pDNA was determined. RESULTS: Nanomaterials characterized by similar hydrodynamic properties were successfully internalized and stored into mesenchymal stem cells. Transfection by SAMN@pDNA occurred and GFP expression was higher than lipofectamine procedure, even in the absence of an external magnetic field. A computational model clarified that transfection efficiency can be ascribed to DNA availability inside cells. CONCLUSIONS: Direct covalent binding of DNA on naked magnetic nanoparticles led to an extremely robust gene delivery tool. Hydrodynamic and chemical-physical properties of SAMN@pDNA were responsible of the successful uptake by cells and of the efficiency of GFP gene transfection. GENERAL SIGNIFICANCE: SAMNs are characterized by colloidal stability, excellent cell uptake, persistence in the host cells, low toxicity and are proposed as novel intelligent DNA nanovectors for efficient cell transfection.
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DNA/química , Técnicas de Transferência de Genes , Nanopartículas/química , Transfecção/métodos , Fenômenos Biofísicos , Coloides/química , DNA/genética , Compostos Férricos/química , Vetores Genéticos , Humanos , Microscopia Eletrônica de Transmissão , Nanopartículas/ultraestrutura , Tamanho da Partícula , Plasmídeos/química , Plasmídeos/genéticaRESUMO
The Tat protein is able to translocate through the plasma membrane and when it is fused with other peptides may acts as a protein transduction system. This ability appears particularly interesting to induce tissue-specific differentiation when the Tat protein is associated to transcription factors. In the present work, the potential of the complex Tat-MyoD in inducing equine peripheral blood mesenchymal stem cells (PB-MSCs) towards the myogenic fate, was evaluated. Results showed that the internalization process of Tat-MyoD happens only in serum free conditions and that the nuclear localization of the fused complex is observed after 15 hours of incubation. However, the supplement of Tat-MyoD only was not sufficient to induce myogenesis and, therefore, in order to achieve the myogenic differentiation of PB-MSCs, conditioned medium from C2C12 cells was added without direct contact. Real Time PCR and immunofluorescence methods evaluated the establishment of a myogenic program. Our results suggest that TAT- transduction of Tat-MyoD, when supported by conditioned medium, represents a useful methodology to induce myogenic differentiation.
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Diferenciação Celular/efeitos dos fármacos , Produtos do Gene tat/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteína MyoD/farmacologia , Animais , Meios de Cultivo Condicionados/farmacologia , Cavalos , Células-Tronco Mesenquimais/fisiologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Transdução de SinaisRESUMO
BACKGROUND: Impression cytology (IC) is a noninvasive technique in which filters are used to sample superficial layers of ocular epithelium. OBJECTIVES: The aim of this study was to compare cytology specimens obtained by IC and cytobrush from healthy canine and feline eyes. METHODS: Dogs and cats were prospectively sampled using polytetrafluorethylene filters on the right eye, and cytobrush on the left eye. Wright-Giemsa-stained specimens were evaluated by 2 observers. Cellularity, preservation, and morphology of cells and presence of goblet and inflammatory cells were scored with a 4-grade scale. Inter-observer agreement and effects of topical anesthesia were analyzed. RESULTS: In 20 canine IC samples, 10 showed good cellularity (score 2-3) and 13 good preservation. Superficial epithelial cells (SEC) were present in 13/20 of IC, while basal-intermediate cells (BIC) were seen in 14/20. In 6/20 and 7/20, goblet and inflammatory cells were noted, respectively. In 20 cats, 15 of IC showed good cellularity and 14 good preservation, and SEC were present in 16/20 of IC and BIC in 17/20. In 13/20 and 3/20 cats, goblet cells and inflammatory cells were noted, respectively. Canine cytobrush specimens appeared well preserved (9/20) and had good cellularity (8/20). In feline cytobrush specimens, good preservation and cellularity were observed in 16/20 and 14/20, respectively. In both species, all cell types were present without a clear separation. There was moderate to fair agreement about cellular morphology in IC between observers. Specimens obtained with and without anesthesia were comparable. CONCLUSION: Impression cytology allowed collection of samples with maintained cytoarchitecture, while cytoplasmatic and nuclear details were often difficult to evaluate.
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Citodiagnóstico/veterinária , Manejo de Espécimes/veterinária , Animais , Gatos , Túnica Conjuntiva/citologia , Citodiagnóstico/métodos , Citoplasma , Cães , Células Epiteliais/citologia , Feminino , Células Caliciformes/citologia , MasculinoRESUMO
BACKGROUND AIMS: Several cytokines and growth factors play an essential role in skin regeneration and epithelial-like stem cells (EpSCs) have beneficial effects on wound healing in horses. However, there are no reports available on the expression of these growth factors and cytokines after EpSC therapy. METHODS: Wounds of 6 cm(2) were induced in the gluteus region of 6 horses and treated with (i) autologous EpSCs, (ii) allogeneic EpSCs, (iii) vehicle treatment or (iv) untreated control. Real time polymerase chain reaction was performed on tissue biopsies taken 1 and 5 weeks after these treatments to evaluate mRNA expression of interferon (IFN)-γ, interleukin (IL)-6, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF)-1 and epidermal keratin (eKER). RESULTS: One week after treatments, mRNA levels of IL-6 (P = 0.012) and VEGF (P = 0.008) were higher in allogeneic EpSC-treated wounds compared with controls. Also, mRNA levels of IGF-1 were higher at 1 week in both autologous (P = 0.027) and allogeneic (P = 0.035) EpSC-treated wounds. At week 5, all EpSC- and vehicle-treated wounds demonstrated significantly higher IFN-γ, VEGF and eKER mRNA expression compared with controls and compared with their respective levels at week 1. CONCLUSIONS: Equine wounds treated with allogeneic EpSCs demonstrate a significant increase in mRNA expression of IL-6, VEGF and IGF-1 in the acute phase. In the longer term, an increase in IFN-γ, VEGF and eKER mRNA was detected in the wounds treated with allogenic EpSCs, autologous EpSCs or their vehicle.
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Biomarcadores/metabolismo , Células Epiteliais/transplante , Transplante de Células-Tronco/métodos , Cicatrização/genética , Animais , Biomarcadores/análise , Citocinas/genética , Citocinas/metabolismo , Epiderme/metabolismo , Células Epiteliais/metabolismo , Cavalos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Regeneração/genética , Pele/metabolismo , Células-Tronco/metabolismo , Transplante AutólogoRESUMO
Tendons regenerate poorly due to a dense extracellular matrix and low cellularity. Cellular therapies aim to improve tendon repair using mesenchymal stem cells and tenocytes; however, a current limitation is the low proliferative potential of tenocytes in cases of severe trauma. The purpose of this study was to develop a method useful in veterinary medicine to improve the differentiation of Peripheral Blood equine mesenchymal stem cells (PB-MSCs) into tenocytes. PB-MSCs were used to study the effects of the addition of some growth factors (GFs) as TGFß3 (transforming growth factor), EGF2 (Epidermal growth factor), bFGF2 (Fibroblast growth factor) and IGF-1 (insulin-like growth factor) in presence or without Low Level Laser Technology (LLLT) on the mRNA expression levels of genes important in the tenogenic induction as Early Growth Response Protein-1 (EGR1), Tenascin (TNC) and Decorin (DCN). The singular addition of GFs did not show any influence on the mRNA expression of tenogenic genes whereas the specific combinations that arrested cell proliferation in favour of differentiation were the following: bFGF2 + TGFß3 and bFGF2 + TGFß3 + LLLT. Indeed, the supplement of bFGF2 and TGFß3 significantly upregulated the expression of Early Growth Response Protein-1 and Decorin, while the use of LLLT induced a significant increase of Tenascin C levels. In conclusion, the present study might furnish significant suggestions for developing an efficient approach for tenocyte induction since the external administration of bFGF2 and TGFß3, along with LLLT, influences the differentiation of PB-MSCs towards the tenogenic fate.
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Diferenciação Celular/efeitos dos fármacos , Cavalos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Traumatismos dos Tendões/veterinária , Tendões/citologia , Animais , Proliferação de Células , Células Cultivadas , Decorina/genética , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Terapia com Luz de Baixa Intensidade , Traumatismos dos Tendões/terapiaRESUMO
OBJECTIVE: The aim of this preliminary study was to investigate the effect of Multiwave Locked System (MLS(®)), a particular model of low-level laser, in the acute phase of collagenase-induced tendon lesions in six adult sheep randomly assigned to two groups. BACKGROUND DATA: Tendon injuries are common among human athletes and in sport horses, require a long recovery time, and have a high risk of relapse. Many traditional treatments are not able to repair the injured tendon tissue correctly. In recent years, the use of low-level laser therapy (LLLT) produced interesting results in inflammatory modulation in different musculoskeletal disorders. METHODS: Group 1 received 10 treatments of MLS laser therapy at a fluence of 5 J/cm(2) on the left hindlimb. Group 2 received 10 treatments of MLS laser therapy at a fluence of 2.5 J/cm(2) on the left hindlimb. In every subject in both groups, the right hindlimb was considered as the control leg. RESULTS: Clinical follow-up and ultrasonography examinations were performed during the postoperative period, and histological examinations were performed at day 30 after the first application of laser therapy. In particular, results from histological examinations indicate that both treatments induced a statistically significant cell number decrease, although only in the second group did the values return to normal. Moreover, the MLS laser therapy dose of 2.5 J/cm(2) (group 2) caused a significant decrease of vessel area. CONCLUSIONS: In this study, clinical and histological evaluation demonstrated that a therapeutic dose <5 J/cm(2) furnished an anti-inflammatory effect, and induced a decrease of fibroblasts and vessel area. Overall, our results suggest that MLS laser therapy was effective in improving collagen fiber organization in the deep digital flexor tendon.
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Terapia com Luz de Baixa Intensidade/métodos , Tendinopatia/radioterapia , Animais , Feminino , Projetos Piloto , Ovinos , Tendinopatia/patologia , Tendões/patologiaRESUMO
Strain injuries of tendons are the most common orthopedic injuries in athletic subjects, be they equine or human. When the tendon is suddenly damaged, an acute inflammatory phase occurs whereas its repetitive overloading may cause chronic injuries. Currently the criteria used for grading injuries are general and subjective, and therefore a reliable grading method would be an improvement. The main purpose of this study was to assess qualitatively the histological pattern of Masson trichrome stain in healthy and injured tendons; indeed, the known "paradox" of Masson staining was used to create an evaluation for the matrix of tendons, following experimental lesions and natural repair processes. A statistically significant difference of aniline-staining between healthy and lesioned tendons was observed. Overall, we think that the Masson staining might be regarded as an informative tool in discerning the collagen spatial arrangement and therefore the histological characteristics of tendons.
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Compostos Azo , Corantes , Amarelo de Eosina-(YS) , Técnicas Histológicas/métodos , Verde de Metila , Traumatismos dos Tendões/patologia , Tendões/patologia , Animais , Contagem de Células , Colágeno/metabolismo , Transtornos Traumáticos Cumulativos/patologia , Membro Posterior/patologia , Processamento de Imagem Assistida por Computador , Células-Tronco Mesenquimais , Ovinos , CicatrizaçãoRESUMO
Collagen has become a key-molecule in cell culture studies and in the tissue engineering field. Industrially, the principal sources of collagen are calf skin and bones which, however, could be associated to risks of serious disease transmission. In fact, collagen derived from alternative and riskless sources is required, and marine organisms are among the safest and recently exploited ones. Sea urchins possess a circular area of soft tissue surrounding the mouth, the peristomial membrane (PM), mainly composed by mammalian-like collagen. The PM of the edible sea urchin Paracentrotus lividus therefore represents a potential unexploited collagen source, easily obtainable as a food industry waste product. Our results demonstrate that it is possible to extract native collagen fibrils from the PM and produce suitable substrates for in vitro system. The obtained matrices appear as a homogeneous fibrillar network (mean fibril diameter 30-400 nm and mesh < 2 µm) and display remarkable mechanical properties in term of stiffness (146 ± 48 MPa) and viscosity (60.98 ± 52.07 GPa·s). In vitro tests with horse pbMSC show a good biocompatibility in terms of overall cell growth. The obtained results indicate that the sea urchin P. lividus can be a valuable low-cost collagen source for mechanically resistant biomedical devices.
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Materiais Biocompatíveis/química , Colágeno/química , Paracentrotus/química , Animais , Fenômenos Biomecânicos , Bovinos , Contagem de Células , Proliferação de Células , Colágeno/ultraestrutura , Humanos , Indicadores e Reagentes , Teste de Materiais , Mercaptoetanol/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Ratos , Suínos , Resistência à Tração , ViscosidadeRESUMO
The major goal of regenerative medicine is to determine experimental techniques that take maximal advantage of reparative processes that occur naturally in the animal body. Injection of mesenchymal stem cells into the core of a damaged tendon represents such an approach. Decellularization of native tendons as potential targets and seeding protocols are currently under investigation. The aim of our study was to manufacture a recellularized biocompatible scaffold from cadaveric tissue for use in total or partial tendon injuries. Results showed that it was possible to introduce proliferating cells into the core of a decellularized tendon to treat the scaffold with a collagen gel. The method was effective in maintaining scaffold extracellular matrix and for expressing collagen type I and cartilage oligomeric matrix protein by injecting mesenchymal stem cells.
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Tecido Adiposo/citologia , Colágeno/farmacologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Regeneração/efeitos dos fármacos , Tendões/fisiologia , Alicerces Teciduais/química , Proteína de Matriz Oligomérica de Cartilagem/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Colorimetria , DNA/metabolismo , Feminino , Géis/farmacologia , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Coloração e Rotulagem , Tendões/efeitos dos fármacosRESUMO
Besides the presence of somatic stem cells in hair follicles and dermis, the epidermis also contains a subpopulation of stem cells, reflecting its high regenerative capacity. However, only limited information concerning epidermis-derived epithelial-like stem/progenitor cells (EpSCs) is available to date. Nonetheless, this stem cell type could prove itself useful in skin reconstitution after injury. After harvesting from equine epidermis, the purified cells were characterized as EpSCs by means of positive expression for CD29, CD44, CD49f, CD90, Casein Kinase 2ß, p63, and Ki67, low expression for cytokeratin (CK)14 and negative expression for CD105, CK18, Wide CK, and Pan CK. Furthermore, their self-renewal capacity was assessed in adhesion as well as in suspension. Moreover, the isolated cells were differentiated toward keratinocytes and adipocytes. To assess the regenerative capacities of EpSCs, six full-thickness skin wounds were made: three were treated with EpSCs and platelet-rich-plasma (EpSC/PRP-treated), while the remaining three were administered carrier fluid alone (PRP-treated). The dermis of EpSC/PRP-treated wounds was significantly thinner and exhibited more restricted granulation tissue than did the PRP-treated wounds. The EpSC/PRP-treated wounds also exhibited increases in EpSCs, vascularization, elastin content, and follicle-like structures. In addition, combining EpSCs with a PRP treatment enhanced tissue repair after clinical application.
Assuntos
Diferenciação Celular , Epiderme/metabolismo , Queratinócitos/metabolismo , Regeneração , Células-Tronco/metabolismo , Animais , Antígenos de Diferenciação/metabolismo , Células Epidérmicas , Epiderme/transplante , Cavalos , Queratinócitos/citologia , Transplante de Células-Tronco , Células-Tronco/citologia , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologia , Ferimentos e Lesões/terapiaRESUMO
In order to treat frequently occurring conditions such as traumatic rupture or over-strain tendinopathies, the techniques of tissue engineering and cell-based therapies have become an accepted modus operandi since other available remedies appear to be ineffective in restoring the original structure and function of the injured tissue. However, the mechanisms accounting for the effectiveness of novel regenerative approaches in treating equine tendon and ligament injuries remain poorly characterised. In this review we summarize and discuss the most significant results of our research regarding bioscaffold technology for treating complete tendon tears and the use of adult stem cells for treating tendon lesions induced by over-strain.
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Células-Tronco Adultas , Transplante de Células-Tronco Mesenquimais/métodos , Traumatismos dos Tendões/veterinária , Engenharia Tecidual/métodos , Animais , Traumatismos dos Tendões/terapia , Engenharia Tecidual/tendências , Alicerces TeciduaisRESUMO
Tendon injuries, degenerative tendinopathies, and overuse tendinitis are common in races horses. Novel therapies aim to restore tendon functionality by means of cell-based therapy, growth factor delivery, and tissue engineering approaches. This study examined the use of autologous mesenchymal stromal cells derived from peripheral blood (PB-MSCs), platelet-rich plasma (PRP) and a combination of both for ameliorating experimental lesions on deep digital flexor tendons (DDFT) of Bergamasca sheep. In particular, testing the combination of blood-derived MSCs and PRP in an experimental animal model represents one of the few studies exploring a putative synergistic action of these treatments. Effectiveness of treatments was evaluated at 30 and 120 days comparing clinical, ultrasonographic, and histological features together with immunohistochemical expression of collagen types 1 and 3, and cartilage oligomeric matrix protein (COMP). Significant differences were found between treated groups and their corresponding controls (placebo) regarding tendon morphology and extracellular matrix (ECM) composition. However, our results indicate that the combined use of PRP and MSCs did not produce an additive or synergistic regenerative response and highlighted the predominant effect of MSCs on tendon healing, enhanced tissue remodeling and improved structural organization.
Assuntos
Transplante de Células-Tronco Mesenquimais , Plasma Rico em Plaquetas , Traumatismos dos Tendões/terapia , Cicatrização/efeitos dos fármacos , Animais , Colágeno Tipo I/biossíntese , Colágeno Tipo III/biossíntese , Colagenases , Sinergismo Farmacológico , Proteínas da Matriz Extracelular/biossíntese , Feminino , Glicoproteínas/biossíntese , Membro Posterior/diagnóstico por imagem , Membro Posterior/patologia , Proteínas Matrilinas , Carneiro Doméstico , Traumatismos dos Tendões/induzido quimicamente , Traumatismos dos Tendões/patologia , Ultrassonografia , Cicatrização/fisiologiaRESUMO
RET is a tyrosine kinase receptor, and transduces signaling by family of glial cell line-derived neurotrophic factor ligands (GFLs). RET is involved in the development of enteric nervous system, of sympathetic, parasympathetic, motor and sensory neurons. RET exists in two main isoforms originated by differential splicing, RET9 and RET51; phylogenetic studies have shown that the RET gene is conserved across vertebrates. The aim of this study was to investigate the RET expression within the brain of zebrafish, using immunohistochemistry, western blotting and RT-PCR. In homogenate brains both RET protein and mRNA were observed. RET immunoreactivity was widespread in neurons and neural processes of all the major regions of the brain. These results demonstrate the occurrence of RET and suggest an involvement of GDNF family ligands in the brain of adult zebrafish.