Ionic Detergent Under Pressure-Vacuum as an Innovative Strategy to Generate Canine Tracheal Scaffold for Organ Engineering.

Decellularized scaffolds applied in tissue engineering offer improvements, supplying the elevated necessity for organs and tissues for replacement. However, obtaining a functional trachea for autotransplantation or allotransplantation is tricky due to the organ anatomical and structural complexity. Most tracheal decellularization protocols are lengthy, expensive, and could damage the tracheal extracellular matrix (ECM) architecture and functionality. Here, we aimed to evaluate the effectiveness of 3 different decellularization protocols combined with chemical and physical methods to obtain acellular canine tracheal scaffolds. Six adult dog tracheas were incised (tracheal segments) resulting in 28 rings for control tissue and 84 rings for decellularization (5-7 mm thick). Subsequently, decellularized tracheal scaffolds were microscopically/macroscopically characterized by histological analysis (Hematoxylin-Eosin, Masson's trichrome, Picrosirius red, Alcian blue, and Safranin O), immunohistochemistry for ECM components, scanning electron microscopy, and genomic DNA quantification. After decellularization, the tracheal tissue revealed reduced genomic DNA, and maintenance of ECM components preserved (structural proteins, adhesive glycoproteins, glycosaminoglycans and proteoglycans), suggesting ECM integrity and functionality. Comparatively, the combined ionic detergent with high vacuum pressure decellularization protocol revealed superior genomic DNA decrease (13.5 ng/mg) and improvement on glycosaminoglycans and proteoglycans preservation regarding the other decellularized trachea scaffolds and native tissue. Our results indicate that the 3 chemical/physical protocols reduce the decellularization time without ECM proteins damage. Notwithstanding, the use of ionic detergent under vacuum pressure was able to generate an innovative strategy to obtain acellular canine tracheal scaffolds with the highest levels of adhesive proteins that support its potentiality for recellularization and future tissue engineering application.

Ursolic acid increases strength in mdx mice model and may decrease fibrosis deposition by tgf-ß downregulation / El ácido ursólico aumenta la resistencia en el modelo de ratones mdx y podría disminuir la deposición de fibrosis mediante la regulación decreciente de tgf-ß

SUMMARY: Dystrophin disfunction results in sarcolemma destabilization, leading muscle cell damage by continuous degeneration cycles and limited regeneration. In muscle dystrophy, caused by dystrophin dysfunction, inflammation, necrosis and fibrosis are pathophysiological muscle function loss characteristics. As a genetic disease, this muscle dystrophy has no cure, however, advances in drug therapy using glucocorticoids can decrease the disease progression. Subsequently, alternative therapies were studied, such as ursolic acid (UA), that inhibits muscle atrophy and increases muscle mass and strength. Herein, we used 10 mg/kg daily supplementation in mdx mice for 4 weeks to evaluate serum creatine phosphokinase (CPK), muscle strength (Kondziela test), muscular organization (histology) and expression of fibrosis related genes (TGF-ß, TNF-α, mstn and ostn). UA supplementation increased muscle morphological organization, motor strength and decreased muscular TGF-ß expression. Altogether, the gene expression profile, histological organization and strength could suggest that UA treatment did not stop the fibrogenesis but decreased its progress.

RESUMEN: La disfunción de la distrofina resulta en la desestabilización del sarcolema, llevando al daño de las células musculares por ciclos continuos de degeneración y regeneración limitada. En la distrofia muscular, debido a la disfunción de la distrofina, la inflamación, la necrosis y la fibrosis, son características fisiopatológicas de la pérdida de la función muscular. Como enfermedad genetica no es possible remediar esta distrofia muscular, sin embargo, los avances en la terapia de medicamentos con glucocorticoides pueden disminuir la progresión de la enfermedad. Se estudiaron terapias alternativas, como el ácido ursólico (UA), que inhibe la atrofia muscular y aumenta la masa y la fuerza muscular. En este estudio, utilizamos una suplementación diaria de 10 mg / kg en ratones mdx durante 4 semanas para evaluar la creatina fosfoquinasa (CPK) sérica, la fuerza muscular (prueba de Kondziela), la organización muscular (histología) y la expresión de genes relacionados con la fibrosis (TGF-ß, TNF- α, mstn y ostn). La suplementación con AU aumentó la organización morfológica muscular, la fuerza motora y la disminución de la expresión muscular de TGF-ß. El perfil de expresión génica, la organización histológica y la fuerza simultáneamente podrían sugerir que el tratamiento con AU no detuvo la fibrogénesis sino que disminuyó su progreso.

Comparison between placental and skeletal muscle ECM: in vivo implantation.

PURPOSE OF THE STUDY: Several tissues have been decellularized and their extracellular matrices used as allogeneic or xenogeneic scaffolds, either in orthotopic or heterotopic implantations, for tissue engineering purposes. Placentas have abundant matrix, extensive microvascular structure, immunomodulatory properties, growth factors and are discarded after birth, representing an interesting source of extracellular matrix. This study aimed at comparing decellularized canine placentas and murine skeletal muscles to regenerate skeletal muscles in a rat model. MATERIALS AND METHODS: Muscle pockets were created at the posterior limbs of male Wistar rats, where the muscle- and placenta-derived extracellular matrices were implanted. Macroscopic, histological, and immunohistochemical analyses were performed after 3, 15, and 45 days of surgeries. RESULTS: On the third day, intense inflammatory reaction, with macrophages (CD163+) and proliferative cells (PCNA+) being observed in control group and adjacent to the decellularized matrices. The percentage of proliferative cells was higher in placenta than in muscle matrices. Macrophages CD163+ high were higher in muscles than in placentas, whereas CD163+ low were higher in placentas than in muscle ECM, at days 3 and 15. Placental matrices were not completely degraded at day 15, as opposed to the muscular ones. After 45 days, both matrices were resorbed and morphologically normal myofibers, with reduction of cell infiltration, were observed. CONCLUSIONS: These results demonstrated that xenogeneic placental ECM, implanted heterotopically (representing a biologically critical and challenging microenvironment), induced local inflammatory reactions similar to the allogeneic muscle ECM, implanted orthotopically. Thus, placenta-derived extracellular matrix must be further explored in regenerative medicine.

Equine yolk sac: a stem cells source / Saco vitelino equino: una fuente de células madre

SUMMARY: Mesenchymal stem cells are characterized by in vitro high proliferation and multilineage potential maintenance. This study aimed to isolate and characterize equine YS mesenchymal stem cells and compare these with amniotic membranes. The yolk sac (YS) and amniotic membranes (AM) were obtained from 20 pregnant mares with gestational age around 30 days. Cells were cultured in α-MEM supplemented with 15 % FBS, 1 % antibiotic solution, 1 % L-glutamine and 1 % nonessential amino acids. To cell characterization we used cytogenetic analysis, fibroblast colony-forming unit assays, cell growth curves, immunophenotyping, flow cytometry, differentiation assays and teratoma formation. Results: Both cell sources presented fibroblastoid and epithelioid-like format. The YS cells have lower colony formation potential then AM ones, 3 versus 8 colonies per 103 plated cells. However, YS cells grew progressively while AM cells showed steady. Both, the YS and amnion cells immunolabeled for Oct-4, Nanog, SSEA-3, cytokeratin 18, PCNA, and vimentin. In addition, presented mesenchymal, hematopoietic, endothelial and pluripotency markers in flow cytometry. Discussion: Both cell sources presented high plasticity and differed into osteogenic, adipogenic, and chondrogenic lineages, and no tumor formation in nude mice was observed. The results suggest that horse YS may be useful for cell therapy such as amnion-derived cells.

RESUMEN: Las células madre mesenquimales se caracterizan por una alta proliferación in vitro y un mantenimiento potencial de múltiples líneas. Este estudio tuvo como objetivo aislar y caracterizar las células madre mesenquimales del saco vitelino equinas y compararlas con las membranas amnióticas. Se obtuvo el saco vitelino (SV) y las membranas amnióticas (MA) de 20 yeguas preñadas con edad gestacional de aproximadamente 30 días. Las células se cultivaron en α -MEM suplementado con 15 % de FBS, 1 % de solución antibiótica, 1 % de L-glutamina y 1 % de aminoácidos no esenciales. Para la caracterización celular utilizamos análisis citogenéticos, ensayos de unidades de colonias de fibroblastos, curvas de crecimiento celular, inmunofenotipaje, citometría de flujo, ensayos de diferenciación y formación de teratomas. Ambas fuentes celulares presentaron formato fibroblastoideo y epitelioide. Las células SV tienen un potencial de formación de colonias más bajo que las de MA, 3 versus 8 colonias por 103 células en placa. Sin embargo, las células SV crecieron progresivamente mientras que las células MA se mostraron estables. Tanto las células YS como las células amnios están inmunomarcadas para Oct-4, Nanog, SSEA-3, citoqueratina 18, PCNA y vimentina. Además, presentó marcadores mesenquimales, hematopoyéticos, endoteliales y pluripotenciales en citometría de flujo. Ambas fuentes celulares presentaron alta plasticidad y diferían en linajes osteogénicos, adipogénicos y condrogénicos, y no se observó formación de tumores en ratones. Los resultados sugieren que el SV de caballo puede ser útil para la terapia celular, como las células derivadas de amnios.

Rabbit vomeronasal organ-derivered cells have mesenchymal profile and neuronal commitment / Las células derivadas del órgano vomeronasal del conejo tienen perfil mesenquimatoso y compromiso neuronal

SUMMARY: The vomeronasal organ (VNO) is an accessory organ involved on the olfactory pathway, that detects pheromones and emits signals in order to modulate social and reproductive behavior. The VNO stem cells replace neurons throughout life. The aim of this study was to isolate and characterize cells derived from the vomeronasal organ from New Zealand rabbits. Five male rabbits with 120 days were used for cell isolation and culture. Results: VNO-derived cells presented labelling for proliferation (PCNA), undifferentiated profile (Nanog), neuronal (GFAP), mesenchymal stem cells (CD73, CD90 and CD105 and Stro-1). Also, presence of cytoskeletal (Vimentin, b-tubulin and CK-18) and absence of hematopoietic markers (CD34, CD117 and CD45) both by immunofluorescence and flow cytometry. By PCR it was possible to verify the expression of some undifferentiated profile (Oct-4), neuronal (Nestin) and mesenchymal (CD73, CD105 and Vimentin) genes. Functionally, VNO-derived cells differentiate in vitro into adipocytes, osteocytes and chondrocytes, and presented no tumorigenic potential when injected to Balb/c nu/nu mice. In conclusion, the rabbit VNO-derived cells have a profile that could be supportive to VNO olfactory/neuroreceptor epithelium by delivering factors to epithelial turnover or even by differentiation into epithelial cells to replacement of commissural epithelium.

RESUMEN: El órgano vomeronasal (OVN) es un órgano accesorio de la vía olfatoria, que detecta feromonas y emite señales que afectan la modulación del comportamiento social y reproductivo. Las células madre OVN reemplazan las neuronas durante toda la vida. El objetivo de este estudio fue aislar y caracterizar células derivadas del órgano vomeronasal de conejos raza Nueva Zelanda. Para el aislamiento y el cultivo celular se utilizaron cinco conejos machos con una edad de 120 días. Las células del OVN presentaron etiquetado para la proliferación (PCNA), un perfil indiferenciado (Nanog), neuronal (GFAP), células madre mesenquimales (CD73, CD90 y CD105 y Stro-1). Además, se ob- servó presencia de citoesqueleto (Vimentina, β-tubulina y CK-18) y ausencia de marcadores hematopoyéticos (CD34, CD117 y CD45) tanto por inmunofluorescencia como por citometría de flujo. Me- diante PCR fue posible verificar la expresión de algunos genes de perfil indiferenciado (Oct-4), neuronal (Nestin) y mesenquimatoso (CD73, CD105 y Vimentin). Las células derivadas del OVN se diferencian in vitro en adipocitos, osteocitos y condrocitos, y no presentan un potencial tumorigénico al ser infiltrados en ratones Balb / c nu / nu. En conclusión, las células derivadas de OVN de conejo tienen un perfil que podría ser compatible con el epitelio olfatorio / neurorreceptor de OVN transmitiendo factores al recambio epitelial o incluso mediante la diferenciación en células epiteliales para reemplazar el epitelio comisural.

Key characteristics of the ovary and uterus for reproduction with particular reference to poly ovulation in the plains viscacha (Lagostomus maximus, chinchillidae).

One of the dogmas of mammalian reproduction states is that primordial germ cells in females are restricted to the intrauterine phase, and that only a small portion of oocytes is available for ovulation during the adult life. Among the rare exceptions to this rule is the plains viscacha. This specie polyovulates up to 800 oocytes per cycle, from which 10 to 12 are implanted, but only 1-2 conceptuses survive. To better understand the key mechanisms of this pattern of embryonic to uterine interactions, we analyzed 19 female genital systems by means of gross morphology, histology, stereology and immunohistochemistry. Data showed that a specialized, highly convoluted structure of the ovarian cortex developed during the intrauterine phase as a prerequisite for the massive super-ovulation, likely associated with the inhibition of apoptosis and continued proliferation of germ cells, as well as maintenance of several corpora lutea during the adult life. In addition, specializations of uterine vasculature and musculature were demonstrated. Altogether, these key morphological characteristics evolved in order to contribute as compensatory or controlling mechanism for polyovulation and polyimplantation that led these species into becoming an unique enigma in reproductive biology, and a potential animal model to provide explanations regarding to developmental specializations.

Comparison between placental and skeletal muscle ECM: in vivo implantation

Purpose of the study: Several tissues have been decellularized and their extracellular matrices used as allogeneic or xenogeneic scaffolds, either in orthotopic or heterotopic implantations, for tissue engineering purposes. Placentas have abundant matrix, extensive microvascular structure, immunomodulatory properties, growth factors and are discarded after birth, representing an interesting source of extracellular matrix. This study aimed at comparing decellularized canine placentas and murine skeletal muscles to regenerate skeletal muscles in a rat model. Materials and Methods: Muscle pockets were created at the posterior limbs of male Wistar rats, where the muscle- and placenta-derived extracellular matrices were implanted. Macroscopic, histological, and immunohistochemical analyses were performed after 3, 15, and 45 days of surgeries. Results: On the third day, intense inflammatory reaction, with macrophages (CD163+) and proliferative cells (PCNA+) being observed in control group and adjacent to the decellularized matrices. The percentage of proliferative cells was higher in placenta than in muscle matrices. Macrophages CD163+ high were higher in muscles than in placentas, whereas CD163+ low were higher in placentas than in muscle ECM, at days 3 and 15. Placental matrices were not completely degraded at day 15, as opposed to the muscular ones. After 45 days, both matrices were resorbed and morphologically normal myofibers, with reduction of cell infiltration, were observed. Conclusions: These results demonstrated that xenogeneic placental ECM, implanted heterotopically (representing a biologically critical and challenging microenvironment), induced local inflammatory reactions similar to the allogeneic muscle ECM, implanted orthotopically. Thus, placenta-derived extracellular matrix must be further explored in regenerative medicine.

Pericytes in the Placenta: Role in Placental Development and Homeostasis.

The placenta is the most variable organ, in terms of structure, among the species. Besides it, all placental types have the same function: production of viable offspring, independent of pregnancy length, litter number, or invasion level. The angiogenesis is a central mechanism for placental functionality, due to proper maternal-fetal communication and exchanges. Much is known about the vasculature structure, but little is known about vasculature development and cellular interactions. Pericytes are perivascular cells that were described to control vasculature stability and permeability. Nowadays there are several new functions discovered, such as lymphocyte modulation and activation, macrophage-like phagocytic properties, tissue regenerative and repair processes, and also the ability to modulate stem cells, majorly the hematopoietic. In parallel, placental tissues are known to be a particularly immune microenvironment and a rich stem cell niche. The pericyte function plethora could be similar in the placental microenvironment and could have a central role in placental development and homeostasis.

Perivascular cell αv integrins as a target to treat skeletal muscle fibrosis.

Fibrosis following injury leads to aberrant regeneration and incomplete functional recovery of skeletal muscle, but the lack of detailed knowledge about the cellular and molecular mechanisms involved hampers the design of effective treatments. Using state-of-the-art technologies, Murray et al. (2017) found that perivascular PDGFRß-expressing cells generate fibrotic cells in the skeletal muscle. Strikingly, genetic deletion of αv integrins from perivascular PDGFRß-expressing cells significantly inhibited skeletal muscle fibrosis without affecting muscle vascularization or regeneration. In addition, the authors showed that a small molecule inhibitor of αv integrins, CWHM 12, attenuates skeletal muscle fibrosis. From a drug-development perspective, this study identifies a new cellular and molecular target to treat skeletal muscle fibrosis.

Macrophage-derived GPNMB accelerates skin healing.

Healing is a vital response important for the re-establishment of the skin integrity following injury. Delayed or aberrant dermal wound healing leads to morbidity in patients. The development of therapies to improve dermal healing would be useful. Currently, the design of efficient treatments is stalled by the lack of detailed knowledge about the cellular and molecular mechanisms involved in wound healing. Recently, using state-of-the-art technologies, it was revealed that macrophages signal via GPNMB to mesenchymal stem cells, accelerating skin healing. Strikingly, transplantation of macrophages expressing GPNMB improves skin healing in GPNMB-mutant mice. Additionally, topical treatment with recombinant GPNMB restored mesenchymal stem cells recruitment and accelerated wound closure in the diabetic skin. From a drug development perspective, this GPNMB is a new candidate for skin healing.

Central Nervous System and Vertebrae Development in Horses: a Chronological Study with Differential Temporal Expression of Nestin and GFAP.

The neural system is one of the earliest systems to develop and the last to be fully developed after birth. This study presents a detailed description of organogenesis of the central nervous system (CNS) at equine embryonic/fetal development between 19 and 115 days of pregnancy. The expression of two important biomarkers in the main structure of the nervous system responsible for neurogenesis in the adult individual, and in the choroid plexus, was demonstrated by Nestin and glial fibrillary acid protein (GFAP) co-labeling. In the 29th day of pregnancy in the undifferentiated lateral ventricle wall, the presence of many cells expressing Nestin and few expressing GFAP was observed. After the differentiation of the lateral ventricle wall zones at 60 days of pregnancy, the subventricular zone, which initially had greater number of Nestin+ cells, began to show higher numbers of GFAP+ cells at 90 days of pregnancy. A similar pattern was observed for Nestin+ and GFAP+ cells during development of the choroid plexus. This study demonstrates, for the first time, detailed chronological aspects of the equine central nervous system organogenesis associated with downregulation of Nestin and upregulation of GFAP expression.

Ultrastructural analysis of the spermatogenesis in the guinea pig (Cavia porcellus) / Análise ultraestrutural da espermatogênese do porquinho-da-Índia (Cavia porcellus)

Understanding of the reproductive function is essential for both, the establishment of appropriate management systems, and for the use of new species as animal models. In this study, we used light and electron microscopy to characterize the sexual development stages of the guinea pig (Cavia porcellus) in specimens of 30, 45 and 90 days of age. We observed the differentiation of spermatocytes only through transmission electron microscopy in the leptotene, zygotene and pachytene phases of meiosis, in 30-day-old animals. During puberty, there was differentiation of the germinative epithelium and formation of the acrosome. Spermatozoa, however, were not detected. Thus, we could infer that puberty happens after 45 days of age. Sexual maturity was evident in 90-day-old specimens. Our results showed that changes in the testicular germinative epithelium during the postnatal sexual development in guinea pig led to morphological changes, including the ones related to the development of Leydig and Sertoli cells, which are directly related to puberty. In this work, we provide new morphological subsidies for a better understanding of reproductive parameters of this species, enabling its use as an animal model in the field of the reproductive biology.(AU)

A função reprodutiva é um fator de vital compreensão tanto para o estabelecimento de sistemas apropriados de manejo, quanto para o uso de novas espécies como modelos animais. Neste estudo através da microscopia de luz e eletrônica caracterizou-se a fase de desenvolvimento sexual do porquinho-da-Índia (Cavia porcellus) em espécimes de 30, 45 e 90 dias de desenvolvimento. Nos animais de 30 dias, a diferenciação dos espermatócitos foi visualizada somente na microscopia eletrônica de transmissão em leptóteno, zigóteno e paquíteno. Durante a puberdade, houve diferenciação do epitélio germinativo, formação do acrossoma, porém não foram evidenciados espermatozóides, assim, infere-se que a puberdade acontece a partir dos 45 dias de idade. A maturidade sexual foi evidente aos 90 dias de idade. Nossos resultados mostraram que ao longo do desenvolvimento sexual pós-natal do porquinho-da-Índia, mudanças no epitélio germinativo testicular levam há alterações morfológicas, inclusive com relação ao desenvolvimento das células de Sertoli e de Leydig, as quais estão diretamente relacionadas com a puberdade. Assim, novos subsídios morfológicos são fornecidos para um melhor entendimento dos parâmetros reprodutivos desta espécie, a fim de viabilizar sua utilização como modelo animal no campo da biologia reprodutiva.(AU)

The use of animal models for stroke research: a review.

Stroke has been identified as the second leading cause of death worldwide. Stroke is a focal neurologic deficit caused by a change in cerebral circulation. The use of animal models in recent years has improved our understanding of the physiopathology of this disease. Rats and mice are the most commonly used stroke models, but the demand for larger models, such as rabbits and even nonhuman primates, is increasing so as to better understand the disease and its treatment. Although the basic mechanisms of stroke are nearly identical among mammals, we here discuss the differences between the human encephalon and various animals. In addition, we compare common surgical techniques used to induce animal models of stroke. A more complete anatomic knowledge of the cerebral vessels of various model species is needed to develop more reliable models for objective results that improve knowledge of the pathology of stroke in both human and veterinary medicine.