Search

Background There is not an ideal biomaterial for tissue-engineered skin substitutes (TESSs), and most of the studies or existing therapies use xenogeneic origin natural biomaterials or biosynthetic scaffolds. Objective To analyse clinical, histological integration and homeostasis parameters of a human TESS manufactured with fibrin-hyaluronic acid biomaterial (HA-Skin), grafted in immunodeficient mice for 8 weeks, and compared with the gold standard treatment (Autograft), a human TESS manufactured with fibrin-agarose biomaterial (AG-Skin) and secondary wound healing dressings. Methods Human TESSs and autografts were implanted into BALB/c mice after surgical excision. Secondary wound healing approach was achieved with biosynthetic collagen wound dressing (Biobrane® ) and fibrin-hyaluronic acid or fibrin-agarose biomaterial without cells (Total N = 44). Clinical integration and homeostasis parameters were evaluated every two weeks for two months. Histological and immunohistochemical analyses were performed four and eight weeks after grafting. Results HA-Skin, AG-Skin and Autograft groups showed a proper clinical integration and epithelization eight weeks later. Scar evaluation revealed better results for Autograft and HA-Skin. Homeostasis analysis indicated similar values of transepidermal water loss and elasticity between HA-Skin (6.42 ± 0.75 g/h/m2 , 0.42 ± 0.08 AU), Autograft (6.91 ± 1.28 g/h/m2 , 0.40 ± 0.08 AU) and healthy mouse skin (6.40 ± 0.43 g/h/m2 , 0.35 ± 0.03 AU). Histological results showed that human TESSs and autografts presented better skin structuration and higher expression of cytokeratins. Conclusions This study suggests that human TESS based on fibrin-hyaluronic acid biomaterial could be suitable for clinical application in the treatment of several dermatological pathologies (wound healing).

Background & Aim The establishment of human keratinocytes’ culture (KTs) to obtain human Tissue Engineered Skin Substitutes is under research nowadays. Best results have been achieved culturing KTs on a feeder layer of irradiated 3T3 murine fibroblasts (i3T3s). Nevertheless, molecular effects of murine cells on KTs is unknown and for this reason, irradiated human fibroblasts (iHFs) have been studied. The objective of this study is to evaluate the effect of different irradiation doses on cell reducing power and cell proliferation of both fibroblasts. Methods, Results & Conclusion Methods 3T3s were obtained from a commercial vial of 3T3-Swiss Albino. HFs were isolated from human skin biopsies. After culturing and final recovery, both cell types were irradiated to doses of 50 and 100 Gy to constitute the feeder. Non-irradiated cells were used as control. Each condition was cultured 15 days in two 24-well plates (2 cm2/well). In each plate, cell densities cultured in triplicate were: 7,500 cells/cm2 and 10,000 cells/cm2. One plate was used for reducing power analysis; using a dilution of PrestoBlue™ (10%). The other plate was used for cell proliferation analysis; using Trypan Blue solution. N for each plate was 3 and a value of p Results Analysis of each type of feeder (Figs. 1-2) revealed significant differences at Days 10 and 15, between both irradiation doses and non-irradiated cells. In the case of iHFs, irradiation dose had a significant effect on cell reducing power, mainly at Day 15. Analysis of i3T3s revealed that the effect of irradiation on reducing power was extremely significant from Day 3 to 15, but no dose-dependent. A comparative analysis (Fig. 3) indicated that the number of non-irradiated cells after 15 days was higher in the case of 3T3s than HFs (644,620 cells vs 143,740 cells), but cell reducing power was lower in the case of murine fibroblasts (72.33% vs 80.67%). In the case of irradiated cells, iHFs were more resistant to irradiation in both, cell reducing power (27.5% vs 4.92%) and cell proliferation (5,962 cells vs 1,351 cells) at Day 15. Conclusion No differences exist considering initial cell density or irradiation dose in both feeders. Comparative analysis revealed that irradiation process is more effective in 3T3s than in HFs. This study needs to be completed with the incorporation of KTs on the different feeder conditions analyzed. Supported by PI17/02083, SAS PI-0458-2016 and PhD fellowship FI18/00269–Alvaro Sierra-Sanchez).

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.

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=GSE86584, Blindness 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.

Agradecimientos: Servicio de Medicina Nuclear del Complejo Hospitalario Universitario de Granada, Objetivos: 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.

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.

Background & Aim The aim of this study was evaluated the epidermal barrier function in an artificial bioengineered skin substitutes (ABSS) based on hyaluronic acid biomaterial. The study was performed evaluating specific skin ointments at different test period. Methods, Results & Conclusion Dermal fibroblasts (DF) and Keratinocytes (KT) were enzymatically isolated from 9 cm2 skin biopsies. After 4-5 weeks in culture, DF were part of dermal fibrin-hyaluronic acid stroma. KT (obtained using feeder-free method) were seeded on the surface to establish ABSS. Flow cytometry assay was carried out to phenotypically evaluate of keratinocytes: CD49f (integrin α6) and CD90 (fibroblast cell surface marker). Cell viability (LIVE/DEAD® assay), structural integrity (histological evaluation) and epidermal barrier function (transepidermal water loss, TEWL: Tewameter®) were measured after 5, 15 and 30 minutes to apply beclomethasone, mupirocin and vaseline. Acellular ABSS were used as controls. Specific cell culture parameters were evaluated after final recovery of keratinocytes. The cell expansion rate was significantly higher (p≤0.001%) with the CNT-57S media were supplemented with human platelet lysate. CD49f percentage was higher in keratinocytes with CNT-57S (95.2%). The CD90 percentage was lower in keratinocytes expanded with CNT-57S human lysate supplemented (1.60%). A viability assay was carried out in ABSS at first (85.1%) and second week (87.8%). Finally, barrier function evaluation of ABSS was carried out by measuring TEWL in the first and in the second week of maintenance. TEWL values in acellular ABSS were higher than in cellular ABSS, although the difference was not significant. Cells improved ABSS barrier function, thus favouring the reduction of water loss. Regarding the evaluation period, significant differences of TEWL were observed, decreasing the values in the second week. This may be due to the growth of the epithelial layer in cellular ABSS, which contributes to improvement of the barrier function. TEWL measures also decreased over the measurement time, although the differences were not significant. No difference was observed with respect to the type of ointment. In conclusion, keratinocytes culture was optimized using animal component-free and feeder-free method. A novel way to evaluate keratinocytes contribution to epidermal barrier function has been development in vitro.

ResumenEn Costa Rica, el 41,3% de los hogares utilizan sistemas de agua caliente sanitaria (ACS), que en su gran mayoría funcionan a partir de energía eléctrica mediante dispositivos de calentamiento en línea, empleando para esto el efecto Joule. Estos sistemas representan a escala nacional un consumo aproximado de más de 250 GWh al año. Para el presente trabajo se recopilaron datos de irradiación solar y temperatura media mensual en cada uno de los distritos del país. Con esta información se elaboró una herramienta de softwareque calcula la fracción solar de una instalación doméstica de ACS empleando el método F-Chart. Además, se analizó la rentabilidad financiera de una instalación solar de ACS para una vivienda promedio, comprobándose la necesidad de establecer una serie de políticas e incentivos con el fin de lograr una masificación del uso de la tecnología solar térmica en el sector residencial. Finalmente, se formuló una estrategia de implantación de la tecnología en el país, cubriendo aspectos de reglamentación, capacitación del cuerpo técnico nacional y creación de leyes que regulen el sector. El objetivo es crear herramientas que permitan introducir los sistemas solares de ACS en Costa Rica para sustituir los equipos eléctricos de calentamiento de agua, como una forma de contribuir al ahorro energético nacional. AbstractIn Costa Rica, 41, 3% of the households are equipped with a Domestic Hot Water System (DHW), which operate in a large majority from electricity. These systems represent an estimated national consumption over 250 GWh per year. In this work, insolation data and monthly average temperature were compiled, for each district of the country. With this data, a software tool was developed, that calculates the solar fraction of a DHW installation using the F-Chart method. The financial performance of a solar DHW system for an average home was analyzed, proving the need of policies and incentives to achieve mass use of solar thermal technology in the residential sector. Finally, an implantation strategy of the technology was proposed for Costa Rica, covering regulatory issues, training of a national technical staff, and creation of laws governing the sector. The goal is to create tools to introduce solar DHW systems in Costa Rica, to replace electric water heaters, as a way of contributing to national energy saving.

INTRODUCTION The shrinkage of surgical specimens (SS) is known in human skin (HS) but has not been studied in an artificial skin (AS) or mouse skin (MS). OBJECTIVES To quantify the degree of shrinkage of SS and establish its timing in HS and an in vitro and animal model to explore the possible causes of this phenomenon. METHODOLOGY We collected 100 SS of HS, 50 SS of AS synthesized with fibrin-agarose biomaterials and 21 SS of MS. The width and length of specimens were measured before the surgical excision (pre-SE), at 5 minutes postsurgery (ex vivo), and after 24 hours of fixation in formalin (postfixation). Histological staining was performed to analyze the differences between HS, AS, and MS that may explain the differences in shrinkage. RESULTS Between pre-SE and postfixation, the width and length shrank by 16.1% and 17.1% in HS, 14.5% and 8.5% in AS, and 26.5% and 23.1% in MS (P < 0.01), respectively. Shrinkage largely occurred between pre-SE and ex vivo. Cells and interstitial fibers were scant in AS and abundant in MS. CONCLUSIONS Almost all of the shrinkage occurred during the first 5 minutes postsurgery. According to the AS model findings, 53.6% of SS shrinkage would be explained by the action of dermal fibers and other cellular components of the dermis.

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 PI13/02576

The porcellanid crab Petrolisthes armatus, with a known geographic distribution covering a wide range of latitudes, was selected to conduct a comparative study on egg production and reproductive output between two populations from Pacific and Atlantic coasts. Ovigerous females were collected between September and November 2005 in Punta Morales, Pacific coast of Costa Rica (n = 137) and from March 2005 to July 2006 in Araca region, Sao Paulo, Brazil (n = 46). The mean size of females was statistically different between both populations with larger females from Brazil than Costa Rica (7.9 ± 1.31 and 6.6 ± 0.95 mm CW, respectively). Likewise, egg production was significantly different in both populations and started in Pacific Costa Rica at a smaller female size than in Brazil. Specimens from Pacific Costa Rica produced roughly three times more eggs than equally sized females from Brazil, while newly extruded embryos were larger in Brazil than in Costa Rica (0.045 and 0.039 mm3, respectively). Egg volume increased during embryogenesis by 112.8 and 164.5% in Costa Rican and Brazilian populations, respectively. The egg water content increased steadily in both populations; however, eggs produced in Brazil contained always more water than those from the same developmental stage in Costa Rica. Average reproductive output (based on dry weight) was substantially lower in Brazil (0.031) than in Costa Rica (0.065). Our results confirm an impressive intraspecific plasticity of reproductive features in an intertidal decapod. The observed phenotypical variability might be related to local environmental conditions as well as to the location of the studied population concerning its geographic range of distribution.

In Costa Rica, 41,3% of the households are equipped with a Domestic Hot Water System (DHW), which operate in a large majority from electricity. These systems represent an estimated national consumption over 250 GWh per year. In this work, insolation data and monthly average temperature were compiled, for each district of the country. With this data, a software tool was developed, that calculates the solar fraction of a DHW installation using the F-Chart method. The financial performance of a solar DHW system for an average home was analyzed, proving the need of policies and incentives to achieve mass use of solar thermal technology in the residential sector. Finally, an implantation strategy of the technology was proposed for Costa Rica, covering regulatory issues, training of a national technical staff, and creation of laws governing the sector. The goal is to create tools to introduce solar DHW systems in Costa Rica, to replace electric water heaters, as a way of contributing to national energy saving. En Costa Rica, el 41,3% de los hogares utilizan sistemas de agua caliente sanitaria (ACS), que en su gran mayoría funcionan a partir de energía eléctrica mediante dispositivos de calentamiento en línea, empleando para esto el efecto Joule. Estos sistemas representan a escala nacional un consumo aproximado de más de 250 GWh al año. Para el presente trabajo se recopilaron datos de irradiación solar y temperatura media mensual en cada uno de los distritos del país. Con esta información se elaboró una herramienta de software que calcula la fracción solar de una instalación doméstica de ACS empleando el método F-Chart. Además, se analizó la rentabilidad financiera de una instalación solar de ACS para una vivienda promedio, comprobándose la necesidad de establecer una serie de políticas e incentivos con el fin de lograr una masificación del uso de la tecnología solar térmica en el sector residencial. Finalmente, se formuló una estrategia de implantación de la tecnología en el país, cubriendo aspectos de reglamentación, capacitación del cuerpo técnico nacional y creación de leyes que regulen el sector. El objetivo es crear herramientas que permitan introducir los sistemas solares de ACS en Costa Rica para sustituir los equipos eléctricos de calentamiento de agua, como una forma de contribuir al ahorro energético nacional.

IntroductionThere is a need to find alternatives to the use of human donor corneas in transplants because of the limited availability of donor organs, the incidence of graft complications, as well as the inability to successfully perform corneal transplant in patients presenting limbal deficiency, neo-vascularized or thin corneas, etc. We have designed a clinical trial to test a nanostructured fibrin-agarose corneal substitute combining allogeneic cells that mimics the anterior human native cornea in terms of optical, mechanical and biological behaviour.Methods and analysisThis is a phase I-II, randomised, controlled, open-label clinical trial, currently ongoing in ten Spanish hospitals, to evaluate the safety and feasibility, as well as clinical efficacy evidence, of this bioengineered human corneal substitute in adults with severe trophic corneal ulcers refractory to conventional treatment, or with sequelae of previous ulcers. In the initial phase of the trial (n=5), patients were sequentially recruited, with a safety period of 45 days, receiving the bioengineered corneal graft. In the second phase of the trial (currently ongoing), subjects are block randomised (2:1) to receive either the corneal graft (n=10), or amniotic membrane (n=5), as the control treatment. Adverse events, implant status, infection signs and induced neovascularization are evaluated as determinants of safety and feasibility of the bioengineered graft (main outcomes). Study endpoints are measured along a follow-up period of 24 months, including 27 post-implant assessment visits according to a decreasing frequency. Intention to treat, and per protocol, and safety analysis will be performed.Ethics and disseminationThe trial protocol received written approval by the corresponding Ethics Committee and the Spanish Regulatory Authority and is currently recruiting subjects. On completion of the trial, manuscripts with the results of phases I and II of the study will be published in a peer-reviewed journal.Trial registrationCT.gov identifier:NCT01765244(Jan2013). EudraCT number: 2010-024290-40 (Dec2012).