Chitosan conduits enriched with fibrin-collagen hydrogel with or without adipose-derived mesenchymal stem cells for the repair of 15-mm-long sciatic nerve defect

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 PI20-0318Fondo Europeo de Desarrollo Regional ERDF-FEDER European UnionPlan Andaluz de Investigacion, Desarrollo e Innovacion (PAIDI2020), Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades, Junta de Andalucia, Espana P18-RT-5059Programa Operativo FEDER Andalucia 2014-2020, Universidad de Granada, Junta de Andalucia, Espana A-CTS-498-UGR18European Commissio

Comprehensive ex vivo and in vivo preclinical evaluation of novel chemo enzymatic decellularized peripheral nerve allografts

As a reliable alternative to autografts, decellularized peripheral nerve allografts (DPNAs) should mimic the complex microstructure of native nerves and be immunogenically compatible. Nevertheless, there is a current lack of decellularization methods able to remove peripheral nerve cells without significantly altering the nerve extracellular matrix (ECM). The aims of this study are firstly to characterize ex vivo, in a histological, biochemical, biomechanical and ultrastructural way, three novel chemical-enzymatic decellularization protocols (P1, P2 and P3) in rat sciatic nerves and compared with the Sondell classic decellularization method and then, to select the most promising DPNAs to be tested in vivo. All the DPNAs generated present an efficient removal of the cellular material and myelin, while preserving the laminin and collagen network of the ECM (except P3) and were free from any significant alterations in the biomechanical parameters and biocompatibility properties. Then, P1 and P2 were selected to evaluate their regenerative effectivity and were compared with Sondell and autograft techniques in an in vivo model of sciatic defect with a 10-mm gap, after 15 weeks of follow-up. All study groups showed a partial motor and sensory recovery that were in correlation with the histological, histomorphometrical and ultrastructural analyses of nerve regeneration, being P2 the protocol showing the most similar results to the autograft control group

Ex Vivo Generation and Characterization of Human Hyaline and Elastic Cartilaginous Microtissues for Tissue Engineering Applications

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 PI20/0318Junta de Andalucia PI-0257-2017 PE-03952019Junta de Andalucia P18-RT-5059University of Granada A-CTS-498-UGR18Junta de Andalucia A-CTS-498-UGR18FEDER-ERDF fund

Ejercicios de transversalización para una propuesta de didáctica del diseño. Caso taller integrador de diseño visual en tiempos de pandemia

A raíz de la cuarentena decretada a inicios de 2020, para contener la pandemia de Covid-19, las instituciones educativas debieron volcar los procesos presenciales a clases mediadas por las nuevas tecnologías y herramientas virtuales. Para responder a este desafío, el equipo docente de Diseño Visual de la Universidad de Caldas planteó una propuesta pedagógica en la enseñanza del diseño que aplicó en el taller integrador realizado durante el primer periodo de 2020 al estudiantado de séptimo semestre. La transversalización es el denominador común de esta didáctica del diseño que consistió en integrar la mayoría de las asignaturas del semestre en un mismo espacio, bajo un modelo curricular del programa que permitió alimentar y complementar contenidos entre docentes y aportó a los conocimientos en un proceso integral proyectual bajo una metodología práctica y propositiva. La propuesta se consolidó como un laboratorio de estrategias didácticas mediante el desarrollo de 10 proyectos ideados por el grupo de estudiantes, y se implementó a partir del Design Thinking, de donde resultó una estrategia innovadora de enseñanza que supone una tendencia viable para la enseñanza del diseño. Las condiciones del periodo académico resultaron propicias para lograr un proceso verdaderamente transversal que arrojó, entre sus resultados, una mejor apropiación de los conceptos y contenidos requeridos para el ejercicio de la disciplina, al permitir confrontar y relacionar las temáticas de cada asignatura para la resolución de un problema de diseño específico

Comprehensive ex vivo and in vivo preclinical evaluation of novel chemo enzymatic decellularized peripheral nerve allografts

As a reliable alternative to autografts, decellularized peripheral nerve allografts (DPNAs) should mimic the complex microstructure of native nerves and be immunogenically compatible. Nevertheless, there is a current lack of decellularization methods able to remove peripheral nerve cells without significantly altering the nerve extracellular matrix (ECM). The aims of this study are firstly to characterize ex vivo, in a histological, biochemical, biomechanical and ultrastructural way, three novel chemical-enzymatic decellularization protocols (P1, P2 and P3) in rat sciatic nerves and compared with the Sondell classic decellularization method and then, to select the most promising DPNAs to be tested in vivo. All the DPNAs generated present an efficient removal of the cellular material and myelin, while preserving the laminin and collagen network of the ECM (except P3) and were free from any significant alterations in the biomechanical parameters and biocompatibility properties. Then, P1 and P2 were selected to evaluate their regenerative effectivity and were compared with Sondell and autograft techniques in an in vivo model of sciatic defect with a 10-mm gap, after 15 weeks of follow-up. All study groups showed a partial motor and sensory recovery that were in correlation with the histological, histomorphometrical and ultrastructural analyses of nerve regeneration, being P2 the protocol showing the most similar results to the autograft control group.Spanish "Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion TecnologicaSpanish Government FIS PI17-0393 FIS PI20-0318Fondo Europeo de Desarrollo RegionalERDF-FEDER European Union P18-RT-5059Plan Andaluz de Investigacion, Desarrollo eInnovacion (PAIDI 2020)Consejeria de Transformacion Economica, Industria, Conocimiento y UniversidadesJunta de Andalucia PI-0086-2020ERDF-FEDER, theEuropean Union CPP2021-009070Ministerio de Ciencia e Innovacion, Union Europea (NextGeneration EU)Agencia Estatal de Investigacion, Espan

Nanostructured fibrin-based hydrogel membranes for use as an augmentation strategy in achilles tendon surgical repair in rats

Hydrogels are polymeric biomaterials characterised by their promising biological and biomechanical properties, which make them potential alternatives for use in tendon repair. The aim of the present study was to generate in vitro, and determine the therapeutic efficacy in vivo, of novel nanostructured fibrin-based hydrogels to be used as an augmentation strategy for the surgical repair of rat Achilles tendon injuries. Fibrin, fibrin-agarose and fibrin-collagen nanostructured hydrogels (NFH, NFAH and NFCH, respectively) were generated and their biomechanical properties and cell-biomaterial interactions characterised ex vivo. Achilles tendon ruptures were created in 24 adult Wistar rats, which were next treated with direct repair (control group) or direct repair augmented with the generated biomaterials (6 rats/group). After 4 and 8 weeks, the animals were euthanised for macroscopical and histological analyses. Biomechanical characterisation showed optimal properties of the biomaterials for use in tendon repair. Moreover, biological analyses confirmed that tendon-derived fibroblasts were able to adhere to the surface of the generated biomaterials, with high levels of viability and functionality. In vivo studies demonstrated successful tendon repair in all groups. Lastly, histological analyses disclosed better tissue and extracellular matrix organisation and alignment with biomaterial-based augmentation strategies than direct repair, especially when NFAH and NFCH were used. The present study demonstrated that nanostructured fibrin-collagen hydrogels can be used to enhance the healing process in the surgical repair of tendon ruptures.The study was supported by the Spanish Society of Orthopaedics and Traumatology (SECOT), the Spanish Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica, Ministerio de Economía y Competitividad (Instituto de Salud Carlos III), the European Regional Development Fund (ERDF-FEDER) Grant number FIS PI20-0318 and the Grant number P18-RT-5059 from the Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020), Consejería de Transformación Económica, Industria, Conocimiento y Universidades, Andalusian Regional Government, Spain. The authors are grateful to Dr Ariane Ruyffelaert for her advice on the English version of the manuscript and to Karen Shashok for editing the revised manuscript. The authors are also grateful to Amalia de la Rosa Romero and Concepción López Rodríguez (Experimental Unit, University Hospital Virgen de las Nieves, Granada, Spain) and Fabiola Bermejo Casares (Department of Histology, University of Granada, Spain) for their technical assistance

Generation of a Biomimetic Substitute of the Corneal Limbus Using Decellularized Scaffolds

Patients with severe limbal damage and limbal stem cell deficiency are a therapeutic challenge. We evaluated four decellularization protocols applied to the full-thickness and half-thickness porcine limbus, and we used two cell types to recellularize the decellularized limbi. The results demonstrated that all protocols achieved efficient decellularization. However, the method that best preserved the transparency and composition of the limbus extracellular matrix was the use of 0.1% SDS applied to the half-thickness limbus. Recellularization with the limbal epithelial cell line SIRC and human adipose-derived mesenchymal stem cells (hADSCs) was able to generate a stratified epithelium able to express the limbal markers p63, pancytokeratin, and crystallin Z from day 7 in the case of SIRC and after 14–21 days of induction when hADSCs were used. Laminin and collagen IV expression was detected at the basal lamina of both cell types at days 14 and 21 of follow-up. Compared with control native limbi, tissues recellularized with SIRC showed adequate picrosirius red and alcian blue staining intensity, whereas limbi containing hADSCs showed normal collagen staining intensity. These preliminary results suggested that the limbal substitutes generated in this work share important similarities with the native limbus and could be potentially useful in the future.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), Grants FIS PI20/0317 and ICI21-00010, cofinanced by FEDER funds (European Union). This work was also supported by grant PI-0086-2020 from Consejería de Salud y Familias, Junta de Andalucía, Spain, and grant B-CTS-504-UGR20 (Proyectos de I+D+i en el marco del Programa Operativo FEDER Andalucía 2014–2020) from the University of Granada, Consejería de Transformación Económica, Industria, Conocimiento y Universidades, Junta de Andalucía, and European Union (cofinanced by FEDER funds)

Detergent-based decellularized peripheral nerve allografts: An in vivo preclinical study in the rat sciatic nerve injury model

Este estudio ha sido financiado por el "Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica, Ministerio de Economía y Competitividad (Instituto de Salud Carlos III), y cofinanciado por el "Fondo Europeo de Desarrollo Regional (FEDER) Unión Europea", Subvención nº FIS PI17-0393, y por el "Programa Operativo FEDER Andalucía 2014-2020, I +D +I, Frontera" Subvención nº A-CTS-488-UGR18. Todo el estudio se realizó en las instalaciones del Grupo de Ingeniería Tisular (CTS-115) de la Universidad de Granada y en la Unidad Experimental del Hospital Universitario Virgen de las Nieves de Granada, España. Este estudio forma parte de la Tesis Doctoral de Jesús Chato-Astrain.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.Instituto de Salud Carlos III: FIS PI17/0393, FIS PI17-0393FEDER Andalucía 2014-2020, I +D +I, A-CTS-488-UGR1

Generation of a novel human dermal substitute functionalized with antibiotic‑loaded nanostructured lipid carriers (NLCs) with antimicrobial properties for tissue engineering

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), EUInstituto de Salud Carlos III AC17/00013Centro para el Desarrollo Tecnológico Industrial -CDTI 00108589Spanish GovernmentJunta de Andalucía PE-0395-2019Fundacion Benefica Anticancer San Francisco Javier y Santa Candida, Granada, SpainDepartment of Economic Development and Infrastructure of the Basque Government budget, through the HAZITEK business R + D support program ZE-2017/00014European Union (EU) OTRI.35A-0

Evaluation of the awareness of novel advanced therapies among family medicine residents in Spain

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<0.00017). Family medicine residents perceived that procedures to implement advanced therapies are well established, especially their application. However, they felt their cognitive background was insufficient to respond efficiently to the expectations generated by these new therapeutic tools, especially in the regulatory framework. High awareness of the risks and limitations of these treatments was reflected by residents’ preference for clinically tested therapies. Although they appropriately situated treatment with these therapies within hospital care, they associated the biofabrication of novel products with research centers, although these therapeutic tools can be produced in different facilities. These results are potentially useful for designing future training programs and health policies for family medicine residents, and suggest the need to implement specific training programs in advanced therapies at the conceptual, procedural and attitudinal level.This work was supported by CTS-115 (Tissue Engineering Group), Junta de Andalucia