2. Universidad Cardenal Herrera-CEU

Introduction: Intra-articular infiltration of plasma rich in growth factors (PRGF) and adipose mesenchymal stromal cells (AMSCs) are known to inhibit osteoarthritis progression. However, in severely a􀀀ected patients, the treatment cannot reach the deeper layers of the articular cartilage; thus, its potential is limited. To overcome this limitation, intra-osseous infiltrations have been suggested. The purpose of this study is to assess the impact of intra- osseous infiltration therapies on serum biomarkers of osteoarthritis and to assess cartilage regeneration macroscopically. Materials and methods: A total of 80 rabbits were divided into four groups based on the intra-osseous treatment administered on the day of surgery: control, PRGF, AMSCs and a combination of PRGF + AMSCs. In addition, all groups received a single intra-articular administration of PRGF on the same day. Serum biomarker levels were measured before infiltration and 28-, 56-, and 84-days post infiltration, and macroscopical assessment was conducted at 56- and 84-days follow-up post infiltration. Results: In the PRGF + AMSCs group, significantly lower concentrations of hyaluronic acid and type II collagen cleavage neoepitope were recorded at all time points during the study, followed by PRGF, AMSCs and control groups. Regarding macroscopical assessment, lower scores were obtained in PRGF + AMSCs group at all study times. Discussion: The results suggest that the combination of intra-articular PRGF with intra-osseous PRGF or AMSCs achieves better results in rabbits with acute chondral defects and that intra-osseous infiltration is a safe procedure.

The regulation of early events in mammalian embryonic development is a complex process. In the early stages, pluripotency, cellular differentiation, and growth should occur at specific times and these events are regulated by different genes that are expressed at specific times and locations. The genes related to pluripotency and cellular differentiation, and growth factors that determine successful embryonic development are different (or differentially expressed) among mammalian species. Some genes are fundamental for controlling pluripotency in some species but less fundamental in others, for example, Oct4 is particularly relevant in bovine early embryonic development, whereas Oct4 inhibition does not affect ovine early embryonic development. In addition, some mechanisms that regulate cellular differentiation do not seem to be clear or evolutionarily conserved. After cellular differentiation, growth factors are relevant in early development, and their effects also differ among species, for example, insulin-like growth factor improves the blastocyst development rate in some species but does not have the same effect in mice. Some growth factors influence genes related to pluripotency, and therefore, their role in early embryo development is not limited to cell growth but could also involve the earliest stages of development. In this review, we summarize the differences among mammalian species regarding the regulation of pluripotency, cellular differentiation, and growth factors in the early stages of embryonic development.

Platelet-rich plasma showed good results in tissue healing when first used in human medicine. After that, its use spread to veterinary medicine. However, there is no standardized method for manual collection of platelet – rich plasma in the canine species. The objectives of this study were to standardize a protocol to obtain platelet – rich plasma (PRP) with high concentration of platelets and transforming growth factor β1 (TGF – β1) without the presence of erythrocytes and leukocytes; and to relate the presence of TGF – β1 with the amount of platelets. For this purpose, there were obtained two blood samples separated one week between each other, from eleven healthy Beagles. Blood samples were centrifuged using different protocols: protocol A (one centrifuge, 210 g and 10 minutes), protocol B (double centrifuge, first one 210 g and 10 minutes, second one 210 g and 15 minutes) and protocol C (one centrifuge, 475 g and 8 minutes). Three plasma fractions were obtained through these protocols: a platelet-rich fraction, a platelet-poor fraction and whole blood. The content of leukocytes, erythrocytes and platelets was measured in the whole blood and plasma rich and poor fractions of the protocol A, B and C. The TGF – β1 concentration was measured in the platelet rich and poor fraction of A and B protocols. The results showed a higher concentration of platelets and TGF – β1 in protocol A. In conclusion, this study offers an economical and reproducible method for obtaining PRP in the canine specie.

Myopia is one of the commonest eye pathologies that could affect 2.56 billion people by 2020. Today high myopia is a leading cause of blindness worldwide due to associated ocular illness. Nevertheless, the cellular bases for these diseases to develop are unclear in many areas. We conducted a prospective study of oxidative stress and growth factors in human myopic and non myopic eyes in an attempt to increase our understanding of the underlying physiopathological conditions to adequately early diagnose, prevent and treat the retina problem that derives from myopia. Aqueous humor samples were obtained from 41 patients being operated for cataracts in our hospital. Axial length, refractive status and complete ophthalmologic examination were recorded. The VEGF and HGF levels were determined by an ELISA kit. Total antioxidant capacity and total nitrites/nitrate levels were established with a lab kit. We show for the first time an increase in the total nitrite levels in high myopia. We also propose for the first time the concurrence of three factors: myopia, oxidative stress, and oxidative stress together with growth factors in the same group of patients. In this way, it would not be accurate to envision high myopia as a type of normal myopia, but one with more diopters or longer axial length.

El óptimo crecimiento fetal depende de la eficiente función placentaria. El factor de crecimiento placentario (PlFG) favorece la angiogénesis y vascularización de la placenta, asegurando el efectivo flujo sanguíneo placentario esencial para mantener el transporte activo de moléculas y la síntesis de hormonas esteroideas. Por otro lado, los componentes del eje somatotropo (GH e IGF-1), la ACTH, el CORT y las catecolaminas influyen notablemente sobre el desarrollo placentario y el crecimiento fetal. Sin embargo, hasta la actualidad estos mecanismos permanecen desconocidos en la yegua. Por este motivo, los objetivos planteados en la presente investigación han sido los siguientes: 1) Establecer valores de referencia para las concentraciones de neurotransmisores (ADR, NORADR, 5-HT y DA), hormonas hipofisarias (ACTH y GH), placentarias (A4, DHEA, T, E1S y P4), adrenales (CORT) y PlFG e IGF-1 en yeguas gestantes sanas; 2) Examinar si la gestación en la yegua PRE induce modificaciones laboratoriales en las dinámicas de los componentes anteriormente descritos, y 3) Analizar si los cambios hormonales de GH, ACTH, andrógenos, E1S y P4 están implicados fisiológicamente en las modificaciones analíticas que experimentan los diversos neurotransmisores y ambos factores de crecimiento en la yegua gestante PRE. Se ha estudiado un total de 33 yeguas PRE con edades comprendidas entre 5 y 17 años, durante la gestación. Se obtuvieron muestras de sangre venosa por las mañanas con una frecuencia mensual. Las muestras fueron almacenadas en tubos de vidrio con activadores de la coagulación y gránulos PS para desuerado. Las concentraciones séricas de ADR, NORADR, DA y 5-HT fueron determinadas mediante técnicas inmunoenzimáticas (EIA) de competición validadas específicamente para la especie equina (3-CAt EIA; Demeditec, Diagnostics GmbH, Germany; Serotonin-EIA; Demeditec, Diagnostics GmbH, Germany). La cuantificación de ACTH y GH fue realizada utilizando EIA de competición estreptavidina-biotina (Phoenix peptide ACTH human Fluorescent Immunoassay Kit FEK-001-01). Los niveles de IGF-1 se analizaron mediante ELISA de competición (DRG® InternationalInc, IGF-1 equino, EIA-3982, USA). La detección del PlGF se realizó utilizando EIA de tipo sandwich (Demeditec DEE01). Las concentraciones de A4, T, DHEA, E1S, P4 y CORT fueron determinadas mediante técnicas EIA utilizando anticuerpos policlonales obtenidos y caracterizados en el Departamento de Fisiología Animal de la Facultad de Veterinaria de la Universidad Complutense de Madrid. El inicio de la gestación representa un incremento fisiológico de las concentraciones de ADR, NORADR, DA, P4 y ACTH. Esta elevación de las catecolaminas ADR y NORADR alcanza un pico de secreción máximo simultáneo al incremento de 5-HT, GH, IGF-1, A4, DHEA, E1S y CORT hacia la mitad de la gestación. La preñez tardía constituye un periodo caracterizado por el descenso de NORAD, DA, 5-HT, ACTH y GH e incremento de T. A diferencia de la mujer y otras especies animales en las que mayoritariamente la P4 y el E1S se consideran los principales reguladores de la secreción hipotalámica de NORADR e hipofisaria de GH, la falta de relación entre dichos parámetros sugiere que la síntesis de NORADR, GH e IGF-1 es independiente de la dinámica esteroidea durante la gestación. Aunque las concentraciones de A4, DHEA, T, E1S y P4 muestran un patrón similar al PlFG, la expresión y la síntesis de este factor parecen no estar sometidas a control hormonal de origen placentario. En conclusión, la gestación representa el incremento fisiológico de la actividad de los ejes simpático-adrenal, hipotalámico-hipofisario y somatotropo en la yegua PRE. Los factores y hormonas integrantes en estos ejes junto a las hormonas placentarias y el PlGF podrían ser considerados como marcadores predictivos de funcionalidad placentaria y del avance correcto de la gestación. / The optimal fetal growth depends on the efficient placental function. Placental growth factor (PlFG) favors angiogenesis and vascularization of the placenta, ensuring effective placental blood flow, essential to maintain active transport of molecules and synthesis of steroid hormones. The components of the somatotrope (GH and IGF-1), ACTH, CORT and catecholamines strongly influence on development of the placenta fetal growth. However, these mechanisms remain unknown in the mare. For this reason, the objectives of the present research were: 1) To establish reference values for neurotransmitter concentrations (ADR, NORADR, 5-HT and DA), pituitary hormones (ACTH and GH), placental (DHEA, T, E1S and P4), adrenals (CORT), PlFG and IGF-1 in healthy pregnant mares; 2) Examine whether gestation in the Spanish Purebred mare induces laboratorial modifications in the dynamics of the previously described components, and 3) Analyze whether the hormonal changes of GH, ACTH, androgens, E1S and P4 are physiologically involved in the analytical modifications experienced by the several neurotransmitters and both growth factors in the Spanish Purebred broodmares. A total of 33 Spanish Purebred broodmares with ages between 5 and 17 years, during gestation have been analyzed. Venous blood samples were collected in the mornings on a monthly frequency. Samples were stored in glass tubes with coagulation activators and PS granules to obtaining serum. Serum concentrations of ADR, NORADR, DA and 5-HT were determined using competition immunoenzymatic techniques (EIA) specifically validated for equine species (3-CAt EIA, Demeditec, Diagnostics GmbH, Germany, Serotonin-EIA, Demeditec, Diagnostics GmbH, Germany). Quantification of ACTH and GH was performed using streptavidin-biotin competition EIA (Phoenix peptide ACTH human Fluorescent Immunoassay Kit FEK-001-01). IGF-1 levels were analyzed by competition ELISA (DRG® InternationalInc, equine IGF-1, EIA-3982, USA). Detection of PlGF was performed using sandwich EIA (Demeditec DEE01). The concentrations of A4, DHEA, T, E1S, P4 and CORT were determined using EIA techniques using polyclonal antibodies obtained and characterized in the Department of Physiology of the Faculty of Veterinary of the Complutense University of Madrid. The onset of gestation represents a physiological increase on ADR, NORADR, DA, P4 and ACTH concentrations. This elevation on catecholamines ADR and NORADR reaches a peak of maximal secretion simultaneously with the increase of 5-HT, GH, IGF-1, A4, DHEA, E1S and CORT towards the middle of gestation. Late pregnancy is a period characterized by the decrease of NORAD, DA, 5-HT, ACTH and GH and increase of T. Unlike women and other animal species in which P4 and E1S are considered the main regulators of NORADR hypothalamic and GH pituitary secretion, the lack of relationship between these parameters suggests that the synthesis of NORADR, GH and IGF-1 is independent of the steroid dynamics during pregnancy. Although the concentrations of A4, DHEA, T, E1S and P4 show a pattern similar to PlFG, the expression and synthesis of this factor appear not to be subject to placental hormonal control. In conclusion, gestation represents the physiological increase of the activity of the sympathetic-adrenal, hypothalamic-pituitary and somatotrope axes in the PRE mare. Factors and hormones in these axes together placental hormones and PlGF could be considered as predictive markers of placental function and correct progression of gestation.