Suchergebnisse

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.

AbstractFibrosis of the injured muscles is a problem of recovery from trauma and denervation. The aim of the work was to investigate the interconnection of matrix metalloproteinase-9 (ММР-9) activity in denervated muscles with fibrosis and to estimate its role in nerve restoration by the epineurial suture, fibrin-based glue, and polyethylene glycol hydrogel. The activity of matrix metalloproteinases was estimated by gelatin zymography. Collagen density in muscles was determined histochemically. An increased level of the active MMP-9 is associated with the fibrous changes in the denervated skeletal muscles and after an epineurial suture. The use of fibrin glue and polyethylene glycol hydrogel resulted in a lower level of collagen and ММР-9 activity, which may be a therapeutic target in the treatment of neuromuscular lesions, and has value in fibrosis analysis following microsurgical intervention for peripheral nerve reconstruction.

In this work, enzymatically active polysaccharide-based hydrogels and aerogels have been developed. To this end, a thermostable β-galactosidase (TmLac) enzyme from Thermotoga maritima embedded in nanoflowers' format was used to evaluate the capacity of the hydrogel matrices to preserve the hydrolytic activity of the enzyme and the reusability of the hydrogels formed. Commercial agar, unpurified agar and agarose were compared as supporting materials. Although the developed hydrogel capsules can be used at high temperature (75 °C) and reused for the digestion of lactose to a greater extent than the free nanoflowers, loaded hydrogel capsules behaved differently depending on the type of polysaccharide used. Commercial agar was the most promising one since these hydrogel capsules could be reused, maintaining the structural integrity and reaching higher enzymatic activity (after seven cycles at 75 °C) than the free TmLac-Ca2+ nanoflowers. To facilitate handling and storage, aerogels were developed by freeze-drying the hydrogel capsules. Aerogels of agarose and unpurified agar underwent structural changes during freeze-drying that adversely affected their subsequent use, losing their integrity after being rehydrated. However, commercial agar aerogels were successfully developed and reused thanks to the existing interactions with TmLac-Ca2+ nanoflowers (confirmed by FTIR), which resulted in better capsule integrity and enzyme protection. The hydrolytic activity of enzymatically active aerogels based on commercial agar was in the same range of the free TmLac and TmLac-Ca2+ nanoflowers, being significantly higher to their counterparts in the hydrated form (hydrogels based on commercial agar). ; This research was supported by grants from Spain's 'Secretaría de Estado de Investigación, Desarrollo e Innovación' (AGL2016-75245-R), Agencia Estatal de Investigación (AEI, Grant PCI2018-092886) and cofunded by the European Union's Horizon 2020 research and innovation programme (ERA-Net SUSFOOD2). MJF was supported by a Ramon y Cajal contract (RYC2014-15842) from the Spanish Ministerio de Economia; Industria y Competitividad. ; Peer reviewed

Protein therapeutics have a major role in medicine, being used to treat diverse pathologies. Their three-dimensional structures offer high specificity and lower toxicity than small organic compounds but also make them less stable limiting their in vivo half-life. Protein-analogs obtained by recombinant DNA technology or by chemical modification and / or the use of drug delivery vehicles have been developed to improve or modulate the in vivo pharmacological activity of proteins. Nevertheless, strategies to improve the shelf-life of protein pharmaceuticals have been less explored which challenge the preservation of their activity. Herein, we present a methodology that simultaneously increases the stability of proteins and modulates the release profile, and implemented with human insulin as a proof of concept. Two novel thermally stable insulin composite crystal formulations intended for the therapeutic treatment of diabetes are reported. These composite crystals have been obtained by crystallizing insulin in agarose and Fmoc-AA (fluorenylmethoxycarbonyl-dialanine) hydrogels. This process affords composite crystals, in which, hydrogel fibers are occluded inside the crystals. Insulin in both crystalline formulations remains unaltered at 50 C for 7 days. Differential Scanning Calorimetry, High Performance Liquid Chromatography, mass spectrometry and in vivo studies have shown that insulin does not degrade after the heat treatment. The nature of the hydrogel modifies the physicochemical properties of the crystals. Crystals grown in Fmoc-AA hydrogel are more stable and have a slower dissolution rate than crystals grown in agarose. This methodology paves the way for the development of more stable protein pharmaceuticals overcoming some of the exiting limitations. ; The research leading to these results has received funding from the "la Caixa" Banking Foundation" CaixaImpulse program, EIT-Health PocPlus program and from the Ministry of Economy and Competitiveness of Spain, acknowledged through the following projects: BIO2016-74875-P, BFU2014-57736-P, AGL2014-58883-R, SAF2017-88457-R, AGL2017-85270-R and FIS2017-85954-R co-funded by Fondo Europeo de Desarrollo Regional, ERDF, European Union and FEDER/Junta de Andalucía-Consejería de Transformación Económica, Industria, Conocimiento y Universidades (Spain) projects P18-FR-3533, P12-FQM-2721, P12-FQM-790, CTS235 and CTS164. MA-A and MCM-T, were supported by fellowships from the Ministry of Education. CIBERehd is funded by Instituto de Salud Carlos III.

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

This manuscript provides an overview of the in vitro and in vivo studies reported in the literature focusing on seaweed polysaccharides based hydrogels that have been proposed for applications in regenerative medicine, particularly, in the field of cartilage tissue engineering. For a better understanding of the main requisites for these specific applications, the main aspects of the native cartilage structure, as well as recognized diseases that affect this tissue are briefly described. Current available treatments are also presented to emphasize the need for alternative techniques. The following part of this review is centered on the description of the general characteristics of algae polysaccharides, as well as relevant properties required for designing hydrogels for cartilage tissue engineering purposes. An in-depth overview of the most well known seaweed polysaccharide, namely agarose, alginate, carrageenan and ulvan biopolymeric gels, that have been proposed for engineering cartilage is also provided. Finally, this review describes and summarizes the translational aspect for the clinical application of alternative systems emphasizing the importance of cryopreservation and the commercial products currently available for cartilage treatment. ; Authors report no declarations of interest. Authors thank the Portuguese Foundation for Science and Technology (FCT) for the PhD fellowship of Elena G. Popa (SFRH/BD/64070/2009) and research project (MIT/ECE/0047/2009). The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no ...

Purpose: Human cornea substitutes generated by tissue engineering currently require limbal stem cells for the generation of orthotypical epithelial cell cultures. We recently reported that bioengineered corneas can be fabricated in vitro from a heterotypical source obtained from Wharton's jelly in the human umbilical cord (HWJSC). Methods: Here, we generated a partial thickness cornea model based on plastic compression nanostructured fibrin-agarose biomaterials with cornea epithelial cells on top, as an orthotypical model (HOC), or with HWJSC, as a heterotypical model (HHC), and determined their potential in vivo usefulness by implantation in an animal model. Results: No major side effects were seen 3 and 12 months after implantation of either bioengineered partial cornea model in rabbit corneas. Clinical results determined by slit lamp and optical coherence tomography were positive after 12 months. Histological and immunohistochemical findings demonstrated that in vitro HOC and HHC had moderate levels of stromal and epithelial cell marker expression, whereas in vivo grafted corneas were more similar to control corneas. Conclusion: These results suggest that both models are potentially useful to treat diseases requiring anterior cornea replacement, and that HHC may be an efficient alternative to the use of HOC which circumvents the need to generate cornea epithelial cell cultures. ; Spanish Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica (I CD Ci) from the Spanish Ministry of Science and Innovation: Instituto de Salud Carlos III FIS PI17/0391 FIS PI14/0955 ; European Union (EU) ; Spanish Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica (I CD Ci) from the Spanish Ministry of Science and Innovation: Ministry of Science, Innovation and Universities of Spain PGC2018-101904-A-I00 RTC-2017-6696-1

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