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Contrary to the adult central nervous system (CNS), the peripheral nervous system (PNS) has an intrinsic ability to regenerate that, among others, passes by expressing regeneration-associated genes such as kinesin family members. We here show that Kinesin family motor protein 4a (KIF4A), associated to neurodevelopmental disorders and thought for long to be only embryonically expressed, is highly abundant in axons and Schwann cells of adult rat CNS and rat and human PNS. Moreover,Kif4ais up-regulated in injured PNS neurons, being detected in their nuclei and regrowing axons, consistent with its functions as a chromokinesin and in the axonal transport of e.g. β1-integrin and L1CAM. Interestingly,Kif4ais also highly up-regulated in Schwann cells transdifferentiating into a proliferative repair phenotype at the injured distal nerve stumps. A role forKif4ain cultured Schwann cells proliferation was confirmed, withKif4amRNA expression being ∼6-fold higher in proliferating versus growth-arrested Schwann cells, andKif4aknockdown impairing Schwann cells’ proliferation. To our knowledge, this is the first description of KIF4A expression in adult nervous systems, up-regulation in neuroregeneration and pro-neuroregenerative roles, including promoting Schwann cells proliferation. KIF4A dual role in axonal regeneration, through neurons and glia, places as an attractive target for future neuroregeneration therapies.

The scapholunate interosseous ligament (SLIL) plays a fundamental role in stabilizing the wrist bones, and its disruption is a frequent cause of wrist arthrosis and disfunction. Traditionally, this structure is considered to be a variety of fibrocartilaginous tissue and consists of three regions: dorsal, membranous and palmar. Despite its functional relevance, the exact composition of the human SLIL is not well understood. In the present work, we have analyzed the human SLIL and control tissues from the human hand using an array of histological, histochemical and immunohistochemical methods to characterize each region of this structure. Results reveal that the SLIL is heterogeneous, and each region can be subdivided in two zones that are histologically different to the other zones. Analysis of collagen and elastic fibers, collagens types I, III and IV, proteoglycans, glycoproteins and versican confirmed that the different regions can be subdivided in two zones that have their own structure and composition. The first part of the dorsal region (zone D1) resembles the control tendons and ligaments, whereas the rest of the SLIL are more similar to the control articular cartilage, especially the first part of the membranous region (zone M1). Cells showing a chondrocyte-like phenotype as determined by S100 were more abundant in M1, whereas the zone containing more CD73-positive stem cells was D2. These results confirm the heterogeneity of the human SLIL and could contribute to explain why certain zones of this structure are more prone to structural damage and why other zones have specific regeneration potential.

Hollow conduits of natural or synthetic origins have shown acceptable regeneration results in short nerve gap repair; however, results are still not comparable with the current gold standard technique “autografts”. Hollow conduits do not provide a successful regeneration outcome when it comes to critical nerve gap repair. Enriching the lumen of conduits with different extracellular materials and cells could provide a better biomimicry of the natural nerve regenerating environment and is expected to ameliorate the conduit performance. In this study, we evaluated nerve regeneration in vivo using hollow chitosan conduits or conduits enriched with fibrin-collagen hydrogels alone or with the further addition of adipose-derived mesenchymal stem cells in a 15 mm rat sciatic nerve transection model. Unexpected changes in the hydrogel consistency and structural stability in vivo led to a failure of nerve regeneration after 15 weeks. Nevertheless, the molecular assessment in the early regeneration phase (7, 14, and 28 days) has shown an upregulation of useful regenerative genes in hydrogel enriched conduits compared with the hollow ones. Hydrogels composed of fibrin-collagen were able to upregulate the expression of soluble NRG1, a growth factor that plays an important role in Schwann cell transdifferentiation. The further enrichment with adipose-derived mesenchymal stem cells has led to the upregulation of other important genes such as ErbB2, VEGF-A, BDNF, c-Jun, and ATF3., Spanish "Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica, Ministerio de Economia y Competitividad (Instituto de Salud Carlos III) FIS PI14-1343 FIS PI17-0393 FIS PI20-0318, Fondo Europeo de Desarrollo Regional ERDF-FEDER European Union, Plan Andaluz de Investigacion, Desarrollo e Innovacion (PAIDI2020), Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades, Junta de Andalucia, Espana P18-RT-5059, Programa Operativo FEDER Andalucia 2014-2020, Universidad de Granada, Junta de Andalucia, Espana A-CTS-498-UGR18, European Commission

Histochemical and fluorescence-based techniques enable the specific identification of myelin by bright-field or fluorescence microscopy. In this chapter, we describe four histological methods for the evaluation of myelin on peripheral nerve tissue sections. The first method combines the Luxol fast blue (LFB) technique with a modified Picrosirius staining contrasted with Harris hematoxylin, called MCOLL. This method simultaneously stains myelin, collagen fibers, and cell nuclei, thus giving an integrated overview of the histology, collagen network, and myelin content of the tissue in paraffin-embedded or cryosectioned samples. Secondly, we describe the osmium tetroxide method, which provides a permanent positive reaction for myelin as well as other lipids present in the tissue. The third method is the immunofluorescence-based detection of myelin proteins that allows to combine information about their expression status with other proteins of interest. Finally, the FluoroMyelin™ stains enable a fast detection of the myelin content that can be easily implemented in immunofluorescence staining panels for cryosectioned tissues. Together, this chapter provides a variety of methods to accurately identify myelin in different experimental approaches.

Human Wharton's jelly stem cells (HWJSC) can be efficiently isolated from the umbilical cord, and numerous reports have demonstrated that these cells can differentiate into several cell lineages. This fact, coupled with the high proliferation potential of HWJSC, makes them a promising source of stem cells for use in tissue engineering and regenerative medicine. However, their real potentiality has not been established to date. In the present study, we carried out a systematic review to determine the multilineage differentiation potential of HWJSC. After a systematic literature search, we selected 32 publications focused on the differentiation potential of these cells. Analysis of these studies showed that HWJSC display expanded differentiation potential toward some cell types corresponding to all three embryonic cell layers (ectodermal, mesodermal, and endodermal), which is consistent with their constitutive expression of key pluripotency markers such as OCT4, SOX2, and NANOG, and the embryonic marker SSEA4. We conclude that HWJSC can be considered cells in an intermediate state between multipotentiality and pluripotentiality, since their proliferation capability is not unlimited and differentiation to all cell types has not been demonstrated thus far. These findings support the clinical use of HWJSC for the treatment of diseases affecting not only mesoderm-type tissues but also other cell lineages. Impact statement Human Wharton's jelly stem cells (HWJSC) are mesenchymal stem cells that are easy to isolate and handle, and that readily proliferate. Their wide range of differentiation capabilities supports the view that these cells can be considered pluripotent. Accordingly, HWJSC are one of the most promising cell sources for clinical applications in advanced therapies.

Nerve autograft is the gold standard technique to repair critical nerve defects, but efficient alternatives are needed. The present study evaluated the suitability of our novel Roosens-based (RSN) decellularized peripheral nerve allografts (DPNAs) in the repair of 10-mm sciatic nerve defect in rats at the functional and histological levels after 12 weeks. These DPNAs were compared with the autograft technique (AUTO) and Sondell (SD) or Hudson (HD) based DPNAs. Clinical and functional assessments demonstrated a partial regeneration in all operated animals. RSN-based DPNAs results were comparable with SD and HD groups and closely comparable with the AUTO group without significant differences (p > .05). Overall hematological studies confirmed the biocompatibility of grafted DPNAs. In addition, biochemistry revealed some signs of muscle affection in all operated animals. These results were confirmed by the loss of weight and volume of the muscle and by muscle histology, especially in DPNAs. Histology of repaired nerves confirmed an active nerve tissue regeneration and partial myelination along with the implanted grafts, being the results obtained with HD and RSN-based DPNAs comparable with the AUTO group. Finally, this in vivo study suggests that our novel RSN-based DPNAs supported a comparable tissue regeneration, along the 10-mm nerve gap, after 12-week follow-up to HD DPNAs, and both were superior to SD group and closely comparable with autograft technique. However, further improvements are needed to overcome the efficacy of the nerve autograft technique.

Spanish Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica (I+D+I) of the Spanish Ministry of Science and Innovation (Instituto de Salud Carlos III), Grant/Award Number: FIS PI18/331, FIS PI21/00980, FIS PI18/332 and ICI19/00024; Consejeria de Salud y Familias, Junta de Andalucia, Spain, Grant/Award Number: PI-0442--2019; FEDER funds, European Union, Objective: The aim of this study was to generate novel models of bioartificial human oral mucosa with increased vascularization potential for future use as an advanced therapies medicinal product, by using different vascular and mesenchymal stem cell sources. Background: Oral mucosa substitutes could contribute to the clinical treatment of complex diseases affecting the oral cavity. Although several models of artificial oral mucosa have been described, biointegration is a major issue that could be favored by the generation of novel substitutes with increased vascularization potential once grafted in vivo. Methods: Three types of mesenchymal stem cells (MSCs) were obtained from adipose tissue, bone marrow, and dental pulp, and their in vitro potential was evaluated by inducing differentiation to the endothelial lineage using conditioning media. Then, 3D models of human artificial oral mucosa were generated using biocompatible fibrin-agarose biomaterials combined with human oral mucosa fibroblasts and each type of MSC before and after induction to the endothelial lineage, using human umbilical vein endothelial cells (HUVEC) as controls. The vascularization potential of each oral mucosa substitute was assessed in vitro and in vivo in nude mice. Results: In vitro induction of MSCs kept in culture was able to increase the expression of VEGF, CD31, and vWF endothelial markers, especially in bone marrow and dental pulp-MSCs, and numerous proteins with a role in vasculogenesis become overexpressed. Then, in vivo grafting resulted in a significant increase in blood vessels formation at the interface area between the graft and the host tissues, with significantly positive expression of VEGF, CD31, vWF, and CD34 as compared to negative controls, especially when pre-differentiated MSCs derived from bone marrow and dental pulp were used. In addition, a significantly higher number of cells committed to the endothelial lineage expressing the same endothelial markers were found within the bioartificial tissue. Conclusion: Our results suggest that the use of pre-differentiated MSCs could contribute to a rapid generation of a vascular network that may favor in vivo biointegration of bioengineered human oral mucosa substitutes., Spanish Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica (I+D+I) of the Spanish Ministry of Science and Innovation (Instituto de Salud Carlos III) FIS PI18/331 FIS PI21/00980 FIS PI18/332 ICI19/00024, Junta de Andalucia PI-0442-2019, European Commission

espanolLa membrana de la cascara del huevo ha sido considerada durante mucho tiempo como un material de desecho. Sin embargo, estudios recientes han demostrado su utilidad para diferentes propositos, como las aplicaciones biologicas. En el presente trabajo, se ha realizado un estudio para identificar los componentes principales de la membrana de la cascara del huevo y para determinar la posibilidad de usarlo como un biomaterial en ingenieria tisular. Para ello, se cultivaron fibroblastos en una membrana de cascara de huevo y luego se realizaron analisis histologicos e histoquimicos. Los resultados muestran no solo la presencia de colageno y proteoglicanos, sino tambien la viabilidad de las celulas dentro de la membrana. Estos resultados revelan el potencial de la membrana de la cascara del huevo para ser utilizado como biomaterial en ingenieria tisular. EnglishThe eggshell membrane has been long time considered as a waste material. However, recent studies have shown its usefulness for different purposes such as biological applications. In the present work, a study has been carried out to identify the eggshell membrane main components and to determine its feasibility for being used as a biomaterial in tissue engineering. Fibroblasts were cultured in eggshell membrane and then histological and histochemical analysis were performed. Results show not only the presence of collagen and proteoglycans, but also the viability of cells inside the membrane. These results revealed the eggshell membrane potential used as a biomaterial in tissue engineering.

This study was supported by grants FIS PI17/0393 and PI20/0318 from the Spanish Ministry of Science and Innovation (Instituto de Salud Carlos III); grants PI-0257-2017 and PE-0395- 2019 from Consejería de Salud y Familias, Junta de Andalucía, España; grant P18-RT-5059 from Consejería de Economía, Conocimiento, Empresas y Universidad, Junta de Andalucía, España; grant A-CTS-498-UGR18 from the University of Granada and Junta de Andalucía, España. It was co-funded by FEDER-ERDF funds., Authors are grateful to Fabiola Bermejo Casares for the technical histological assistance. Special thanks to Ariane Ruyffelaert for her critical review and proofreading service. This work forms part of the doctoral thesis conducted by David Sánchez Porras (Doctoral Program in Biomedicine, Doctoral School, University of Granada, Spain)., Considering the high prevalence of cartilage-associated pathologies, low self-repair capacity and limitations of current repair techniques, tissue engineering (TE) strategies have emerged as a promising alternative in this field. Three-dimensional culture techniques have gained attention in recent years, showing their ability to provide the most biomimetic environment for the cells under culture conditions, enabling the cells to fabricate natural, 3D functional microtissues (MTs). In this sense, the aim of this study was to generate, characterize and compare scaffold-free human hyaline and elastic cartilage-derived MTs (HC-MTs and EC-MTs, respectively) under expansion (EM) and chondrogenic media (CM). MTs were generated by using agarose microchips and evaluated ex vivo for 28 days. The MTs generated were subjected to morphometric assessment and cell viability, metabolic activity and histological analyses. Results suggest that the use of CM improves the biomimicry of the MTs obtained in terms of morphology, viability and extracellular matrix (ECM) synthesis with respect to the use of EM. Moreover, the overall results indicate a faster and more sensitive response of the EC-derived cells to the use of CM as compared to HC chondrocytes. Finally, future preclinical in vivo studies are still needed to determine the potential clinical usefulness of these novel advanced therapy products., Instituto de Salud Carlos III Spanish Government FIS PI17/0393 PI20/0318, Junta de Andalucia PI-0257-2017 PE-03952019, Junta de Andalucia P18-RT-5059, University of Granada A-CTS-498-UGR18, Junta de Andalucia A-CTS-498-UGR18, FEDER-ERDF funds

espanolIntroduccion. El presente trabajo describe y evalua la implementacion de un modelo de aula invertida en la materia de Histologia para el aprendizaje de ingenieria tisular en el grado de Farmacia con el objetivo de incorporar dichos conocimientos ante su escasa presencia en dicho grado. Sujetos y metodos. El modelo consiste en intercalar en el curso ordinario de Histologia del grado de Farmacia un modulo de autoaprendizaje inverso. Participan 110 alumnos que responden a un cuestionario sobre ambitos conceptuales, procedimentales y actitudinales al comienzo y al final del proceso. Los resultados se analizan estadisticamente. Resultados. Los resultados muestran valores superiores en la evaluacion final con respecto a la evaluacion inicial. Esta diferencia fue estadisticamente significativa (p Conclusion. Los valores mas elevados observados en los componentes actitudinal y procedimental, tras la implementacion del modelo propuesto, ponen de relieve la necesidad de incrementar el componente conceptual en la formacion de la ingenieria tisular en el curriculo de farmacia EnglishIntroduction. This paper describes and evaluates the implementation of a flipped learning model in the discipline of histology for learning tissue engineering contents in the Pharmacy degree, with the aim of incorporating this knowledge for the scarce presence of such matter in that degree. Subjects and methods. The model consists of inserting in the ordinary course of Histology of the pharmacy degree an inverse self-learning module. A questionnaire on conceptual, procedural and attitudinal fields was answered by the 110 students involved in the study at the beginning and end of the process. Results were statistically analysed. Results. The results after the implementation of the flipped learning model show statistically significant higher values (p Conclusion. The highest values observed in the attitudinal and procedural components, after the implementation of the proposed model, highlight the need to increase the conceptual component in the formation of tissue engineering in the pharmacy curriculum

Because cartilage has limited regenerative capability, a fully efficient advanced therapy medicinal product is needed to treat severe cartilage damage. We evaluated a novel biomaterial obtained by decellularizing sturgeon chondral endoskeleton tissue for use in cartilage tissue engineering. In silico analysis suggested high homology between human and sturgeon collagen proteins, and ultra-performance liquid chromatography confirmed that both types of cartilage consisted mainly of the same amino acids. Decellularized sturgeon cartilage was recellularized with human chondrocytes and four types of human mesenchymal stem cells (MSC) and their suitability for generating a cartilage substitute was assessed ex vivo and in vivo. The results supported the biocompatibility of the novel scaffold, as well as its ability to sustain cell adhesion, proliferation and differentiation. In vivo assays showed that the MSC cells in grafted cartilage disks were biosynthetically active and able to remodel the extracellular matrix of cartilage substitutes, with the production of type II collagen and other relevant components, especially when adipose tissue MSC were used. In addition, these cartilage substitutes triggered a pro-regenerative reaction mediated by CD206- positive M2 macrophages. These preliminary results warrant further research to characterize in greater detail the potential clinical translation of these novel cartilage substitutes., Spanish Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+i) of the Spanish Ministry of Economy and Competitiveness (Instituto de Salud Carlos III), Grant FIS PI20/0317, FEDER funds (European Union), Consejería de Salud y Familias, Junta de Andalucía, Spain - PI-0257-2017

Acellular nerve allografts (ANGs) represent a promising alternative in nerve repair. Our aim is to improve the structural and biomechanical properties of biocompatible Sondell (SD) and Roosens (RS) based ANGs using genipin (GP) as a crosslinker agent ex vivo. The impact of two concentrations of GP (0.10% and 0.25%) on Wistar rat sciatic nerve-derived ANGs was assessed at the histological, biomechanical, and biocompatibility levels. Histology confirmed the differences between SD and RS procedures, but not remarkable changes were induced by GP, which helped to preserve the nerve histological pattern. Tensile test revealed that GP enhanced the biomechanical properties of SD and RS ANGs, being the crosslinked RS ANGs more comparable to the native nerves used as control. The evaluation of the ANGs biocompatibility conducted with adipose-derived mesenchymal stem cells cultured within the ANGs confirmed a high degree of biocompatibility in all ANGs, especially in RS and RS-GP 0.10% ANGs. Finally, this study demonstrates that the use of GP could be an efficient alternative to improve the biomechanical properties of ANGs with a slight impact on the biocompatibility and histological pattern. For these reasons, we hypothesize that our novel crosslinked ANGs could be a suitable alternative for future in vivo preclinical studies., Spanish "Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica, Ministerio de Economia y Competitividad (Instituto de Salud Carlos III) FIS PI14-1343 FIS PI17-0393 FIS PI20-0318, Fondo Europeo de Desarrollo Regional ERDF-FEDER European Union, Plan Andaluz de Investigacion, Desarrollo e Innovacion (PAIDI 2020), Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades, Junta de Andalucia, Espana P18-RT-5059, Programa Operativo FEDER Andalucia 2014-2020, Universidad de Granada, Junta de Andalucia, Espana A-CTS-498UGR18, ERDF-FEDER, the European Union

This work was supported by the Spanish Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica (I+D+i) of the Spanish Ministry of Science and Innovation (Instituto de Salud Carlos III), Grants FIS PI21/0980, FIS PI18/0331, FIS PI18/0332, FIS PI20/0317, FIS PI20/318, and ICI19-00024, co-financed by FEDER funds (European Union). It was also supported by grant PE-0395-2019 from Consejeria de Salud y Familias, Junta de Andalucia, Spain, and grants B-CTS-450-UGR20 and A-CTS-498-UGR18 (Proyectos de I+D+i en el marco del Programa Operativo FEDER Andalucia 2014-2020) from the University of Granada, Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades, Junta de Andalucia and European Union and P18-RT-5059 from Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades, Junta de Andalucia. Co-financed by FEDER funds (European Union)., The embryonic development of the human umbilical cord (hUC) is complex, and different regions can be identified in this structure. The aim of this work is to characterize the hUC at in situ and ex vivo levels to stablish their potential use in vascular regeneration. Human umbilical cords were obtained and histologically prepared for in the situ analysis of four hUC regions (intervascular—IV, perivascular—PV, subaminoblastic—SAM, and Wharton’s jelly—WH), and primary cell cultures of mesenchymal stem cells (hUC-MSC) isolated from each region were obtained. The results confirmed the heterogeneity of the hUC, with the IV and PV zones tending to show the higher in situ expression of several components of the extracellular matrix (collagens, proteoglycans, and glycosaminoglycans), vimentin, and MSC markers (especially CD73), although isolation and ex vivo culture resulted in a homogeneous cell profile. Three vascular markers were positive in situ, especially vWF, followed by CD34 and CD31, and isolation and culture revealed that the region associated with the highest expression of vascular markers was IV, followed by PV. These results confirm the heterogeneity of the hUC and the need for selecting cells from specific regions of the hUC for particular applications in tissue engineering., Spanish Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica (I+D+i) of the Spanish Ministry of Science and Innovation (Instituto de Salud Carlos III) FIS PI21/0980 FIS PI18/0331 FIS PI18/0332 FIS PI20/0317 FIS PI20/318 ICI19-00024, European Commission, Junta de Andalucia PE-0395-2019, University of Granada B-CTS-450-UGR20 CTS-498-UGR18, Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades, Junta de Andalucia, European Commission Junta de Andalucia P18-RT-5059

Background: Treatment of patients affected by severe burns is challenging, especially due to the high risk of Pseudomonas infection. In the present work, we have generated a novel model of bioartificial human dermis substitute by tissue engineering to treat infected wounds using fibrin-agarose biomaterials functionalized with nanostructured lipid carriers (NLCs) loaded with two anti-Pseudomonas antibiotics: sodium colistimethate (SCM) and amikacin (AMK). Results: Results show that the novel tissue-like substitutes have strong antibacterial effect on Pseudomonas cultures, directly proportional to the NLC concentration. Free DNA quantification, WST-1 and Caspase 7 immunohistochemical assays in the functionalized dermis substitute demonstrated that neither cell viability nor cell proliferation were affected by functionalization in most study groups. Furthermore, immunohistochemistry for PCNA and KI67 and histochemistry for collagen and proteoglycans revealed that cells proliferated and were metabolically active in the functionalized tissue with no differences with controls. When functionalized tissues were biomechanically characterized, we found that NLCs were able to improve some of the major biomechanical properties of these artificial tissues, although this strongly depended on the type and concentration of NLCs. Conclusions: These results suggest that functionalization of fibrin-agarose human dermal substitutes with antibioticloaded NLCs is able to improve the antibacterial and biomechanical properties of these substitutes with no detectable side effects. This opens the door to future clinical use of functionalized tissues., NanoGSkin project of EuroNanoMed-III (ERA-NET Cofund scheme of the Horizon 2020 Research and Innovation Framework Programme), EU, Instituto de Salud Carlos III AC17/00013, Centro para el Desarrollo Tecnológico Industrial -CDTI 00108589, Spanish Government, Junta de Andalucía PE-0395-2019, Fundacion Benefica Anticancer San Francisco Javier y Santa Candida, Granada, Spain, Department of Economic Development and Infrastructure of the Basque Government budget, through the HAZITEK business R + D support program ZE-2017/00014, European Union (EU) OTRI.35A-07

AimsAchilles tendon injuries are a frequent problem in orthopaedic surgery due to their limited healing capacity and the controversy surrounding surgical treatment. In recent years, tissue engineering research has focused on the development of biomaterials to improve this healing process. The aim of this study was to analyze the effect of tendon augmentation with a nanostructured fibrin-agarose hydrogel (NFAH) or genipin cross-linked nanostructured fibrin-agarose hydrogel (GP-NFAH), on the healing process of the Achilles tendon in rats.MethodsNFAH, GP-NFAH, and MatriDerm (control) scaffolds were generated (five in each group). A biomechanical and cell-biomaterial-interaction characterization of these biomaterials was then performed: Live/Dead Cell Viability Assay, water-soluble tetrazolium salt-1 (WST-1) assay, and DNA-released after 48 hours. Additionally, a complete section of the left Achilles tendon was made in 24 Wistar rats. Animals were separated into four treatment groups (six in each group): direct repair (Control), tendon repair with MatriDerm, or NFAH, or GP-NFAH. Animals were euthanized for further histological analyses after four or eight weeks post-surgery. The Achilles tendons were harvested and a histopathological analysis was performed.ResultsTensile test revealed that NFAH and GP-NFAH had significantly higher overall biomechanical properties compared with MatriDerm. Moreover, biological studies confirmed a high cell viability in all biomaterials, especially in NFAH. In addition, in vivo evaluation of repaired tendons using biomaterials (NFAH, GP-NFAH, and MatriDerm) resulted in better organization of the collagen fibres and cell alignment without clinical complications than direct repair, with a better histological score in GP-NFAH.ConclusionIn this animal model we demonstrated that NFAH and GP-NFAH had the potential to improve tendon healing following a surgical repair. However, future studies are needed to determine the clinical usefulness of these engineered strategies. Cite this article: Bone Joint J 2020;102-B(8):1095–1106.

Objetivos: El objetivo de este trabajo es la evaluación diagnóstica de las competencias conceptuales, procedimentales y actitudinales en estudiantes de Medicina previo a la realización de un modelo de aprendizaje+servicio sobre hemodonación. Métodos: 161 estudiantes de Medicina participaron en este estudio realizando una encuesta de valoración de 15 competencias cocneptuales, procedimentales y actitudinales justo antes de la rotación por diferentes estaciones de aprendizaje + servicio en un Centro Regional de Transfusión Sanguínea. Las valoraciones fueron analizadas como evaluación diagnóstica previa. Resultados: 12 de las 15 competencias evaluadas obtuvieron una puntuación superior a 3, por lo que las compentecias previas fueron bastante buenas en el alumnado. Las comptencias de tipo actitudinal fueron las más valoradas (4.29 ± 0.54), siendo significativamente superiores a las procedimentales (3.50 ± 0.65); y estas, a su vez, superiores a las de tipo conceptual (2.78 ± 0.77). Las competencias conpcetuales fueron peor valiradas debido a la inclusión de algunas nociones técnicas que los estudiantes deberán aprender. Conclusiones: Los participantes en el estudio mostraron un nivel elevado de competencias a nivel procedimental y actitudinal previa a la relización de un aprendizaje+servicio sobre hemodonación. La adquisición de los nuevos conceptos permitirán reforzar aquellas actitudes y procedimientos que derivan de dichos conceptos, dotando al alumno de una educación sanitaria más completa que la provista por otros métodos pedagógicos tradicionales., Aim: The objective of this work is the diagnostic evaluation of conceptual, procedural and attitudinal skills in medical students previous to the realization of a model of service-learning on blood donation. Methods: 161 medical students participated in this study by conducting a survey to assess 15 conceptual, procedural and attitudinal competencies just before rotation through different servicelearning stations in a Regional Blood Transfusion Center. The assessments were analyzed as a previous diagnostic evaluation. Results: 12 of the 15 competencies evaluated obtained a score higher than 3, so that the previous competences were high in the students. Attitudinal skills were the most valued (4.29 ± 0.54), being significantly higher than procedural skills (3.50 ± 0.65); and these, in turn, were higher than conceptual skills (2.78 ± 0.77). The conceptual competences were less valued due to the inclusion of some technical notions that the students are still to learn. Conclusions: The participants in this study showed a high level of procedural and attitudinal competencies before the performance of a service-learning on blood donation. The acquisition of the new concepts will allow to strengthen those derived attitudes and procedures from those concepts, providing the student with a more complete health education than that provided by other traditional pedagogical methods.

En el artículo se identifican los componentes afectivo–motivacionales para el aprendizaje de la ciencia en estudiantes de farmacia al condicionar la incidencia de los distintos componentes el proceso de aprendizaje. Se realiza el estudio mediante un cuestionario específico observándose variaciones en la autodeterminación, la autoeficacia, la motivación intrínseca y la motivación a corto y largo plazo con diferencias entre ambos géneros., In the article the affective-motivational components for science learning in pharmacy students are identified because the learning process is conditioned by these components. The study was carried out using a specific questionnaire. The results showed variations in self-determination, self-efficacy, intrinsic motivation, and short and long-term motivation, with differences between both genders.

Significance: Recent generation of bioengineered human skin allowed the efficient treatment of patients with severe skin defects. However, the optical and biomechanical properties of these models are not known. Aim: Three models of bioengineered human skin based on fibrin-agarose biomaterials (acellular, dermal skin substitutes, and complete dermoepidermal skin substitutes) were generated and analyzed. Approach: Optical and biomechanical properties of these artificial human skin substitutes were investigated using the inverse adding-doubling method and tensile tests, respectively. Results: The analysis of the optical properties revealed that the model that most resembled the optical behavior of the native human skin in terms of absorption and scattering properties was the dermoepidermal human skin substitutes after 7 to 14 days in culture. The time-course evaluation of the biomechanical parameters showed that the dermoepidermal substitutes displayed significant higher values than acellular and dermal skin substitutes for all parameters analyzed and did not differ from the control skin for traction deformation, stress, and strain at fracture break. Conclusions: We demonstrate the crucial role of the cells from a physical point of view, confirming that a bioengineered dermoepidermal human skin substitute based on fibrin-agarose biomaterials is able to fulfill the minimal requirements for skin transplants for future clinical use at early stages of in vitro development., Ministry of Science, Innovation and Universities of Spain PGC2018-101904-A-I0, Instituto de Salud Carlos III (ISCIII), Ministry of Science, Innovation and Universities, through AES 2017 AC17/00013, Instituto de Salud Carlos III (ISCIII), Ministry of Science, Innovation and Universities within EuroNanoMed framework, EU AC17/00013, University of Granada A.TEP.280.UGR18, Junta de Andalucía PE-0395-2019, Fundación Benéfica Anticancer San Francisco Javier y Santa Cándida, Granada, Spain, OTRI.35A-07

espanolLos biomateriales juegan un papel esencial en el desarrollo de nuevos sustitutos tisulares ya que proporcionan el entorno tridimensional esencial para promover la adhesion, migracion y proliferacion celular. En este sentido, los biomateriales naturales aportan moleculas biologicamente activas que tipicamente promueven la adhesion y el crecimiento optimo de las celulas. La gelatina es un biomaterial cuyo alto contenido de colageno y cuya red tridimensional interconectada podria contribuir a la generacion de tejidos artificiales similares a los tejidos nativos. En este contexto, diferentes tecnicas de entrecruzamiento quimico han sido utilizadas con el fin de mejorar las propiedades fisicas y mecanicas de los biomateriales de uso en Ingenieria Tisular. El glutaraldehido (GA) es un entrecruzante quimico bien conocido que puede proporcionar materiales con una mejora sustancial en las propiedades de traccion. El objetivo de este articulo es determinar la concentracion optima de GA en hidrogeles de gelatina para determinar la posible aplicacion de estos nuevos biomateriales entrecruzados en la clinica traslacional. A este respecto, se obtuvieron resultados interesantes que podrian ser utiles para disenar andamios con propiedades controladas segun su grado de entrecruzamiento que facilitaria la produccion de productos mas similares a los tejidos nativos. Los hidrogeles de gelatina entrecruzados con GA al 5% mostraron patrones morfologicos adecuados que sugieren una posible aplicacion para la regeneracion del tejido cardiovascular. EnglishBiomaterials play a key role in the development of new tissue substitutes as they provide the essential 3D environment to promote cell adhesion, migration and proliferation. In this sense natural biomaterials offer biologically active molecules which typically promote excellent cells adhesion and growth. Gelatin is a biomaterial whose high collagen content and its interbranching 3D network could certainly contribute to the construction of more native-like tissues. In this context, different techniques of chemical cross-linking have been used in order to improve the physical and mechanical properties of biomaterials for use in Tissue Engineering. Glutaraldehyde (GA) is a well-known chemical crosslinker that can provide materials with substantial improvement in tensile properties. The aim of this article is to test different GA concentrations to crosslink gelatin hydrogels to evaluate the potential application of these new crosslinked biomaterials according to the specific properties of the different tissues in the translational clinic. In this regard interesting findings were obtained that could be helpful to design controlled-properties scaffolds regarding its crosslinked degree that would facilitate the production of more suitable tissue-like products. The proposed 5% GA crosslinked gelatin hydrogels shown morphological patterns and meet the requirements of a first macroscopic and microscopic evaluation which suggests a potential application for the regeneration of cardiovascular tissue.

Number of publications has been widely used as a measure of research output, especially academic and university research. Number of publications in tissue engineering (TE) has increased year by year since early 1990s. However, after an exponential growth phase, recently publications increase at lower rates, suggesting a consolidation process in which reviews become a relevant and high-evidence document type. The aim of this study is to perform a scientometric evaluation of published literature reviews on TE to assess the status of scientific evolution and confirm the consolidation of TE as a research area. Published reviews on TE from 1991 to 2018 were retrieved from Web of Science core collection and this corpus of knowledge was analyzed by growth rate, research area, source title, and citation. Our results revealed that TE can be considered a consolidating area as it leaves the forefront stage of a gompertzian growth curve model. Original research/review ratio is lineally decreasing during the past decade. The emergence of reviews serves to confirm and refute hypothesis and build up a more reliable theoretical framework as well as a guide for future educational approaches. Distribution assessment of categories and journals indicates the multidisciplinary profile of this area focused on the design and development of new tissues. Biomedical sciences become relevant productors of reviews as they need to support TE innovations with high evidence leading to a safer and more efficient treatment of current injuries and diseases. Impact statement Scientometric analysis of published reviews about tissue engineering (TE) suggests that TE can be considered a consolidating area as it leaves the forefront stage of a gompertzian growth curve model. Biomedical sciences become relevant productors of reviews as they need to support TE innovations with high evidence leading to a safer and more efficient treatment of current injuries and diseases.

Advanced therapies are increasingly demanded by patients with the intent of treating some incurable conditions. Because family medicine professionals play an important role as health educators, their residency programs should incorporate new knowledge related to advanced therapies. To successfully implement these programs, how family medicine residents perceive these therapies should be investigated. The main components of perception, i.e. conceptual, procedural and attitudinal, refer to knowledge, skills and feelings, respectively. We designed a specific questionnaire to assess the components of perceptions of advanced therapies in 300 medical residents enrolled in the Spanish National Family Medicine Residency Program. Each component consisted of 4 or 5 topics and each topic contained 6 items. Respondents scored highest in the procedural component (average 4.12±1.00), followed by the attitudinal (3.94±1.07) and conceptual component (3.04±1.43). Differences among the three components were statistically significant (p, This work was supported by CTS-115 (Tissue Engineering Group), Junta de Andalucia.

Mesenchymal stem cells (MSCs) can differentiate toward epithelial cells and may be used as an alternative source for generation of heterotypical artificial human skin substitutes, thus, enhancing their development and translation potential to the clinic. The present study aimed at comparing four types of heterotypical human bioengineered skin generated using MSCs as an alternative epithelial cell source. Adipose-tissue-derived stem cells (ADSCs), dental pulp stem cells (DPSCs), Wharton’s jelly stem cells (WJSCs) and bone marrow stem cells (BMSCs) were used for epidermal regeneration on top of dermal skin substitutes. Heterotypic human skin substitutes were evaluated before and after implantation in immune-deficient athymic mice for 30 d. Histological and genetic studies were performed to evaluate extracellular matrix synthesis, epidermal differentiation and human leukocyte antigen (HLA) molecule expression. The four cell types differentiated into keratinocytes, as shown by the expression of cytokeratin 10 and filaggrin 30 d post-grafting; also, they induced dermal fibroblasts responsible for the synthesis of extracellular fibrillar and non-fibrillar components, in a similar way among each other. WJSCs and BMSCs showed higher expression of cytokeratin 10 and filaggrin, suggesting these cells were more prone to epidermal regeneration. The absence of HLA molecules, even when the epithelial layer was differentiated, supports the future clinical use of these substitutes – especially ADSCs, DPSCs and WJSCs – with low rejection risk. MSCs allowed the generation of bioengineered human skin substitutes with potential clinical usefulness. According to their epidermal differentiation potential and lack of HLA antigens, WJSCs should preferentially be used., Spanish Ministry of Economy and Competitiveness (Instituto de Salud Carlos III), FIS PI15/2048 (co-financed by ERDF-FEDER, European Union), award number AC17/00013 (NanoGSkin) by ISCIII thorough AES 2017 and within the EuroNanoMed framework and PE-0393-201

This study evaluates the cognitive structure and social behavior of tissue engineering (TE) based on a science mapping analysis. Understanding the terms and topics that play a key role in the development of TE can help administrative authorities to better plan funding. Moreover, a better knowledge of collaborative networks in TE and the identification of potential new opportunities for collaboration may enhance synergies in scientific activities to implement future approaches to therapy.

Objective: The current study aimed to design a histological method to determine the presence and organization of the collagen network in sturgeon notochord. Methods: Serial sections of sturgeon notochord (Acipenser naccarii) were used and assigned to two different experimental groups: Hyaluronidase pre-treatment (HP) in an alcohol acid solution (1% HCl in 70% alcohol solution) for 15 min and hyaluronidase solution in a 2 µg/ ml concentration (pre-heated at 37º C), and control (CTR) group, without pre-treatment. Then, the ECM was assessed by two histochemical methods: Picrosirius Red (PR) staining for 30 min with Sirius red (0.1% of Sirius red in saturated aqueous picric acid), for collagen bundle staining, and Alcian Blue (AB) staining for glycosaminoglycans detection. Results: Samples analyzed in this study showed positive histochemical reaction for collagen fibers in both experimental groups. Referring to PR staining, the CTR group presented a larger and homogeneous reaction was observed in the entire samples, whereas HP group presented a more definite and intense pattern of collagen network. Also, this more intense signal in HP group matched with an increase of birefringence in polarized microscopy images of PR. However, HP group showed a lower intense and more heterogeneous signal when was compared with CTR group in AB staining. Conclusion: Using a simple histological example, our study illustrates the capability of a hyaluronidase pre-treatment to enhance picrosirius red staining in sturgeon notochord trough light and polarized microscopy., Objetivo: El presente estudio tiene por objetivo diseñar un método histológico para determinar la presencia y organización de la red de colágeno en la notocorda del esturión. Métodos: Secciones seriada de la notocorda de esturión (Acipenser naccarii) fueron utilizadas y se asignaron a dos grupos experimentales diferentes: pretratamiento de hialuronidasa (HP) en una solución de alcohol ácido (HCl al 1% en solución de alcohol al 70%) durante 15 minutos y posteriormente a una solución de hialuronidasa en una concentración de 2 μg / ml (precalentada a 37º C) y grupo de control (CTR), sin tratamiento previo. Luego, la ECM se evaluó mediante dos métodos histoquímicos: tinción Picrosirius Red (PR) durante 30 minutos con rojo Sirio (0,1% de rojo Sirio en una solución saturada de ácido pícrico), para la tinción de colágeno; y tinción con Alcian Blue (AB) para detección de glicosaminoglicanos. Resultados: Las muestras analizadas en este estudio mostraron una reacción histoquímica positiva para las fibras de colágeno en ambos grupos experimentales. Con respecto a la tinción PR, el grupo CTR presentó una reacción mayor y más homogénea en toda la superficie de las muestras, mientras que el grupo HP presentó un patrón red de colágeno más definido e intenso. Además, esta señal más intensa en el grupo HP coincidió con un aumento de la birrefringencia en las imágenes de microscopía polarizada de PR. Sin embargo, el grupo HP mostró una señal menos intensa y más heterogénea cuando se comparó con el grupo CTR en la tinción AB. Conclusión: Utilizando un ejemplo histológico simple, nuestro estudio ilustra la capacidad de un pretratamiento de hialuronidasa para mejorar la tinción de picrosirius en la notocorda del esturión a través de la luz y la microscopía polarizada.

Integration-defective lentiviral vectors (IDLVs) have become an important alternative tool for gene therapy applications and basic research. Unfortunately, IDLVs show lower transgene expression as compared to their integrating counterparts. In this study, we aimed to improve the expression levels of IDLVs by inserting the IS2 element, which harbors SARs and HS4 sequences, into their LTRs (SE-IS2-IDLVs). Contrary to our expectations, the presence of the IS2 element did not abrogate epigenetic silencing by histone deacetylases. In addition, the IS2 element reduced episome levels in IDLV-transduced cells. Interestingly, despite these negative effects, SE-IS2-IDLVs outperformed SE-IDLVs in terms of percentage and expression levels of the transgene in several cell lines, including neurons, neuronal progenitor cells, and induced pluripotent stem cells. We estimated that the IS2 element enhances the transcriptional activity of IDLV LTR circles 6- to 7-fold. The final effect the IS2 element in IDLVs will greatly depend on the target cell and the balance between the negative versus the positive effects of the IS2 element in each cell type. The better performance of SE-IS2-IDLVs was not due to improved stability or differences in the proportions of 1-LTR versus 2-LTR circles but probably to a re-positioning of IS2-episomes into transcriptionally active regions. This study is financed by the ISCIII Health Research Fund (Spain)and the European Regional Development Fund (FEDER) throughresearch grants PI12/01097 and PI15/02015 (F.M.) and CD09/0020,PI15/00794, and CPII15/00032 (P.A.); the ISCIII Cellular TherapyNetwork (TerCel, RD12/0019/0006) (F.M.); a Juan de la Cierva fellowship (JCI_2012_12666; to R.M.); the CICE and CS of the Juntade Andalucía FEDER/European Cohesion Fund (FSE) for Andalucía2007-2013 through research grants P09-CTS-04532, PI-57069, PI-0001/2009, and PAIDI-Bio-326 (F.M.) and PI-0160/2012 and PI-0014-2016 (K.B.);the Ministerio de Economía, Industria y competi-tividad through research grant SAF2017-89745-R (J.L.G.-P.); aRYC contract. (RYC-2015-18382; to P/J.R.); an FPU fellowship(FPU16/05467; to M.T.-M.); a PEJ contract (PEJ-2014-A-46314; to A.B.C.-G. and PEJ-2014-A-17105 S.S.-H.); the European Research (ERC-Consolidator ERC-STG-2012-233764); and a privatedonation from Ms. Francisca Serrano (Trading y Bolsa para Torpes,Granada, Spain).We also wish to tand a private donation from Ms. Francisca Serrano (Trading y Bolsa para Torpes, Granada, Spain).We also wish to thank Michael O’Shea for proofreading the article. J.L.G.-P’s lab is supported by MINECO-FEDER (SAF2017-89745-R). Sí

Scleral defects can result as a consequence of trauma, infectious diseases or cancer and surgical repair with allogeneic scleral grafts can be required. However, this method has limitations and novel alternatives are needed. Here, the efficacy of acellular nanostructured fibrin-agarose hydrogel-based substitutes (NFAH) in the repair of scleral defects in rabbits was studied. For this, scleral defects of 5-mm diameter were made on 18 adult-male New Zealand rabbits and repaired with acellular NFAH, NFAH crosslinked with genipin (NFAH-GP) or glutaraldehyde (NFAH-GA), allogeneic scleral grafts as control (C-CTR) or not repaired (negative control N-CTR) (n = 3 each). Macroscopic and histological analyses were performed after 40-days. Macroscopy confirmed the repair of all defects in a comparable manner than the C-CTR. Histology showed no degradation nor integration in C-CTR while NFAH-GP and NFAH-GA biomaterials were encapsulated by connective and inflammatory tissues with partial biodegradation. The NFAH were fully biodegraded and replaced by a loose connective tissue and sclera covering the defects. This in vivo study demonstrated that the NFAH are a promising biocompatible and pro-regenerative alternative to the use of allogeneic cadaveric grafts. However, large defects and long-term studies are needed to demonstrate the potential clinical usefulness of these substitutes.