RESUMO
Tendinopathies are poorly understood diseases for which treatment remains challenging. Relevant in vitro models to study human tendon physiology and pathophysiology are therefore highly needed. Here we propose the automated 3D writing of tendon microphysiological systems (MPSs) embedded in a biomimetic fibrillar support platform based on cellulose nanocrystals (CNCs) self-assembly. Tendon decellularized extracellular matrix (dECM) was used to formulate bioinks that closely recapitulate the biochemical signature of tendon niche. A monoculture system recreating the cellular patterns and phenotype of the tendon core was first developed and characterized. This system was then incorporated with a vascular compartment to study the crosstalk between the two cell populations. The combined biophysical and biochemical cues of the printed pattern and dECM hydrogel were revealed to be effective in inducing human-adipose-derived stem cells (hASCs) differentiation toward the tenogenic lineage. In the multicellular system, chemotactic effects promoted endothelial cells migration toward the direction of the tendon core compartment, while the established cellular crosstalk boosted hASCs tenogenesis, emulating the tendon development stages. Overall, the proposed concept is a promising strategy for the automated fabrication of humanized organotypic tendon-on-chip models that will be a valuable new tool for the study of tendon physiology and pathogenesis mechanisms and for testing new tendinopathy treatments.
RESUMO
The importance of the hoof to the horse health is clear, and the current knowledge regarding the cellular aspects of hoof keratinocytes is poor. Studies on equine keratinocyte culture are scarce. Developing keratinocyte cultures in vitro is a condition for studies on molecular biology, cell growth and differentiation. Some methods have already been established, such as those for skin keratinocyte culture. However, few methodologies are found for lamellar keratinocytes. The objective of this study was to standardize the equine hoof keratinocyte isolation and cultivation, and then characterize the cell immunophenotype. For this, the primary culture method used was through explants obtained from three regions of the equine hoof (medial dorsal, dorsal, and lateral dorsal). After the cell isolation and cultivation, the cell culture and its explants were stained with anti-pan cytokeratin (pan-CK) (AE1/AE3), vimentin (V9), p63 (4A4), and Ki-67 (MIB-1) antibodies. Cells were grown to third passage, were positive for pan-CK, p63 and Ki-67, and few cells had vimentin positive expression. As for the explants, the epidermal laminae were not stained for vimentin or Ki-67. However, some cells presented positive pan-CK and p63 expression. This study demonstrated the viability of lamellar explants of equine hooves as a form of isolating keratinocytes in primary cultures, as well as characterized the proliferation ability of such keratinocytes in monolayers.(AU)
É notória a importância do casco na saúde dos equinos, mas o conhecimento em nível celular é pouco entendido. Estudos envolvendo o cultivo de queratinócitos equinos são escassos. Sabe-se que o desenvolvimento de cultivos de queratinócitos in vitro é uma condição para estudos sobre a biologia molecular, crescimento e diferenciação celular. Alguns métodos já estão estabelecidos, como para cultivo de queratinócitos de pele, mas poucas metodologias são encontradas para queratinócitos lamelares. O objetivo desse estudo foi padronizar o cultivo de queratinócitos provenientes de casco equino visando futuramente associar ao estudo da medicina regenerativa para assim estabelecer um modelo experimental in vitro e indicar o uso criterioso de terapias regenerativas para a laminite equina. Desta forma, o cultivo em monocamada e a caracterização de queratinócitos lamelares foram realizados. Para isso, o método de cultura primária utilizado foi através de explantes obtidos de três regiões do casco (dorso-medial, dorsal e dorso-lateral). As células foram caracterizadas para os marcadores anti pan-cytokeratin (AE1/AE3), vimentin (V9), p63 (4A4) e Ki-67 (MIB-1) nos cultivos e nos explantes. As células foram cultivadas até terceira passagem, tendo marcação positiva para pan-CK, p63 e Ki-67 e fraca marcação para vimentina. Já as lâminas epidermais não tiveram marcação de vimentin e Ki-67, porém marcaram acentuadamente para pan-CK e p63. Este estudo demonstrou a exiquibilidade do uso de explantes lamelares do casco de equinos, como forma de isolamento de queratinócitos em cultivos primários, bem como caracterizou a habilidade de proliferação desses queratinócitos em monocamada.(AU)
Assuntos
Animais , Cultura Primária de Células/veterinária , Doenças do Pé/veterinária , Casco e Garras/patologia , Doenças dos Cavalos/patologia , Doenças dos Cavalos/terapia , Queratinócitos/citologiaRESUMO
Jugular thrombosis in horses occurs commonly in iatrogenic situations, secondary to endotoxemic clinical condition and disseminated vascular coagulation, potentially leading to death. Thus, hemostatic evaluation becomes necessary and extremely important for monitoring the risks of systemic hypercoagulability and for the efficiency of allopathic and surgical treatment. This paper describes the hemostatic behavior in experimental jugular thrombosis of ten healthy equines, subsequently submitted to two thrombectomy techniques and receiving heparin sodium as anti-rethrombosis therapy. These animals were evaluated for 20 days by thromboelastometry (TEM), platelet count, hematocrit and fibrinogen, at four moments: pre-induction to phlebitis (D0-MPF); three days after thrombophlebitis induction (D3-MFM); 6 days after, - moment of thrombophlebitis - (D9-MT); and 54 (D16) and 126 (D19) hours after thrombectomies (PTM). Thrombectomy was performed via a Vollmar Ring (group 1, n=5) and Fogarty catheter (group 2, n=5). All the animals received heparin (150 UI/kg, SC) every 12 hours, for ten days after the respective thrombectomies. Through the blood samples were evaluated TEM, activated partial thromboplastin time (aPTT) and prothrombin time (PT), dosing of fibrinogen, hematocrit and platelet count at the abovementioned moments. For comparison between groups and moments the t test was applied at 5% significance level. No significant difference was verified between treatment groups at any of the moments. There were reductions in clotting time (CT) and clot formation time (CFT), with increase in maximum lysis (ML) until the moment D9-MT. Evaluation through INTEM® reagent presented prolongations of CT and CFT with reduction of α angle and ML starting from D16 and D19. Similarly, aPTT presented significant differences between moments pre- (D0, 3 and 9) and post- (D16 and 19) anticoagulant and surgical treatment. The platelet numbers were diminished at moments D16 and D19. In evaluation with EXTEM® reagent, prolongation of CT and CFT occurred only between the moments D0 vs. D3 and vs. D9. O PT did not present significant differences. The results obtained demonstrate that experimental jugular thrombophlebitis leads to local clinical alterations, with impairment of tissue and of the extrinsic coagulation pathway (EXTEM® ), but without evidence of systemic hypercoagulability status, since there was no increase of the alpha angle or maximum clot firmness (MCF). Furthermore, TEM was shown useful and more sensitive than conventional coagulation tests (PT, aPTT and fibrinogen) for the monitoring of anticoagulant therapy, as demonstrated in other works.(AU)
A trombose jugular nos equinos ocorre comumente em situações iatrogênicas, secundárias a quadros endotoxêmicos e a coagulação vascular disseminada, podendo levar ao óbito. Por isso, avaliação hemostática se faz necessária e de extrema importância para monitorar os riscos de hipercoagulabilidade sistêmica e também a eficiência do tratamento alopático e cirúrgico. Este trabalho descreve o comportamento hemostático na trombose jugular experimental de dez equinos hígidos, submetidos posteriormente a duas técnicas de trombectomia e recebendo heparina sódica como terapia anti retrombosante. Estes animais foram avaliados durante 20 dias por tromboelastometria (TEM), contagem de plaquetas, hematócrito e fibrinogênio, em quatro momentos: pré-indução à flebite (D0-MPF); três dias após a indução da tromboflebite (D3-MFM); 6 dias após, - momento de tromboflebite - (D9-MT); e 54 (D16) e 126 (D19) horas após as trombectomias (MPT). A trombectomia foi realizada com Anel de Vollmar (grupo 1, n=5) e cateter de Fogarty (grupo 2, n=5). Todos os animais receberam heparina (150 UI/Kg, SC) a cada 12 horas, durante dez dias após as respectivas trombectomias. Através de amostras de sangue, foram avaliadas a TEM, o tempo de tromboplastia parcial ativada (TTPa) e tempo de protrombina (TP), a dosagem de fibrinogênio, hematócrito e contagem de plaquetas nos momentos descritos acima. Para a comparação entre os grupos e momentos foi aplicado teste t, com nível de significância de 5%. Não foi verificada diferença significativa entre os grupos de tratamento em nenhum dos momentos. Houve redução do tempo de coagulação (CT) e do tempo de formação do coágulo (CFT), com aumento da lise máxima (LM) até o momento D9-MT. A avaliação com o reagente intem apresentou prolongamento do CT e do CFT e redução do ângulo α e da LM a partir do D16 e D19. Da mesma forma, o TTPa apresentou diferenças significativas entre os momentos pré (D0, 3 e 9) e pós (D16 e 19) tratamento cirúrgico e anticoagulante. Houve diminuição do número de plaquetas nos momentos D16 e D19. Na avaliação com reagente extem ocorreu apenas o prolongamento do CT e CFT entre os momentos D0 e o D3 e D9. O TP não apresentou diferenças significativas. Os resultados obtidos demonstram que a tromboflebite jugular experimental leva a alterações clínicas locais, com comprometimento tecidual e da via extrínseca da coagulação (extem), porém sem evidências de um estado sistêmico de hipercoagulabilidade, pois não houve aumento do ângulo alfa e da firmeza máxima do coágulo (MCF). Além disso, a TEM se mostrou útil e mais sensível que os testes convencionais de coagulação (TP, TTPa e fibrinogênio) para o acompanhamento da terapia anticoagulante, conforme demonstrado em outros trabalhos.(AU)
Assuntos
Animais , Anticoagulantes/análise , Transtornos Hemostáticos/veterinária , Cavalos , Tromboflebite/veterinária , Trombose/veterinária , Catéteres/veterinária , Técnicas Hemostáticas/veterinária , Trombectomia/veterináriaRESUMO
O plasma rico em plaquetas (PRP) é conhecido por apresentar propriedades anabólicas, anti-inflamatórias e capacidade de gelificação. Atualmente o PRP é considerado eficaz na reparação da cartilagem, sendo sua capacidade de formação de gel indicada para o preenchimento de defeitos condrais. O objetivo desse estudo foi analisar o uso do PRP ativado, no formato de arcabouço, como suporte para o implante de células tronco mesenquimais (CTM), no preenchimento e tratamento de lesões condrais induzidas em equinos. Doze equinos foram submetidos a uma cirurgia artroscópica no tempo zero do experimento (T0), onde foi induzida uma lesão condral de 15 mm de diâmetro na tróclea medial femoral dos membros pélvicos direito. As 12 articulações foram divididas em dois grupos distintos com seis articulações cada (GA e GB). As articulações do GA foram submetidas ao tratamento com o implante de CTM em gel de PRP. As articulações de GB foram o grupo controle do experimento. As CTMs foram extraídas do tecido adiposo e o PRP em gel foi obtido por protocolo de dupla centrifugação seguido da adição de trombina liofilizada. Após cinco meses (T150) foi realizada nova artroscopia para avaliação macroscópica do local, coleta de amostras do tecido de reparação para análises de microscopia eletrônica, sendo realizadas imagens ressonância magnética e tomografia computadorizada no local do implante no GA. Observamos que o gel de PRP associado às CTM demonstrou ser adequado no tratamento de defeitos condrais experimentais dos equinos. GA evidenciou um melhor aspecto macroscópico e microscópico do tecido de reparação, sendo que GB mostrou maior desorganização das fibras colágenas. Nas imagens de ressonância magnética e tomografia computadorizada apenas foi relevante o local da lesão condral. O arcabouço de gel de PRP demonstrou ser apropriado no suporte do tratamento com as CTMs, sendo de fácil aplicação e efetivo, demonstrando resultados promissores na reparação de lesões condrais induzidas.(AU)
The platelet-rich plasma (PRP) is characterized by its anabolic, anti-inflammatory and gelling capability. Nowadays, the PRP is considered effective in the repair of cartilage defects, and its gelling capability is proper to filling chondral defects. So, the aim of this study was to investigate the use of activated PRP as a fibrin gel scaffold, such as support for the use with mesenchymal stem cells (MSC), on the treatment of experimentally chondral articular defects. Twelve horses were subjected to an arthroscopic surgery at time zero of the experiment (T0). A chondral defect of 15 mm diameter was created on the medial femoral trochlea and these 12 joints were divided into two groups each with six joints in each group (GA and GB). The joints of the GA were treated with implantation of MSC and PRP-gel. GB joints were the control group. MSCs were cultivated from adipose tissue and PRP-gel was obtained by double centrifugation protocol followed by addition of lyophilized thrombin. After five months (T150) was performed new arthroscopy for macroscopic evaluation of the defect local, collect samples of tissue repair for electron microscopy assessment and also was implemented a magnetic resonance images and computed tomography on GA. It was observed that the PRP-gel associated with CTMs showed a suitable treatment of experimental chondral defects in horses. GA showed a better macroscopic and microscopic appearance of the tissue repair. GB showed smaller number of chondrocytes and increased collagen fibers disorganization. At the magnetic resonance and computed tomography imaging only the local of chondral defect was viewed. The PRP-gel scaffold was satisfactory to use and support MSCs implantation. It showed an easy handling and it was effective, showing a promising results in the repair of induced chondral defects.(AU)
Assuntos
Animais , Células-Tronco Adultas , Doenças das Cartilagens/terapia , Doenças das Cartilagens/veterinária , Fibrina/uso terapêutico , Cavalos/lesões , Plasma Rico em Plaquetas , Artroscopia/veterinária , Terapia Baseada em Transplante de Células e Tecidos/veterináriaRESUMO
Trombose da veia jugular é problema freqüente na medicina eqüina, implicando muitas vezes em conseqüências fatais. O objetivo deste trabalho foi avaliar em eqüinos a aplicabilidade da trombectomia com cateter de Fogarty, técnica rotineiramente empregada pela medicina humana, no restabelecimento da perviedade vascular. Foram utilizados 10 eqüinos divididos em dois grupos de cinco animais, em que se induziu a trombose da veia jugular direita, através do acesso cirúrgico à veia e aplicação de sutura estenosante e injeção de glicose a 50%. No grupo controle avaliou-se a evolução da tromboflebite sem qualquer tipo de intervenção terapêutica. Os animais do grupo tratado foram submetidos à trombectomia com cateter de Fogarty. Foram avaliados os parâmetros clínicos gerais, regionais, ultra-sonográficos e angiográficos, nos momentos pré-indução (M-PRÉ), indução da trombose (MTI) e 10 dias de evolução da trombose (M10). A técnica empregada induziu a tromboflebite, que obstruiu completamente um segmento da veia jugular de todos os animais. Os animais do grupo controle mantiveram os trombos obstruindo totalmente o lume vascular até o final do período de avaliação, sendo que avaliações regionais mostraram principalmente o edema parotídeo e o ingurgitamento vascular, cranial à tromboflebite da veia jugular. O grupo tratado apresentou as veias jugulares pérvias ao final do experimento, confirmadas pelos exames ultra-sonográficos e angiográficos, com remissão total dos sinais clínicos. Concluiu-se que a técnica da trombectomia com cateter de Fogarty foi eficiente na desobstrução da veia jugular submetida à trombose experimental.
Thrombosis of jugular vein is a common problem in the equine medicine, implying frequently in fatal outcomes. The diagnosis is relatively simple, based on the clinical findings, angiographics images and ultrasonographycs. The therapeutic employed to a large extent of the cases is unsatisfactory. The purpose of this study was to evaluate the applicability of the thrombectomy with Fogarty's catheter in horses. This technique is routinely used in medicine, in the reestablishment of the vascular perviousness. Ten horses were allocated in two groups (five animals each) and induced to an unilateral thrombosis of right jugular vein, through the surgical access and an application of stenotic suture and glucose 50% injection. In the control group evolution of the thrombophlebitis without any therapeutical intervention was evaluated. The animals of the treatment group were submitted to the thrombectomy with Fogarty's catheter. General clinical parameters were analyzed at the moment of the preinduction (MPRE), induction of thrombosis (MTI), and at the 10th day of thrombosis evolution (M10). The procedure induced thrombophlebitis that completely obstructed a segment of the jugular vein in all animals. In the animals of the control group, the thrombus totally obstructed the vascular lumen until the end of the period of evaluation, and parotid edema and vascular dilated, cranial to the thrombophlebitis of jugular vein were observed. The treatment group presented all veins pervious in the end of the experiment, with total remission of the clinical signs, confirmed by angiographic and ultrasonographic examinations. So far, it was concluded that the technique of thrombectomy with Fogarty's catheter was effective in removal of the thrombosis obstruction experimentally induced in the jugular vein.
Assuntos
Animais , Cateterismo , Equidae , Trombectomia/métodos , Tromboflebite/cirurgia , Veias Jugulares/cirurgiaRESUMO
Hereditary equine regional dermal asthenia belongs to a group of inherited, congenital connective tissue dysplasias usually described as hyperelastosis cutis, cutaneous asthenia, dermatosparaxis, or Ehlers-Danlos-like syndrome. This report presents the clinical and histological features of three related Quarter horses affected with regional dermal asthenia. These horses had bilateral asymmetric lesions of the trunk and lumbar regions, where the skin was hyperextensible. Handling of the skin elicited a painful response and superficial trauma led to skin wounds. The skin was thinner than normal in the affected areas, with thickened borders and harder fibrotic masses (pseudotumours). The histopathological findings included thinner and smaller collagen fibrils, and a loose arrangement of collagen fibres in the middle, adventitial and deep dermis. Masson's trichrome and Calleja stains did not reveal any abnormality of collagen and elastic fibres. Electron microscopy showed no abnormalities. As in human patients, pseudotumour histopathological findings included fibroplasia and neovascularization. The pedigree chart of these animals supports an autosomal recessive type of inheritance, which has been suggested by other studies. This is the first report of this disease in Brazil. Its clinical and histological features resemble those described in horses affected with this condition in the United States.