Study of C, N, P and K Release from Residues of Newly Proposed Cover Crops in a Spanish Olive Grove

Cover crops (CC)s are increasingly employed by farmers in olive groves. Spontaneous soil cover is the most commonly used CC. Its continuous utilization changes ruderal flora. It is necessary to study new CCs. Living CCs provide C and nutrients to soil during decomposition. Information on this issue in olive groves is scarce. A 4-year field study involving grab sampling of Brachypodium distachyon, Sinapis alba and spontaneous CC residues was conducted to study C and nutrient release from cover crop residues. Throughout the decomposition cycles, C, N and P release accounted for 40 to 58% of the C, N and P amounts in the residues after mowing. Most K was released (80–90%). Expressed in kg per hectare, the release of C and N in Brachypodium (C: 4602, N: 181, P: 29, K: 231) and Sinapis (C: 4806, N: 152, P: 18, K: 195) was greater than that in spontaneous CC (C: 3115, N: 138, P: 21, K: 256). The opposite results were observed for K. The Rickman model, employed to estimate the amount of C, N and P in residues, yielded a good match between the simulated and measured values. In comparison to spontaneous CC, the newly proposed CCs have a higher potential to provide soil with C and N

Validation of Bact/Alert automathic system in the microbiological control of cell medicinal products of Advanced Therapies

Objetivo. El Control de calidad para demostrar que un producto está libre de agentes microbianos adventicios es un aspecto clave de control de procesos y evaluación de la calidad de todas las preparaciones medicinales celulares y en la ingeniería tisular. El objetivo de este estudio es validar el sistema de detección por hemocultivo BacT / ALERT para el control microbiológico de las células mesenquimales para terapia celular, según la Farmacopea Europea (EU.PH), 2.6.27. “Control microbiológico de productos celulares” (1). Método. Para el cálculo del límite de detección las botellas de hemocultivo fueron inoculadas e incubadas con 4 réplicas de 30 UFC, 5 réplicas de 15 UFC y 5 réplicas de 6 UFC de los microorganismos en ausencia de producto celular. Se llevaron a cabo también experimentos en presencia de producto con 400.000 células mesenquimales. Este método se ha comparado con el método de referencia de Esterilidad de la EU.PH (2). La especificidad se ensayó inoculando 5 réplicas con 400.000 células mesenquimales sin microorganismos. Resultados. Todas las botellas inoculadas con células mesenquimales sin microorganismos permanecieron negativas después de 7 días de incubación. Todas las botellas inoculadas con cepas bacterianas aerobias y anaerobias fueron detectadas como positivas por el sistema, en el caso del límite inferior (6 UFC) en menos de 36 horas. Se detectaron como positivas las botellas inoculadas con Candida albicans (6 UFC) en menos de 48 horas y con Aspergillus niger (6 UFC) en menos de 72 horas. No hubo diferencias notables en el tiempo de detección entre botellas inoculadas con y sin la presencia de células mesenquimales. Conclusión: El sistema de detección de hemocultivos Bact/Alert es un método fiable para la detección de la contaminación microbiana de medicamentos a base de células mesenquimales y cumple los requisitos de la UE PH, 2.6.27, para el control microbiológico de productos celulares.Objective. Quality control to demonstrate that a product is free from adventitious microbial agents is a key aspect of process control and quality evaluation of all cell medicinal preparations and in tisular engineering. Evaluate the validation of the BacT/ALERT Blood Culture System for the microbial control of mesenchymal cells for cell therapy according European Pharmacopoeia (EU.PH), 2.6.27. “Microbiological control of cellular products” (1). Method. Blood culture bottles were challenged with 4 replica of 30 cfu, 5 replica of 15 cfu and 5 replica of 6 cfu of the test microorganisms. Test were also carried out in the presence in each contaminated culture bottle of 400.000 mesenchymal cells. This method has been compared with the reference method for Sterility of the EU.PH (2). Specificity was tested inoculating 5 replicas of broth culture media with 400.000 cells without microorganisms. Results. All bottles challenged with mesenchymal cells without microorganisms remained negative after 7 days of incubation. All inoculated bottles with aerobic and anaerobic bacterial strains were flagged as positive for the system, in case of low inoculum (6 cfu) in less than 36 hours. Candida inoculated bottles (6 cfu) were detected in less than 48 hours and Aspergillus (6 cfu) in less than 72 hours. There were no significant differences in the detection time between bottles inoculated with and without the presence of mesenchymal cells. Conclusion: The BacT/ALERT blood culture detection system and is a reliable method for detection of microbial contamination of mesenchymal cells medicinal products that fulfils the requirements of the EU PH, 2.6.27, for the microbiological control of cellular products

Effects of Multifunctional Margins Implementation on Biodiversity in Annual Crop

The most suitable land for agricultural use has been gradually occupied by crops around the world. Large, uninterrupted croplands have been created, while disproportionate amounts of fertilizers, insecticides, fungicides and herbicides are applied on them. As a result, agricultural activity has a negative impact on biodiversity the ecological intensification of cultivated lands has become necessary. Multifunctional Margins (MFM), the establishment of native flora margins adjacent to croplands, provide a semi-natural habitat for food and wildlife refuge. Three different species mixtures sown in MFM were studied in this paper. The large capacity of six species used in MFM (Borago officinalis, Glebionis coronaria, Coriandrum sativum, Sinapis alba, Trifolium resupinatum and Vicia sativa) was determined. Reductions of up to 65% in the appearance of weeds and increases of 36% in pollinator biodiversity in sown MFM with respect to the MFM of spontaneous flora were observed. The biodiversity of the epigeal fauna increased by 15% in the MFM of spontaneous flora and by 32% in sown MFM, with respect to annual crops

Pilot study of cutaneous tolerability of fibrin-agarose substitutes in healthy volunteers

Objetivos: En el presente estudio se persigue comprobar posibles reacciones adversas, derivadas del uso tópico de láminas de fibrina-agarosa en el antebrazo de voluntarios sanos. Metodología: Se llevó a cabo un estudio experimental en siete voluntarios sanos, cinco varones y dos mujeres, que no presentaban ningún tipo de lesión cutánea visible. En el antebrazo de cada voluntario se colocaron dos láminas de fibrina-agarosa de 4 cm2 . Cada lámina se cubrió con un apósito impregnado y sobre una de las láminas se aplicó pomada antibiótica con mupirocina. Ambas láminas se cubrieron finalmente con un apósito protector y se mantuvieron en contacto directo sobre la piel durante 48 horas. Resultados: Los resultados determinaron que no se detectaron reacciones adversas después de 48 horas de evolución ni en los siguientes 7 días en ningún voluntario. Se observaron diferencias entre las dos láminas implantadas en cada voluntario, ya que al retirar el apósito cubierto con pomada antibiótica, la lámina presentaba un aspecto más hidratado que la que no llevaba pomada antibiótica. Conclusiones: El uso tópico de las láminas de fibrina-agarosa en voluntarios sanos no presenta reacciones adversas del tipo irritación o alergia al aplicarse directamente por vía tópica. Aunque el tamaño muestral del estudio es limitado, sugiere que la combinación de fibrina-agarosa se presenta como el biomaterial idóneo para el desarrollo de un modelo de piel artificial humana.Purpose: This study aims to analyse possible adverse reactions resulting from the topical use of fibrin-agarose substitutes in the forearm of healthy volunteers. Methods: An experimental study was carried out in seven healthy volunteers, five males and two females, who did not have any cutaneous lesion. Two fibrin-agarose substitutes of 4 cm2 were placed in the forearm of each volunteer. Each substitute was covered with an impregnated dressing and one of the substitutes was covered with antibiotic ointment (mupirocin). Both substitutes were finally covered with a protective dressing. The substitutes were maintained for 48 hours. Results: The results determined that no adverse reactions were detected in any volunteer after 48 hours and a week of evolution. Differences were observed between the two substitutes implanted in each volunteer, since when removing the covered dressing with antibiotic ointment, the substitute presented a more hydrated appearance than the one without antibiotic cream. Conclusions: The implant of fibrin-agarose substitutes in healthy volunteers does not present irritation or allergic type adverse reactions when they applied directly topically on the skin. Although the sample size is low, the fibrin-agarose combination is presented as the biomaterial suitable for the development of an artificial human skin model

Epithelial in vitro differentiation of human mesenchymal stem cells (hMSCs) from adipose tissue (AT) and bone marrow (BM): cellular characterization and study of HLA I and II expression

AGRADECIMIENTOS Laboratorio de Citogenética del servicio de Análisis Clínicos del Hospital Universitario Virgen de las Nieves. Servicio de Análisis Clínicos (Sección de Citometría/Biopatología tumoral) del Hos- pital Universitario Virgen de las Nieves.Introducción: Las células troncales mesenquimales derivadas de tejido adiposo o médula ósea constituyen uno de los tratamientos de terapia celular más utilizados en los ensayos clínicos actuales por su capacidad inmunomoduladora. Además, por su potencial de diferenciación a células epiteliales pueden ser utilizadas en ingeniería tisular incorporadas a tejidos artificiales como la piel o córnea, sustituyendo a las células epiteliales autólogas de estos tejidos. Es necesario realizar una correcta caracterización de estas células diferenciadas y estudiar el efecto de la diferenciación en la expresión del HLA de clase I y II. Objetivos: Caracterizar y realizar los controles de calidad GMP en dos líneas de células mesenquimales troncales humanas de distintos orígenes (tejido adiposo y médula ósea) tras diferenciarlas a células epiteliales in vitro, y analizar si se modifica la expresión de los marcadores HLA I y II antes y después del proceso diferenciador. Metodología: Se ha realizado el aislamiento y expansión de las dos líneas celulares de células mesenquimales troncales a partir del tejido fuente y se ha procedido a su diferenciación in vitro a células epiteliales mediante medios de cultivos suplementados con factores de crecimiento específico. Se han realizado controles de calidad siguiendo los requerimientos de las normas de correcta fabricación y se ha estudiado por citometría de flujo la expresión de HLA tipo I y II antes y después del proceso diferenciador. Finalmente se ha comprobado mediante estudio histológico e inmunohistoquímico las características de las células diferenciadas. Resultados: Se han aislado dos líneas de células mesenquimales troncales de tejido adiposo y médula ósea que cumplen los controles de calidad propuestos. Tras el proceso diferenciador in vitro, las células mesenquimales troncales humanas no expresan marcadores HLA (I y II) importantes en la respuesta inmune, pero sí expresan débilmente proteínas relacionadas con los principales estratos epiteliales (CK5, CK6 y CK14). Conclusión: La ausencia de expresión de marcadores de HLA I y II por citometría de flujo en las células diferenciadas favorecería su uso con carácter alogénico en la construcción de piel y córneas humanas por ingeniería de tejidos, sin embargo, son necesarios más estudios que confirmen estos resultados preliminares y protocolos que optimicen el proceso diferenciador in vitro de las células mesenquimales troncales.Background: Human mesenchymal stem cells derived from adipose tissue and bone marrow are one of the most common cell therapy procedures used in recent clinical trials due to their immunomodulation capacity. Furthermore, for their epithelial differentiation potential can be used in tissue engineering, incorporated in artificial tissues such as skin and cornea, replacing autologous epithelial cells. It is necessary to make a correct cellular characterization of differentiated cells and to study the effect in HLA I and II expression. Objetives: Characterization and quality controls under GMP conditions of in vitro differentiated human mesenchymal stem cells from different sources (adipose tissue and bone marrow) to epithelial lineage, and study of HLA I and II expression before and after differentiation. Methods: Isolation and expansion of two human mesenchymal stem cells lines from their tissues of origin and in vitro differentiation to epithelial cells using culture mediums supplemented with specific growth factors. Quality controls according Good Manufacturing Practices have been made and HLA I and II expression before and after differentiation have been studied. Finally, characteristics of differentiated cells have been demonstrated by histological and immunohistochemical analysis. Results: Two human mesenchymal stem cells lines from adipose tissue and bone marrow have been isolated complying with the proposed quality controls. After in vitro differentiation, human mesenchymal stem cells do not express HLA (I and II) markers, which are important in immune response, but weakly express proteins related to main epithelial layers of human skin (CK5, CK6 and CK14). Conclusion: The absence of expression of HLA I and II by flow cytometry in differentiated cells would promote the use of them with allogenic character to construct human skin and cornea by tissue engineering, however, more studies and protocols are required to confirm these preliminary results and to optimize in vitro differentiation of human mesenchymal stem cells.FIS ISC-III and FEDER PI13/0257

Optimization of human keratinocyte culture to develop an artificial human skin model: cell alternatives as feeder layer of Advanced Therapies

Agradecimientos: Servicio de Medicina Nuclear del Complejo Hospitalario Universitario de GranadaObjetivos: En el presente estudio se persigue optimizar el cultivo de queratinocitos para desarrollar un modelo de piel artificial humana. Para ello, se utilizan como capa alimentadora células de origen humano: fibroblastos dérmicos humanos y células mesenquimales troncales derivadas de tejido adiposo. Los resultados obtenidos se comparan con los fibroblastos 3T3, capa alimentadora de origen murino utilizada desde hace décadas. Metodología: Se llevó a cabo un estudio experimental, utilizando células de origen humano y células de origen murino subletalmente irradiadas, como capa alimentadora para el establecimiento del cultivo de queratinocitos. Se evaluó la tasa de expansión celular y la tasa de duplicación en el pase celular de queratinocitos y en la recuperación celular final que se llevó a cabo a las 3 semanas de cultivo; así como el rendimiento celular y la viabilidad celular, que también se evaluaron en el procesamiento inicial. Resultados: Los resultados determinan que los fibroblastos dérmicos humanos irradiados y las células mesenquimales troncales derivadas de tejido adiposo pueden actuar como capa alimentadora promoviendo la adhesión y la expansión celular de los queratinocitos. Los fibroblastos dérmicos humanos proporcionan resultados equiparables a los obtenidos con los fibroblastos 3T3 murinos. Conclusiones: Los fibroblastos dérmicos humanos irradiados proporcionan una capa alimentadora funcional que permite la expansión in vitro de manera eficaz de los queratinocitos que se van a utilizar con fines clínicos para el desarrollo de un modelo de piel artificial humana.Purpose: This study aims to optimize keratinocyte culture to develop an artificial human skin model. For this purpose, human cells are used as feeder layer: human dermal fibroblasts and adipose derived mesenchymal stem cells. The results obtained are compared with 3T3 fibroblasts, murine feeder layer used for decades. Methods: We conducted an experimental study using human and murine sub-lethally irradiated cells as feeder layer for the establishment of keratinocyte culture. Cell expansion rate and doubling rate were evaluated in the keratinocyte cell passage and in the final cell recovery (was carried out at 3 weeks). The yield and viability of keratinocytes were also evaluated in the initial processing. Results: The results determine that irradiated human dermal fibroblasts and irradiated adipose derived mesenchymal stem cells can act as feeder layer promoting adhesion and expansion of keratinocytes. Human dermal fibroblasts provide comparable results to those obtained with murine 3T3 fibroblasts. Conclusions: Irradiated human dermal fibroblasts provide a functional feeder layer which allows effectively in vitro expansion of keratinocytes to be used for clinical purposes for the development of an artificial human skin model

Successful development and clinical translation of a novel anterior lamellar artificial cornea

We thank the Andalusian Public Foundation Progress and Health, through the Andalusian Initiative for Advanced Therapies, for assuming the roles and responsibilities of sponsoring this clinical trial. We thank Dr. Manuel de la Rosa and Dr. Salvador Arias Santiago for providing insight and expertise that assisted the research.The datasets generated and/or analyzed during the current study are available in the Gene Expression Omnibus (GEO) public repository, ref. GSE86584 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE86584Blindness due to corneal diseases is a common pathology affecting up to 23 million individuals worldwide. The tissue‐engineered anterior human cornea, which is currently being tested in a Phase I/II clinical trial to treat severe corneal trophic ulcers with preliminary good feasibility and safety results. This bioartificial cornea is based on a nanostructured fibrin–agarose biomaterial containing human allogeneic stromal keratocytes and cornea epithelial cells, mimicking the human native anterior cornea in terms of optical, mechanical, and biological behavior. This product is manufactured as a clinical‐grade tissue engineering product, fulfilling European requirements and regulations. The clinical translation process included several phases: an initial in vitro and in vivo preclinical research plan, including preclinical advice from the Spanish Medicines Agency followed by additional preclinical development, the adaptation of the biofabrication protocols to a good manufacturing practice manufacturing process, including all quality controls required, and the design of an advanced therapy clinical trial. The experimental development and successful translation of advanced therapy medicinal products for clinical application has to overcome many obstacles, especially when undertaken by academia or SMEs. We expect that our experience and research strategy may help future researchers to efficiently transfer their preclinical results into the clinical settings.This study was supported by the Spanish National Plan for Scientific and Technical Research and Innovation (I + D + I) from the Spanish Ministry of Economy and Competitiveness (Carlos III Institute of Health), grants FIS PI14/0955 and FIS PI17/0391 (both cofinanced by ERDF‐FEDER, European Union); by the Spanish Ministry of Health, Social Policy and Equity, grant EC10‐285; and by preclinical research funds from the Regional Ministry of Health through the Andalusian Initiative for Advanced Therapies

Cover Crop Contributions to Improve the Soil Nitrogen and Carbon Sequestration in Almond Orchards (SW Spain)

Almond (Prunus dulcis Mill. [D.A. Webb]) is the third most widely spread crop in Spain and has traditionally been cultivated in marginal areas and shallow soils under rainfed conditions. However, it recently has been progressively introduced in flat irrigated areas. The implementation of cover crops in the inter-rows of woody crops has been proven as a suitable strategy to reduce the runoff and soil erosion but they also can boost soil quality and health. A field experiment was conducted during two-monitoring seasons to examine the soil nitrogen and carbon sequestration potential of three seeded cover crops [barley (Hordeum vulgare L.), hairy vetch (Vicia villosa Roth), and a mixture of 65% barley and 35% vetch] and a control of spontaneous flora in irrigated almond orchards (SW Spain). Here, we show that barley provided the highest biomass amount, followed by mixture covers, vetch, and the control treatment. Also, vetch covered the soil faster in the growing stage, but its residues were decomposed easier than barley and mixture treatments during the decomposition period after mowing, providing less soil protection when the risk of water erosion with autumn rainfall is high. On the other hand, vetch improved soil nitrate content by over 35% with respect to barley and mixture treatments at 0–20 cm soil depth throughout the studied period. In addition, a greater carbon input to the soil was determined in the barley plot. That is, the mixture and barley cover crops had higher potential for carbon sequestration, augmenting the soil organic carbon by more than 1.0 Mg ha−1 during the study period. Thus, taking into consideration the findings of the present experiment, the establishment of a seeded cover crop would be more advisable than spontaneous flora to mitigate soil erosion, enhancing soil fertility and carbon sequestration in irrigated almond plantations in Mediterranean semi-arid regions

Successful restoration of corneal surface integrity with a tissue-engineered allogeneic implant in severe keratitis patients

Objectives: Corneal diseases are among the main causes of blindness, with approximately 4.6 and 23 million patients worldwide suffering from bilateral and unilateral corneal blindness, respectively. The standard treatment for severe corneal diseases is corneal transplantation. However, relevant disadvantages, particularly in high-risk conditions, have focused the attention on the search for alternatives. Methods: We report interim findings of a phase I-II clinical study evaluating the safety and preliminary efficacy of a tissue-engineered corneal substitute composed of a nanostructured fibrin-agarose biocompatible scaffold combined with allogeneic corneal epithelial and stromal cells (NANOULCOR). 5 subjects (5 eyes) suffering from trophic corneal ulcers refractory to conventional treatments, who combined stromal degradation or fibrosis and limbal stem cell deficiency, were included and treated with this allogeneic anterior corneal substitute. Results: The implant completely covered the corneal surface, and ocular surface inflammation decreased following surgery. Only four adverse reactions were registered, and none of them were severe. No detachment, ulcer relapse nor surgical re-interventions were registered after 2 years of follow-up. No signs of graft rejection, local infection or corneal neovascularization were observed either. Efficacy was measured as a significant postoperative improvement in terms of the eye complication grading scales. Anterior segment optical coherence tomography images revealed a more homogeneous and stable ocular surface, with complete scaffold degradation occurring within 3–12 weeks after surgery. Conclusions: Our findings suggest that the surgical application of this allogeneic anterior human corneal substitute is feasible and safe, showing partial efficacy in the restoration of the corneal surface