Suchergebnisse

Blood loss remains a major concern during surgery and can increase the morbidity of the intervention. The use of topical haemostatic agents to overcome this issue therefore becomes necessary. Fibrin sealants are promising haemostatic agents due to their capacity to promote coagulation, but their effectiveness and applicability need to be improved. We have compared the haemostatic efficacy of a novel nanostructured fibrin‐agarose hydrogel patch, with (c‐NFAH) or without cells (a‐NFAH), against two commercially available haemostatic agents in a rat model of hepatic resection. Hepatic resections were performed by making short or long incisions (mild or severe model, respectively), and haemostatic agents were applied to evaluate time to haemostasis, presence of haematoma, post‐operative adhesions to adjacent tissues, and inflammation factors. We found a significantly higher haemostatic success rate (time to haemostasis) with a‐NFAH than with other commercial haemostatic agents. Furthermore, other relevant outcomes investigated were also improved in the a‐NFAH group, including no presence of haematoma, lower adhesions, and lower grades of haemorrhage, inflammation, and necrosis in histological analysis. Overall, these findings identify a‐NFAH as a promising haemostatic agent in liver resection and likely in a range of surgical procedures. ; This work was supported by preclinical research funds from the Regional Government of Andalusia through the Andalusian Initiative for Advanced Therapies

Blood loss remains a major concern during surgery and can increase the morbidity of the intervention. The use of topical haemostatic agents to overcome this issue therefore becomes necessary. Fibrin sealants are promising haemostatic agents due to their capacity to promote coagulation, but their effectiveness and applicability need to be improved. We have compared the haemostatic efficacy of a novel nanostructured fibrin‐agarose hydrogel patch, with (c‐NFAH) or without cells (a‐NFAH), against two commercially available haemostatic agents in a rat model of hepatic resection. Hepatic resections were performed by making short or long incisions (mild or severe model, respectively), and haemostatic agents were applied to evaluate time to haemostasis, presence of haematoma, post‐operative adhesions to adjacent tissues, and inflammation factors. We found a significantly higher haemostatic success rate (time to haemostasis) with a‐NFAH than with other commercial haemostatic agents. Furthermore, other relevant outcomes investigated were also improved in the a‐NFAH group, including no presence of haematoma, lower adhesions, and lower grades of haemorrhage, inflammation, and necrosis in histological analysis. Overall, these findings identify a‐NFAH as a promising haemostatic agent in liver resection and likely in a range of surgical procedures. ; This work was supported by preclinical research funds from the Regional Government of Andalusia through the Andalusian Initiative for Advanced Therapies. ; Peer reviewed

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

[EN] Matrix-assisted autologous chondrocyte implantation (MACI) has shown promising results for cartilage repair, combining cultured chondrocytes and hydrogels, including alginate. The ability of chondrocytes for MACI is limited by different factors including donor site morbidity, dedifferentiation, limited lifespan or poor proliferation in vitro. Mesenchymal stem cells could represent an alternative for cartilage regeneration. In this study, we propose a MACI scaffold consisting of a mixed alginate-agarose hydrogel in combination with human dental pulp stem cells (hDPSCs), suitable for cartilage regeneration. Scaffolds were characterized according to their rheological properties, and their histomorphometric and molecular biology results. Agarose significantly improved the biomechanical behavior of the alginate scaffolds. Large scaffolds were manufactured, and a homogeneous distribution of cells was observed within them. Although primary chondrocytes showed a greater capacity for chondrogenic differentiation, hDPSCs cultured in the scaffolds formed large aggregates of cells, acquired a rounded morphology and expressed high amounts of type II collagen and aggrecan. Cells cultured in the scaffolds expressed not only chondral matrix-related genes, but also remodeling proteins and chondrocyte differentiation factors. The degree of differentiation of cells was proportional to the number and size of the cell aggregates that were formed in the hydrogels. ; This work was funded by the Ministry of Economy and Competitiveness of the Spanish Government (PID2019-106099RB-C42, MM) and by the Generalitat Valenciana, Spain (PROMETEO/2020/069, CC). CIBER-BBN and CIBER-ER are financed by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and the Instituto de Salud Carlos III, with assistance of the European Regional Development Fund. ; Oliver-Ferrándiz, M.; Milián, L.; Sancho-Tello, M.; Martín De Llano, JJ.; Gisbert-Roca, F.; Martínez-Ramos, C.; Carda, C. (2021). ...

This article belongs to the Special Issue Functional Polymer Gels for Advanced Applications in Biomedicine, Energy and Optoelectronics. ; Dermo-epidermal equivalents based on plasma-derived fibrin hydrogels have been extensively studied for skin engineering. However, they showed rapid degradation and contraction over time and low mechanical properties which limit their reproducibility and lifespan. In order to achieve better mechanical properties, elasticity and biological properties, we incorporated a elastin-like recombinamer (ELR) network, based on two types of ELR, one modified with azide (SKS-N3) and other with cyclooctyne (SKS-Cyclo) chemical groups at molar ratio 1:1 at three different SKS (serine-lysine-serine sequence) concentrations (1, 3, and 5 wt.%), into plasma-derived fibrin hydrogels. Our results showed a decrease in gelation time and contraction, both in the absence and presence of the encapsulated human primary fibroblasts (hFBs), higher mechanical properties and increase in elasticity when SKSs content is equal or higher than 3%. However, hFBs proliferation showed an improvement when the lowest SKS content (1 wt.%) was used but started decreasing when increasing SKS concentration at day 14 with respect to the plasma control. Proliferation of human primary keratinocytes (hKCs) seeded on top of the hybrid-plasma hydrogels containing 1 and 3% of SKS showed no differences to plasma control and an increase in hKCs proliferation was observed for hybrid-plasma hydrogels containing 5 wt.% of SKS. These promising results showed the need to achieve a balance between the reduced contraction, the better mechanical properties and biological properties and indicate the potential of using this type of hydrogel as a testing platform for pharmaceutical products and cosmetics, and future work will elucidate their potential. ; The authors are grateful for the funding from the Spanish Government (FPU15-00448, PID2019-110709RB-100, RED2018-102417-T), Junta de Castilla y León (VA317P18. Infrared2018-UVA06), Interreg. V España Portugal POCTEP (0624_2IQBIONEURO_6_E) and Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León. This research was also funded by Programa de Actividades de I + D entre Grupos de Investigación de la Comunidad de Madrid, S2018/BAA-4480, Biopieltec-CM; by Programa Estatal de I + D + i Orientada a los Retos de la So-ciedad, RTI2018-101627-B-I00; by Programa de Apoyo a la Realización de Proyectos Interdisciplinares de I + D para Jóvenes Investigadores de la Universidad Carlos III de Madrid (project: BIOMASKIN); and by Cátedra Fundación Ramón Areces.

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

WOS: 000314426400021 ; PubMed ID: 23380006 ; We report our efforts in identifying optimal scanning laser microscope parameters to study cells in three-dimensional culture. For this purpose we studied contrast of extracellular matrix (ECM) mimics, as well as signal attenuation, and bleaching of red and green fluorescent protein labeled cells. Confocal backscattering, second harmonic generation (SHG), and autofluorescence were sources of contrast in ECM mimics. All common ECM mimics exhibit contrast observable with confocal reflectance microscopy. SHG imaging on collagen I based hydrogels provides high contrast and good optical penetration depth. Agarose is a useful embedding medium because it allows for large optical penetration and exhibits minimal autofluorescence. We labeled breast cancer cells' outline with DsRed2 and nucleus with enhanced green fluorescent protein (eGFP). We observed significant difference both for the bleaching rates of eGFP and DsRed2 where bleaching is strongest during two-photon excitation (TPE) and smallest during confocal imaging. But for eGFP the bleaching rate difference is smaller than for DsRed2. After a few hundred microns depth in a collagen I hydrogel, TPE fluorescence of DsRed2 becomes twice as strong compared to confocal imaging. In fibrin and agarose gels, the imaging depth will need to be beyond 1 mm to notice a TPE advantage. ; Turkish government's Higher Education CouncilMinistry of National Education - Turkey; National Institutes of Health (NIH)United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [S10RR023737] ; The authors wish to thank the Turkish government's Higher Education Council for supporting this project with a scholarship. The majority of work was conducted on a National Institutes of Health (NIH) sponsored shared instrument (S10RR023737). The framework to investigate novel 3D imaging techniques was provided by NIH R01HL077683. The authors wish to thank the engineers and scientists at Lavision Biotec for creating a modular intravital microscope and sharing their experience in designing and maintaining state of the art microscopy equipment.

WOS: 000314426400021 ; PubMed ID: 23380006 ; We report our efforts in identifying optimal scanning laser microscope parameters to study cells in three-dimensional culture. For this purpose we studied contrast of extracellular matrix (ECM) mimics, as well as signal attenuation, and bleaching of red and green fluorescent protein labeled cells. Confocal backscattering, second harmonic generation (SHG), and autofluorescence were sources of contrast in ECM mimics. All common ECM mimics exhibit contrast observable with confocal reflectance microscopy. SHG imaging on collagen I based hydrogels provides high contrast and good optical penetration depth. Agarose is a useful embedding medium because it allows for large optical penetration and exhibits minimal autofluorescence. We labeled breast cancer cells' outline with DsRed2 and nucleus with enhanced green fluorescent protein (eGFP). We observed significant difference both for the bleaching rates of eGFP and DsRed2 where bleaching is strongest during two-photon excitation (TPE) and smallest during confocal imaging. But for eGFP the bleaching rate difference is smaller than for DsRed2. After a few hundred microns depth in a collagen I hydrogel, TPE fluorescence of DsRed2 becomes twice as strong compared to confocal imaging. In fibrin and agarose gels, the imaging depth will need to be beyond 1 mm to notice a TPE advantage. ; Turkish government's Higher Education CouncilMinistry of National Education - Turkey; National Institutes of Health (NIH)United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [S10RR023737] ; The authors wish to thank the Turkish government's Higher Education Council for supporting this project with a scholarship. The majority of work was conducted on a National Institutes of Health (NIH) sponsored shared instrument (S10RR023737). The framework to investigate novel 3D imaging techniques was provided by NIH R01HL077683. The authors wish to thank the engineers and scientists at Lavision Biotec for creating a modular intravital microscope and sharing their experience in designing and maintaining state of the art microscopy equipment.