Suchergebnisse

Five agarose types (D1LE, D2LE, LM, MS8 and D5) were evaluated in tissue engineering and compared for the first time using an array of analysis methods. Acellular and cellular constructs were generated from 0.3–3%, and their biomechanical properties, in vivo biocompatibility (as determined by LIVE/DEAD, WST-1 and DNA release, with n = 6 per sample) and in vivo biocompatibility (by hematological and biochemical analyses and histology, with n = 4 animals per agarose type) were analyzed. Results revealed that the biomechanical properties of each hydrogel were related to the agarose concentration (p < 0.001). Regarding the agarose type, the highest (p < 0.001) Young modulus, stress at fracture and break load were D1LE, D2LE and D5, whereas the strain at fracture was higher in D5 and MS8 at 3% (p < 0.05). All agaroses showed high biocompatibility on human skin cells, especially in indirect contact, with a correlation with agarose concentration (p = 0.0074 for LIVE/DEAD and p = 0.0014 for WST-1) and type, although cell function tended to decrease in direct contact with highly concentrated agaroses. All agaroses were safe in vivo, with no systemic effects as determined by hematological and biochemical analysis and histology of major organs. Locally, implants were partially encapsulated and a pro-regenerative response with abundant M2- type macrophages was found. In summary, we may state that all these agarose types can be safely used in tissue engineering and that the biomechanical properties and biocompatibility were strongly associated to the agarose concentration in the hydrogel and partially associated to the agarose type. These results open the door to the generation of specific agarose-based hydrogels for definite clinical applications such as the human skin, cornea or oral mucosa. ; Hispanagar SA, Burgos, Spain, through CDTI, Ministry of Science and Innovation, Spain, Programa Operativo Plurirregional de Crecimiento Inteligente (CRIN) IDI-20180052 ; ISCIII thorough AES AC17/00013 ; Junta de Andalucía PE-0395-2019 ; Spanish Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+i) from Ministerio de Ciencia, Innovación y Universidades (Instituto de Salud Carlos III) FIS PI17/0391 ; Fondo Europeo de Desarrollo Regional ERDF-FEDER, European Union PI20/0317

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. ; This research was funded by the Spanish "Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica, Ministerio de Economía y Competitividad (Instituto de Salud Carlos III), Grants No FIS PI14-1343, FIS PI17-0393, FIS PI20-0318 co-financed by the "Fondo Europeo de Desarrollo Regional ERDF-FEDER European Union"; Grant No P18-RT-5059 by "Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020), Consejería de Transformación Económica, Industria, Conocimiento y Universidades, Junta de Andalucía, España"; and Grant A-CTS-498- UGR18 by "Programa Operativo FEDER Andalucía 2014–2020, Universidad de Granada, Junta de Andalucía, España", co-funded by ERDF-FEDER, the European Union. ; Yes

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 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

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).

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

Generation of biocompatible and biomimetic tissue-like biomaterials is crucial to ensure the success of engineered substitutes in tissue repair. Natural biomaterials able to mimic the structure and composition of native extracellular matrices typically show better results than synthetic biomaterials. The aim of this study was to perform an in vivo time-course biocompatibility analysis of fibrin-agarose tissue-like hydrogels at the histological, imagenological, hematological, and biochemical levels. Tissue-like hydrogels were produced by a controlled biofabrication process allowing the generation of biomechanically and structurally stable hydrogels. The hydrogels were implanted subcutaneously in 25 male Wistar rats and evaluated after 1, 5, 9, and 12 weeks of in vivo follow-up. At each period of time, animals were analyzed using magnetic resonance imaging (MRI), hematological analyses, and histology of the local area in which the biomaterials were implanted, along with major vital organs (liver, kidney, spleen, and regional lymph nodes). MRI results showed no local or distal alterations during the whole study period. Hematology and biochemistry showed some fluctuation in blood cells values and in some biochemical markers over the time. However, these parameters were progressively normalized in the framework of the homeostasis process. Histological, histochemical, and ultrastructural analyses showed that implantation of fibrin-agarose scaffolds was followed by a progressive process of cell invasion, synthesis of components of the extracellular matrix (mainly, collagen) and neovascularization. Implanted biomaterials were successfully biodegraded and biointegrated at 12 weeks without any associated histopathological alteration in the implanted zone or distal vital organs. In summary, our in vivo study suggests that fibrin-agarose tissue-like hydrogels could have potential clinical usefulness in engineering applications in terms of biosafety and biocompatibility. ; Spanish Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica (I+D+i) from the Spanish Ministerio de Ciencia, Innovacion y Universidades (Instituto de Salud Carlos III) (ERDF-FEDER, European Union) FIS PI17/0391 PI17/0393 PI18/332 ; ISCIII thorough AES 2017 AC17/00013 ; EuroNanoMed framework AC17/00013 ; Hispanagar, SA, Burgos, Spain, through CDTI, Ministry of Economy and Competitiveness, Spain IDI-20180052 ; Junta de Andalucia CS PI-0257-2017 PE-0395-2019 ; Ministerio de Economia, Industria y Competitividad, MINECO FIS2017-85954-R ; Agencia Estatal de Investigacion, AEI, Spain FIS2017-85954-R ; European Union (EU) FIS2017-85954-R ; National Cordoba University, Argentina Secyt 266 HCS 659/2018 ; Programa Operativo Pluriregional de Crecimiento Inteligente (CRIN) IDI-20180052 ; ERDF-FEDER funds, EU IDI-20180052