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1.
Int J Bioprint ; 9(2): 670, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37065655

RESUMO

In recent years, extrusion-based three-dimensional (3D) bioprinting is employed for engineering cardiac patches (CP) due to its ability to assemble complex structures from hydrogel-based bioinks. However, the cell viability in such CPs is low due to shear forces applied on the cells in the bioink, inducing cellular apoptosis. Herein, we investigated whether the incorporation of extracellular vesicles (EVs) in the bioink, engineered to continually deliver the cell survival factor miR-199a-3p would increase the viability within the CP. EVs from THP-1-derived activated macrophages (MΦ) were isolated and characterized by nanoparticle tracking analysis (NTA), cryogenic electron microscopy (cryo-TEM), and Western blot analysis. MiR-199a-3p mimic was loaded into EVs by electroporation after optimization of applied voltage and pulses. Functionality of the engineered EVs was assessed in neonatal rat cardiomyocyte (NRCM) monolayers using immunostaining for the proliferation markers ki67 and Aurora B kinase. To examine the effect of engineered EVs on 3D-bioprinted CP viability, the EVs were added to the bioink, consisting of alginate-RGD, gelatin, and NRCM. Metabolic activity and expression levels of activated-caspase 3 for apoptosis of the 3D-bioprinted CP were evaluated after 5 days. Electroporation (850 V with 5 pulses) was found to be optimal for miR loading; miR-199a-3p levels in EVs increased fivefold compared to simple incubation, with a loading efficiency of 21.0%. EV size and integrity were maintained under these conditions. Cellular uptake of engineered EVs by NRCM was validated, as 58% of cTnT+ cells internalized EVs after 24 h. The engineered EVs induced CM proliferation, increasing the ratio of cell-cycle re-entry of cTnT+ cells by 30% (Ki67) and midbodies+ cell ratio by twofold (Aurora B) compared with the controls. The inclusion of engineered EVs in bioink yielded CP with threefold greater cell viability compared to bioink with no EVs. The prolonged effect of EVs was evident as the CP exhibited elevated metabolic activities after 5 days, with less apoptotic cells compared to CP with no EVs. The addition of miR-199a-3p-loaded EVs to the bioink improved the viability of 3D-printed CP and is expected to contribute to their integration in vivo.

2.
Gels ; 8(12)2022 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-36547293

RESUMO

Cardiac tissue engineering has emerged as a promising strategy to treat infarcted cardiac tissues by replacing the injured region with an ex vivo fabricated functional cardiac patch. Nevertheless, integration of the transplanted patch with the host tissue is still a burden, limiting its clinical application. Here, a bi-functional, 3D bio-printed cardiac patch (CP) design is proposed, composed of a cell-laden compartment at its core and an extracellular vesicle (EV)-laden compartment at its shell for better integration of the CP with the host tissue. Alginate-based bioink solutions were developed for each compartment and characterized rheologically, examined for printability and their effect on residing cells or EVs. The resulting 3D bio-printed CP was examined for its mechanical stiffness, showing an elastic modulus between 4-5 kPa at day 1 post-printing, suitable for transplantation. Affinity binding of EVs to alginate sulfate (AlgS) was validated, exhibiting dissociation constant values similar to those of EVs with heparin. The incorporation of AlgS-EVs complexes within the shell bioink sustained EV release from the CP, with 88% cumulative release compared with 92% without AlgS by day 4. AlgS also prolonged the release profile by an additional 2 days, lasting 11 days overall. This CP design comprises great potential at promoting more efficient patch assimilation with the host.

3.
J Control Release ; 232: 215-27, 2016 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-27117458

RESUMO

Therapeutic implementation of RNA interference (RNAi) through delivery of short interfering RNA (siRNA) is still facing several critical hurdles, which mostly can be solved through the use of an efficient delivery system. We hereby introduce anionic siRNA nanoparticles (NPs) co-assembled by the electrostatic interactions of the semi-synthetic polysaccharide hyaluronan-sulfate (HAS), with siRNA, mediated by calcium ion bridges. The NPs have an average size of 130nm and a mild (-10mV) negative surface charge. Transmission electron microscopy (TEM) using gold-labeled components and X-ray photoelectron spectroscopy (XPS) demonstrated the spatial organization of siRNA molecules in the particle core, surrounded by a layer of HAS. The anionic NPs efficiently encapsulated siRNA, were stable in physiological-relevant environments and were cytocompatible, not affecting cell viability or homeostasis. Efficient cellular uptake of the anionic siRNA NPs, associated with potent gene silencing (>80%), was observed across multiple cell types, including murine primary peritoneal macrophages and human hepatocellular carcinoma cells. In a clinically-relevant model of acute inflammatory response in IL-6-stimulated human hepatocytes, STAT3 silencing induced by HAS-Ca(2+)-siRNA NPs resulted in marked decrease in the total and activated STAT3 protein levels, as well as in the expression levels of downstream acute phase response genes. Collectively, anionic NPs prove to be an efficient and cytocompatible delivery system for siRNA.


Assuntos
Cálcio/metabolismo , Ácido Hialurônico/administração & dosagem , Nanopartículas/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Células Cultivadas , Fibroblastos/metabolismo , Inativação Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hepatócitos/metabolismo , Humanos , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Fator de Transcrição STAT3/genética
4.
J Control Release ; 203: 150-60, 2015 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-25702963

RESUMO

Gene silencing using small interfering RNA (siRNA) relies on the critical need for a safe and effective carrier, capable of strong but reversible complexation, siRNA protection, cellular uptake, and cytoplasmatic unloading of its cargo. We hypothesized that a delivery platform based on the eletrostatic interactions of siRNA with calcium ions in solution would fulfill these needs, ultimately leading to effective gene silencing. Physical characterization of the calcium-siRNA complexes, using high resolution microscopy and dynamic light scattering (DLS), showed the formation of stable nanosized complexes ~80nm in diameter, bearing mild (~-7mV) negative surface charge. The complexes were extremely stable in the presence of serum proteins or high concentrations of heparin; they maintained their nanosized features in suspension for days; and effectively protected the siRNA from enzymatic degradation. The Ca-siRNA complexes were disintegrated in the presence of Ca-chelating ion exchange resin, thus proving their reversibility. Excellent cytocompatibility of calcium-siRNA complexes was achieved using physiological calcium ion concentrations. The calcium-siRNA complexes successfully induced a very high (~80%) level of gene silencing in several cell types, at both mRNA and protein levels, associated with efficient cellular uptake. Collectively, our results show that the developed delivery platform based on reversible calcium-siRNA interactions offers a simple and versatile method for enhancing the therapeutic efficiency of siRNA.


Assuntos
Cálcio/metabolismo , Complexos de Coordenação/metabolismo , Portadores de Fármacos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Transfecção/métodos , Animais , Cálcio/química , Quelantes de Cálcio/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Complexos de Coordenação/química , Portadores de Fármacos/química , Camundongos Endogâmicos BALB C , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
5.
J Tissue Eng Regen Med ; 9(8): 918-29, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23956230

RESUMO

Biomaterials capable of controlling the release of multiple growth factors (GFs) could potentially promote the integration of co-transplanted neural progenitor cells (NPCs) and stimulate the plasticity and regenerability of the lesioned spinal cord. As a first step towards the employment of such a vehicle for cell therapy, this study examined the capability of an alginate-sulphate/alginate scaffold, able to capture and rigorously control the release of GFs, to promote the expansion and lineage differentiation of NPCs in vitro. Epidermal growth factor (EGF) and fibroblast growth factor-2 (bFGF) were affinity-bound to alginate-sulphate (200 ng/scaffold) and the bioconjugates were mixed with partially calcium-crosslinked alginate. NPCs isolated from 18 day-old rat embryo brains and seeded into the scaffold during preparation were found to proliferate and differentiate within the vehicle. A continuous release of both bFGF and EGF was noted for a period of 21 days. The concentrations of released GFs were sufficient to promote extensive NPC proliferation at initial cultivation times; the number of neurospheres in the scaffold was twice the number found in the 2D cultures supplemented with 20 ng/ml each factor every 3 days. Between days 10-14, when the GF concentrations had substantially declined, extensive cell migration from the neurospheres as well as lineage differentiation were noted in the scaffold; immunocytochemical analyses confirmed the presence of neurons, astrocytes and oligodendrocytes.The scaffold has a potential to serve as cell delivery vehicle, with proven capability to promote cell retention and expansion, while enabling NPC lineage differentiation in situ.


Assuntos
Alginatos/química , Células-Tronco Neurais/citologia , Neurônios/citologia , Alicerces Teciduais , Animais , Astrócitos/citologia , Encéfalo/embriologia , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Movimento Celular , Proliferação de Células , Fator de Crescimento Epidérmico/metabolismo , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Imuno-Histoquímica , Neurônios/metabolismo , Oligodendroglia/citologia , Ratos , Ratos Wistar , Sulfatos/química
6.
Artigo em Inglês | MEDLINE | ID: mdl-25220432

RESUMO

Polysaccharides have emerged as important functional materials because of their unique properties such as biocompatibility, biodegradability, and availability of reactive sites for chemical modifications to optimize their properties. The overwhelming majority of the methods to modify polysaccharides employ random chemical modifications, which often improve certain properties while compromising others. On the other hand, the employed methods for selective modifications often require excess of coupling partners, long reaction times and are limited in their scope and wide applicability. To circumvent these drawbacks, aniline-catalyzed oxime formation is developed for selective modification of a variety of polysaccharides through their reducing end. Notably, it is found that for efficient oxime formation, different conditions are required depending on the composition of the specific polysaccharide. It is also shown how our strategy can be applied to improve the physical and functional properties of alginate hydrogels, which are widely used in tissue engineering and regenerative medicine applications. While the randomly and selectively modified alginate exhibits similar viscoelastic properties, the latter forms significantly more stable hydrogel and superior cell adhesive and functional properties. Our results show that the developed conjugation reaction is robust and should open new opportunities for preparing polysaccharide-based functional materials with unique properties.

7.
Acta Biomater ; 10(7): 3209-16, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24607858

RESUMO

Extended partial hepatectomy may be needed in cases of large hepatic mass, and can lead to fulminant hepatic failure. Macroporous alginate scaffold is a biocompatible matrix which promotes the growth, differentiation and long-term hepatocellular function of primary hepatocytes in vitro. Our aim was to explore the ability of implanted macroporous alginate scaffolds to protect liver remnants from acute hepatic failure after extended partial hepatectomy. An 87% partial hepatectomy (PH) was performed on C57BL/6 mice to compare non-treated mice to mice in which alginate or collagen scaffolds were implanted after PH. Mice were scarified 3, 6, 24 and 48 h and 6 days following scaffold implantation and the extent of liver injury and repair was examined. Alginate scaffolds significantly increased animal survival to 60% vs. 10% in non-treated and collagen-treated mice (log rank=0.001). Mice with implanted alginate scaffolds manifested normal and prolonged aspartate aminotransferases and alanine aminotransferases serum levels as compared with the 2- to 20-fold increase in control groups (P<0.0001) accompanied with improved liver histology. Sustained normal serum albumin levels were observed in alginate-scaffold-treated mice 48 h after hepatectomy. Incorporation of BrdU-positive cells was 30% higher in the alginate-scaffold-treated group, compared with non-treated mice. Serum IL-6 levels were significantly decreased 3h post PH. Biotin-alginate scaffolds were quickly well integrated within the liver tissue. Collectively, implanted alginate scaffolds support liver remnants after extended partial hepatectomy, thus eliminating liver injury and leading to enhanced animal survival after extended partial hepatectomy.


Assuntos
Alginatos , Bandagens , Morte Celular , Hepatectomia , Fígado/patologia , Alicerces Teciduais , Animais , Bromodesoxiuridina/metabolismo , Ácido Glucurônico , Ácidos Hexurônicos , Interleucina-6/sangue , Fígado/metabolismo , Fígado/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
8.
Tissue Eng Part C Methods ; 20(11): 886-94, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24568665

RESUMO

The therapeutic application of autologous cardiac-derived progenitor cells (CPCs) requires a large cell quantity generated under defined conditions. Herein, we investigated the applicability of a three-dimensional (3D) perfusion cultivation system to facilitate the expansion of CPCs harvested from human heart biopsies and characterized by a relatively high percentage of c-kit(+) cells. The cells were seeded in macroporous alginate scaffolds and after cultivation for 7 days under static conditions, some of the constructs were transferred into a perfusion bioreactor, which was operated for an additional 14 days. A robust and highly reproducible human CPC (hCPC) expansion of more than seven-fold was achieved under the 3D perfusion culture conditions, while under static conditions, the expansion of CPCs was limited only to the first 7 days, after which it leveled-off. On day 21 of perfusion cultivation, the expanded cells exhibited a higher expression level of the progenitor marker c-kit, suggesting that the c-kit-positive CPCs are the main cell population undergoing proliferation. The profile of the spontaneous differentiation in the perfused construct was different from that in the static cultivated constructs; genes typical for cardiac and endothelial cell lineages were more widely expressed in the perfused constructs. By contrast, the differentiation to osteogenic (Von Kossa staining and alkaline phosphatase activity) and adipogenic (Oil Red staining) lineages was reduced in the perfused constructs compared with static cultivated constructs. Collectively, our results indicate that 3D perfusion cultivation mode is an appropriate system for robust expansion of human CPCs while maintaining their progenitor state and differentiation potential into the cardiovascular cell lineages.


Assuntos
Técnicas de Cultura Celular por Lotes/instrumentação , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Perfusão/instrumentação , Células-Tronco/citologia , Células-Tronco/fisiologia , Engenharia Tecidual/instrumentação , Reatores Biológicos , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos
9.
Biomaterials ; 32(7): 1838-47, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21112626

RESUMO

Cardiac tissue engineering aims to repair damaged myocardial tissues by applying heart patches created in vitro. Herein, we explored the possible role of a combination of two matrix-attached peptides, the adhesion peptide G(4)RGDY and heparin-binding peptide G(4)SPPRRARVTY (HBP) in cardiac tissue regeneration. Neonatal rat cardiac cells were seeded into unmodified, single peptide or double peptide-attached alginate scaffolds, all having the same physical features of porosity, hydrogel forming and matrix stiffness. The cardiac tissue developed in the HBP/RGD-attached scaffolds revealed the best features of a functional muscle tissue, as judged by all studied parameters, i.e., immunostaining of cardiac cell markers, histology, western blot of protein expressions and metabolic activity. By day 7, well-developed myocardial fibers were observed in these cell constructs. At 14 days the HBP/RGD-attached constructs presented an isotropic myofiber arrangement, while no such arrangement was seen in the other constructs. The expression levels of α-actinin, N-cadherin and Connexin-43, showing preservation and an increase in Connexin-43 expression (Cx-43) with time, further supported the formation a contractile muscle tissue in the HBP/RGD-attached scaffolds. Collectively, the attachment of combinatorial peptides representing different signaling in ECM-cell interactions proved to play a key role, contributing to the formation of a functional cardiac muscle tissue, in vitro.


Assuntos
Alginatos/química , Miocárdio/citologia , Peptídeos/química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Western Blotting , Adesão Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Microscopia Confocal , Microscopia Eletrônica de Varredura , Miocárdio/metabolismo , Oligopeptídeos/metabolismo , Ratos , Sindecanas/metabolismo , Vinculina/metabolismo
10.
Biochemistry ; 48(36): 8731-7, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19653643

RESUMO

The large, catalytic subunits (LSUs; ilvB, ilvG and ilvI, respectively) of enterobacterial acetohydroxyacid synthases isozymes (AHAS I, II and III) have molecular weights approximately 60 kDa and are paralogous with a family of other thiamin diphosphate dependent enzymes. The small, regulatory subunits (SSUs) of AHAS I and AHAS III (ilvN and ilvH) are required for valine inhibition, but ilvN and ilvH can only confer valine sensitivity on their own LSUs. AHAS II is valine resistant. The LSUs have only approximately 15, <<1 and approximately 3%, respectively, of the activity of their respective holoenzymes, but the holoenzymes can be reconstituted with complete recovery of activity. We have examined the activation of each of the LSUs by SSUs from different isozymes and ask to what extent such activation is specific; that is, is effective nonspecific interaction possible between LSUs and SSUs of different isozymes? To our surprise, the AHAS II SSU ilvM is able to activate the LSUs of all three of the isozymes, and the truncated AHAS III SSUs ilvH-Delta80, ilvH-Delta86 and ilvH-Delta89 are able to activate the LSUs of both AHAS I and AHAS III. However, none of the heterologously activated enzymes have any feedback sensitivity. Our results imply the existence of a common region in all three LSUs to which regulatory subunits may bind, as well as a similarity between the surfaces of ilvM and the other SSUs. This surface must be included within the N-terminal betaalphabetabetaalphabeta-domain of the SSUs, probably on the helical face of this domain. We suggest hypotheses for the mechanism of valine inhibition, and reject one involving induced dissociation of subunits.


Assuntos
Acetolactato Sintase/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Cetol-Ácido Redutoisomerase/metabolismo , Subunidades Proteicas/metabolismo , Acetolactato Sintase/química , Acetolactato Sintase/genética , Acetolactato Sintase/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Domínio Catalítico/genética , Escherichia coli/genética , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cetol-Ácido Redutoisomerase/antagonistas & inibidores , Cetol-Ácido Redutoisomerase/química , Cetol-Ácido Redutoisomerase/genética , Peso Molecular , Subunidades Proteicas/química , Subunidades Proteicas/genética , Deleção de Sequência/genética , Valina/química , Valina/fisiologia
11.
Biochim Biophys Acta ; 1764(5): 955-960, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16795146

RESUMO

Acetohydroxyacid synthase I (AHAS I), one of three isozymes in Escherichia coli catalyzing the first common step in the biosynthesis of branched amino acids, is composed of two kinds of subunits. The large catalytic (B) and small regulatory (N) subunits of the holoenzyme dissociate and associate freely and rapidly and are quite different in size, charge and hydrophobicity, so that high resolution purification methods lead to partial separation of subunits and to heterogeneity. We have prepared several linked AHAS I proteins, in which the large subunit B with a hexahistidine-tag at the N-terminus, was covalently joined by a flexible linker, containing several (X) amino acids, to the small subunit N to form His6-BuXN polypeptides. All linked BuXN polypeptides have similar specific activity, sensitivity to valine and substrate specificity as the wild type holoenzyme. The most successful BuXN linked protein (Bu30N-r) was inserted into and expressed in yeast and its catalytic properties were tested.


Assuntos
Acetolactato Sintase/química , Domínio Catalítico , Engenharia de Proteínas , Subunidades Proteicas/química , Acetolactato Sintase/genética , Sequência de Aminoácidos , Sequência de Bases , Domínio Catalítico/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
12.
Biochim Biophys Acta ; 1760(3): 356-63, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16326011

RESUMO

AHAS I is an isozyme of acetohydroxyacid synthase which is apparently unique to enterobacteria. It has been known for over 20 years that it has many properties which are quite different from those of the other two enterobacterial AHASs isozymes, as well as from those of "typical" AHASs which are single enzymes in a given organism. These include a unique mechanism for regulation of expression and the absence of a preference for forming acetohydroxybutyrate. We have cloned the two subunits, ilvB and ilvN, of this Escherichia coli isoenzyme and examined the enzymatic properties of the purified holoenzyme and the enzyme reconstituted from purified subunits. Unlike other AHASs, AHAS I demonstrates cooperative feedback inhibition by valine, and the kinetics fit closely to an exclusive binding model. The formation of acetolactate by AHAS I is readily reversible and acetolactate can act as substrate for alternative AHAS I-catalyzed reactions.


Assuntos
Acetolactato Sintase/metabolismo , Proteínas de Escherichia coli/metabolismo , Acetolactato Sintase/biossíntese , Acetolactato Sintase/genética , Acetona/análogos & derivados , Acetona/metabolismo , Clonagem Molecular , Escherichia coli/enzimologia , Retroalimentação Fisiológica , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , Isomerismo , Cinética , Valina/farmacologia
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