RESUMO
3D scaffolds used to repair damaged tissues should be able to mimic both composition and functions of natural extracellular matrix, which is mainly composed of polysaccharides and proteins. In our previous research new biomimetic sponges, based on blends of alginate with gelatin, were produced and characterized for myocardial tissue engineering applications. It was observed that these scaffolds can potentially function as a promising cardiac extracellular matrix substitute, but a reinforcement is required to improve their suturing properties. Aim of the present work was the development of a suturable biomimetic patch by the inclusion of a synthetic mesh within an alginate/gelatin scaffold. The mesh, produced by dry spinning, was made of eight superimposed layers of polycaprolactone microfibers, each one rotated of 45° with respect to the adjacent one. Reinforced scaffolds were obtained through the use of a mold, specially designed to place the fibrous mesh exactly in the center of the sponge. Both the reinforcement mesh and the reinforced scaffold were characterized. A perfect integration between the mesh and the sponge was observed. The fibrous mesh reduced the capacity of the sponge to absorb water, but the degree of hydrophilicity of the material was still comparable with that of natural cardiac tissue. The reinforced system showed a suitable stability in aqueous environment and it resulted much more resistant to suturing than not reinforced scaffold and even than human arteries. Polycaprolactone mesh was not cytotoxic and the reinforced scaffold was able to support cardiomyocytes adhesion and proliferation. Overall, the obtained results confirmed that the choice to modify the alginate/gelatin sponges through the insertion of an appropriate reinforcement system turned out to be correct in view of their potential use in myocardial tissue engineering.
Assuntos
Alginatos/química , Materiais Biomiméticos/química , Gelatina/química , Alicerces Teciduais , Animais , Adesão Celular , Linhagem Celular , Sobrevivência Celular , Colorimetria , Humanos , Camundongos , Ratos , Engenharia Tecidual/métodosRESUMO
The present work aimed at the production and characterization of small caliber biomimetic and bioactive tubular scaffolds, which are able to favor the endothelialization process, and therefore potentially be suitable for vascular tissue engineering. The tubular scaffolds were produced using a specially designed mold, starting from a gelatin/gellan/elastin (GGE) blend, selected to mimic the composition of the extracellular matrix of native blood vessels. GGE scaffolds were obtained through freeze-drying and subsequent cross-linking. To obtain systems capable of promoting endothelization, the scaffolds were functionalized using two different bioactive peptides, Gly-Arg-Gly-Asp-Ser-Pro (GRGSDP) and Arg-Glu-Asp-Val (REDV). A complete physicochemical, mechanical, functional, and biological characterization of the developed scaffolds was performed. GGE scaffolds showed a good porosity, which could promote cell infiltration and proliferation and a dense external surface, which could avoid bleeding. Moreover, developed scaffolds showed good hydrophilicity, an elastic behavior similar to natural vessels, suitability for sterilization by an ISO accepted treatment, and an adequate suture retention strength. In vitro cell culture tests showed no cytotoxic activity against 3T3 fibroblasts. The functionalization with the REDV peptide favored the adhesion and growth of endothelial cells, while GRGDSP-modified scaffolds represented a better substrate for fibroblasts.
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The use of injectable scaffolds to repair the infarcted heart is receiving great interest. Thermosensitive polymers, in situ polymerization, in situ cross-linking, and self-assembling peptides are the most investigated approaches to obtain injectability.Aim of the present work was the preparation and characterization of a novel bioactive scaffold, in form of injectable microspheres, for cardiac repair. Gellan/gelatin microspheres were prepared by a water-in-oil emulsion and loaded by adsorption with Insulin-like growth factor 1 to promote tissue regeneration. Obtained microspheres underwent morphological, physicochemical and biological characterization, including cell culture tests in static and dynamic conditions and in vivo tests. Morphological analysis of the microspheres showed a spherical shape, a microporous surface and an average diameter of 66 ± 17µm (under dry conditions) and 123 ± 24 µm (under wet conditions). Chemical Imaging analysis pointed out a homogeneous distribution of gellan, gelatin and Insulin-like growth factor-1 within the microsphere matrix. In vitro cell culture tests showed that the microspheres promoted rat cardiac progenitor cells adhesion, and cluster formation. After dynamic suspension culture within an impeller-free bioreactor, cells still adhered to microspheres, spreading their cytoplasm over microsphere surface. Intramyocardial administration of microspheres in a cryoinjury rat model attenuated chamber dilatation, myocardial damage and fibrosis and improved cell homing.Overall, the findings of this study confirm that the produced microspheres display morphological, physicochemical, functional and biological properties potentially adequate for future applications as injectable scaffold for cardiac tissue engineering.
Assuntos
Coração/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/administração & dosagem , Microesferas , Miocárdio/patologia , Alicerces Teciduais , Animais , Materiais Biocompatíveis , Reatores Biológicos , Adesão Celular , Meios de Cultura , Injeções , Insulina/metabolismo , Cinética , Masculino , Microfluídica , Microscopia Eletrônica de Varredura , Infarto do Miocárdio/terapia , Polímeros/química , Ratos , Ratos Wistar , Regeneração , Células-Tronco/citologia , Engenharia Tecidual/métodosRESUMO
Tissue engineering scaffolds should be able to reproduce optimal microenvironments in order to support cell attachment, three-dimensional growth, migration and, regarding fibroblasts, must also promote extracellular matrix production. Various bioactive molecules are employed in the preparation of spongy scaffolds to obtain biomimetic matrices by either surface-coating or introducing them into the bulk composition of the biomaterial. The biomimetic properties of a spongy matrix composed of PVA combined with the natural component gelatine were evaluated by culturing human gingival fibroblasts on the scaffold. Cell adhesion, morphology and distribution within the scaffold were assessed by histology and electron microscopy; viability and metabolic activity as well as extracellular matrix production were analyzed by MTT assay, cytochemistry and immunocytochemistry. Fibroblasts interacted positively with PVA/gelatine. They adhered to the PVA/gelatine matrix in which they had good spreading activity and active metabolism; fibroblasts were also able to produce extracellular matrix molecules (type I collagen, fibronectin and laminin) compared to bi-dimensionally grown cells. The in situ creation of a biological matrix by human fibroblasts together with the ability to produce growth factor TGF-beta1 and the intracellular signal transduction molecule RhoA, suggests that this kind of PVA/gelatine sponge may represent a suitable support for in vitro extracellular matrix production and connective tissue regeneration.
Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/fisiologia , Gelatina , Gengiva/citologia , Álcool de Polivinil , Engenharia Tecidual/métodos , Adesão Celular , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/ultraestrutura , Humanos , Imuno-Histoquímica , Microscopia Eletrônica/métodos , Fator de Crescimento Transformador beta1/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
In this paper a device, based on urease-loaded microspheres, is presented. The first task of this work was the optimization of a procedure for the alginate microspheres realization, having a radius as close as possible to the optimal one necessary to achieve the maximum enzyme exploitation. This optimal radius was calculated theoretically through a mathematical model which describes the concentration of substrate (urea) inside the microspheres on the assumption of a diffusion-reaction mechanism. The enzyme-loaded microspheres were successfully tested in a prototypal device aimed at the depletion of urea from a circulating fluid simulating blood flow: the results showed that urea concentration in the circulating fluid drops down to less than 25% of the initial value after 5 h.
Assuntos
Alginatos/química , Portadores de Fármacos/química , Microesferas , Ureia/metabolismo , Urease/uso terapêutico , Alginatos/uso terapêutico , Enzimas Imobilizadas/administração & dosagem , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/uso terapêutico , Ácido Glucurônico/química , Ácido Glucurônico/uso terapêutico , Ácidos Hexurônicos/química , Ácidos Hexurônicos/uso terapêutico , Humanos , Nefropatias/terapia , Tamanho da Partícula , Ureia/sangue , Urease/administração & dosagem , Urease/metabolismoRESUMO
Tissue engineering has emerged as a viable approach to treat disease or repair damage in tissues and organs. One of the key elements for the success of tissue engineering is the use of a scaffold serving as artificial extracellular matrix (ECM). The ECM hosts the cells and improves their survival, proliferation, and differentiation, enabling the formation of new tissue. Here, we propose the development of a class of protein/polysaccharide-based porous scaffolds for use as ECM substitutes in cardiac tissue engineering. Scaffolds based on blends of a protein component, collagen or gelatin, with a polysaccharide component, alginate, were produced by freeze-drying and subsequent ionic and chemical crosslinking. Their morphological, physicochemical, and mechanical properties were determined and compared with those of natural porcine myocardium. We demonstrated that our scaffolds possessed highly porous and interconnected structures, and the chemical homogeneity of the natural ECM was well reproduced in both types of scaffolds. Furthermore, the alginate/gelatin (AG) scaffolds better mimicked the native tissue in terms of interactions between components and protein secondary structure, and in terms of swelling behavior. The AG scaffolds also showed superior mechanical properties for the desired application and supported better adhesion, growth, and differentiation of myoblasts under static conditions. The AG scaffolds were subsequently used for culturing neonatal rat cardiomyocytes, where high viability of the resulting cardiac constructs was observed under dynamic flow culture in a microfluidic bioreactor. We therefore propose our protein/polysaccharide scaffolds as a viable ECM substitute for applications in cardiac tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 769-781, 2018.
Assuntos
Materiais Biomiméticos/química , Matriz Extracelular/metabolismo , Coração/fisiologia , Polissacarídeos/química , Proteínas/química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Reatores Biológicos , Bovinos , Linhagem Celular , Proliferação de Células , Forma Celular , Módulo de Elasticidade , Hidrólise , Cinética , Microfluídica , Mioblastos/citologia , Ratos , Espectroscopia de Infravermelho com Transformada de Fourier , SuínosRESUMO
INTRODUCTION: Injectable scaffolds are emerging as a promising strategy in the field of myocardial tissue engineering. Among injectable scaffolds, microparticles have been poorly investigated. The goal of this study was the development of novel gelatin/gellan microparticles that could be used as an injectable scaffold to repair the infarcted myocardium. In particular, the effect of particle size on cardiac progenitor cell response was investigated. METHODS: Particles were produced by a water-in-oil emulsion method. Phosphatidylcholine was used as a surfactant. Particles with different diameter ranges (125-300 µm and 350-450 µm) were fabricated using two different surfactant concentrations. Morphological, physicochemical, and functional characterizations were carried out. Cardiac progenitor cell adhesion and growth on microparticles were tested both in static and dynamic suspension culture conditions. RESULTS: Morphological analysis of the produced particles showed a spherical shape and porous surface. The hydrophilicity of particle matrix and the presence of intermolecular interactions between gellan and gelatin were pointed out by the physicochemical characterization. A weight loss of 75 ± 5 % after 90 days of hydrolytic degradation was observed. Injectability through a narrow needle (26 G) and persistence of the microparticles at the injection site were preliminarily verified by ex vivo test. In vitro cell culture tests showed a preservation of rat cardiac progenitor biologic properties and indicated a preferential cell adherence to microparticles with a smaller size. CONCLUSION: Overall, the obtained results indicate that the produced gelatin/gellan microparticles could be potentially employed as injectable scaffolds for myocardial regeneration.
Assuntos
Microesferas , Miocárdio/citologia , Miócitos Cardíacos/citologia , Células-Tronco/citologia , Animais , Materiais Biocompatíveis , Adesão Celular , Proliferação de Células , Células Cultivadas , Emulsões , Gelatina/química , Miócitos Cardíacos/fisiologia , Tamanho da Partícula , Polissacarídeos Bacterianos/química , Porosidade , Ratos , Células-Tronco/fisiologia , Engenharia Tecidual/métodos , Alicerces TeciduaisAssuntos
Alginatos/química , Avidina/química , Materiais Biomiméticos/química , Biotina/química , Gelatina/química , Poríferos/química , Alicerces Teciduais/química , Animais , Materiais Biomiméticos/metabolismo , Adesão Celular , Proliferação de Células , Elasticidade , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Miocárdio/citologia , Polidioxanona/química , Porosidade , Implantação de Prótese , Ratos , Somatomedinas/química , Somatomedinas/metabolismo , Células-Tronco , Propriedades de Superfície , Engenharia Tecidual , ViscosidadeRESUMO
Scaffolds for cardiac patch application must meet stringent requirements such as biocompatibility, biodegradability, and facilitate vascularization in the engineered tissue. Here, a bioactive, biocompatible, and biodegradable electrospun scaffold of poly(glycerol sebacate)-poly(ε-caprolactone) (PGS-PCL) is proposed as a potential scaffold for cardiac patch application. The fibers are smooth bead free with average diameter = 0.8 ± 0.3 µm, mean pore size = 2.2 ± 1.2 µm, porosity = 62 ± 4%, and permeability higher than that of control biological tissue. For the first time, bioactive PGS-PCL fibers functionalized with vascular endothelial growth factor (VEGF) are developed, the approach used being chemical modification of the PGS-PCL fibers followed by subsequent binding of VEGF via amide bonding. The approach results in uniform immobilization of VEGF on the fibers; the concentrations are 1.0 µg cm(-2) for the PGS-PCL (H) and 0.60 µg cm(-2) for the PGS-PCL (L) samples. The bioactive scaffold supports the attachment and growth of seeded myogenic and vasculogenic cell lines. In fact, rat aortic endothelial cells also display angiogenic features indicating potential for the formation of vascular tree in the scaffold. These results therefore demonstrate the prospects of VEGF-functionalized PGS-PCL fibrous scaffold as promising matrix for cardiac patch application.
Assuntos
Materiais Biocompatíveis/química , Polímeros/química , Alicerces Teciduais , Animais , Materiais Biocompatíveis/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Decanoatos/química , Módulo de Elasticidade , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Glicerol/análogos & derivados , Glicerol/química , Proteínas Imobilizadas/química , Proteínas Imobilizadas/farmacologia , Miocárdio/citologia , Permeabilidade , Poliésteres/química , Polímeros/farmacologia , Porosidade , Ratos , Células-Tronco/citologia , Células-Tronco/metabolismo , Resistência à Tração , Engenharia Tecidual , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Mesenchymal stem cells (MSCs) represent a promising source of progenitor cells having the potential to repair and to regenerate diseased or damaged skeletal tissues. Bone marrow (BM) has been the first source reported to contain MSCs. However, BM-derived cells are not always acceptable, due to the highly invasive drawing and the decline in MSC number and differentiative capability with increasing age. Human umbilical cord blood (UCB), obtainable by donation with a noninvasive method, has been introduced as an alternative source of MSCs. Here human UCB-derived MSCs isolation and morpho-functional characterization are reported. Human UCB-derived mononuclear cells, obtained by negative immunoselection, exhibited either an osteoclast-like or a mesenchymal-like phenotype. However, we were able to obtain homogeneous populations of MSCs that displayed a fibroblast-like morphology, expressed mesenchym-related antigens and showed differentiative capacities along osteoblastic and early chondroblastic lineages. Furthermore, this study is one among a few papers investigating human UCB-derived MSC growth and differentiation on three-dimensional scaffolds focusing on their potential applications in regenerative medicine and tissue engineering. UCB-derived MSCs were proved to grow on biodegradable microfiber meshes; additionally, they were able to differentiate toward mature osteoblasts when cultured inside human plasma clots, suggesting their potential application in orthopedic surgery.
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Forma Celular , Sangue Fetal/citologia , Células-Tronco Mesenquimais/citologia , Medicina Regenerativa , Adipogenia , Biomarcadores , Proliferação de Células , Separação Celular , Células Cultivadas , Condrócitos/citologia , Condrogênese , Citometria de Fluxo , Humanos , Imunofenotipagem , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Osteoblastos/citologia , OsteogêneseRESUMO
Tissue engineering has the potential to supply constructs capable of restoring the normal function of native tissue following injury. Poly(L-lactic acid) (PLLA) scaffolds are amongst the most commonly used biodegradable polymers in tissue engineering and previous studies performed on ovine fibroblasts have showed that addition of gelatin creates a favorable hydrophilic microenvironment for the growth of these cells. The attractiveness of using mesenchymal stromal cells (MSCs) in tissue regeneration is that they are able to differentiate into several lines including osteoblasts. In this study, we investigated the ability of gelatin/PLLA sponges to support the adhesion, proliferation, and osteogenic differentiation of human MSCs isolated from the bone marrow of four donors. [Figure: see text].
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Gelatina , Ácido Láctico , Células-Tronco Mesenquimais/citologia , Osteogênese , Polímeros , Engenharia Tecidual/métodos , Diferenciação Celular , Proliferação de Células , Humanos , Poliésteres , Células Estromais/citologiaRESUMO
Biodegradable synthetic polymers such as poly(lactic acid) (PLA) are widely used to prepare scaffolds for cell transplantation and tissue growth, using different techniques set up for the purpose. However the poor hydrophilicity of these polymers represents the main limitation to their use as scaffolds because it causes a low affinity for the cells. An effective way to solve this problem could be represented by the addition of biopolymers that are in general highly hydrophilic. The present work concerns porous biodegradable sponge-like systems based on poly(L-lactic acid) (PLLA) and gelatine. Morphology and porosity characteristics of the sponges were studied by scanning electron microscopy and mercury intrusion porosimetry respectively. Blood compatibility was investigated by bovine plasma fibrinogen (BPF) adsorption test and platelet adhesion test (PAT). The cell culture method was used in order to evaluate the ability of the matrices to work as scaffolds for tissue regeneration. The obtained results indicate that the sponges have interesting porous characteristics, good blood compatibility and above all good ability to support cell adhesion and growth. In fact viable and metabolically active animal cells were found inside the sponges after 8 weeks in culture. On this basis the systems produced seem to be good candidates as scaffolds for tissue regeneration.
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Implantes Absorvíveis , Materiais Biocompatíveis/química , Fibroblastos/citologia , Fibroblastos/fisiologia , Gelatina/química , Regeneração Tecidual Guiada/métodos , Ácido Láctico/química , Polímeros/química , Animais , Células Cultivadas , Regeneração Tecidual Guiada/instrumentação , Teste de Materiais , Poliésteres , Ovinos , Propriedades de SuperfícieRESUMO
Biodegradable synthetic polymers such as poly(lactic acid) are widely used to prepare scaffolds for cell transplantation and tissue growth, using different techniques set up for the purpose. However the poor hydrophilicity of these polymers represents the main limitation to their use as scaffolds because it causes a low affinity for the cells. An effective way to solve this problem could be represented by the addition of biopolymers that are in general highly hydrophilic. The present work concerns porous biodegradable sponge-like systems based on poly(L-lactic acid) and gelatine. Morphology and porosity characteristics of the sponges were studied by scanning electron microscopy and mercury intrusion porosimetry respectively. Blood compatibility was investigated by bovine plasma fibrinogen adsorption test and platelet adhesion test. The cell culture method was used in order to evaluate the ability of the matrices to work as scaffolds for tissue regeneration. The obtained results indicate that the sponges have interesting porous characteristics, good blood compatibility and above all good ability to support cell adhesion and growth. In fact viable and metabolically active animal cells were found inside the sponges after 8 weeks in culture. On this basis the systems produced seem to be good candidates as scaffolds for tissue regeneration.
Assuntos
Materiais Biocompatíveis/química , Gelatina/química , Ácido Láctico/química , Polímeros/química , Implantes Absorvíveis , Animais , Bovinos , Células Cultivadas , Gelatina/ultraestrutura , Regeneração Tecidual Guiada/instrumentação , Adesividade Plaquetária , Poliésteres , Porosidade , OvinosRESUMO
Poly(vinyl alcohol) (PVA) hydrogels prepared by a freeze-thawing procedure were evaluated as matrices for the release of water-insoluble drugs such as dexamethasone. As it is impossible to directly entrap a lipophilic drug into a hydrophilic matrix, a novel mechanism has been designed based on producing biodegradable nanoparticles loaded with the drug, that could then be entrapped into the hydrogels. Nanoparticles were prepared by a solvent evaporation technique using a biodegradable copolymer of poly(lactic acid)-poly(glycolic acid) (PLGA). The effects of several processing parameters on particle properties were investigated. The drug release from free nanoparticles was compared to that from the nanoparticles entrapped into the PVA matrices. It was observed that the release profile of the drug is not significantly affected by the PVA matrix. A correlation was found between the amount of drug released and the PVA concentration in the hydrogels: the percentage of drug released, as a function of time, decreased by increasing PVA concentration, indicating that PVA concentration can be used as a tool in modulating the release of the drug.
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Biodegradable hydrophilic gelatin nanoparticles, containing different initial amounts of methotrexate (MTX), were prepared using a simple solvent evaporation technique based on a single water-in-oil emulsion and stabilized by the use of glutaraldehyde as cross-linking agent. The effects of several parameters on particle size, drug encapsulation efficiency and drug release were investigated. Size and shape of the nanoparticles were examined by scanning electron microscopy. The release of MTX was monitored in vitro and the mechanism of release was studied. Particles with a mean diameter of 100-200 nm were produced, which were able to release MTX following a diffusion-controlled mechanism of release. It was observed that the initial amount of MTX used for sample loading did not have any effect on the pattern of release, while it affected the amount of drug entrapped into the nanoparticles and also both the release rate and the total amount of drug released.
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Hydrogels based on blends of poly(vinyl alcohol) (PVA) with dextran were prepared by a physical cross-linking procedure and used as matrices for the entrapment of biodegradable nanoparticles loaded with dexamethasone. The nanoparticles were prepared, by a solvent evaporation technique, using biodegradable copolymers of poly(lactic acid)-poly(glycolic acid) (PLGA). Size, morphology and surface characteristics of the nanoparticles were evaluated by scanning electron microscopy. The mechanism of drug release from the nanoparticles entrapped into the PVA-based matrices was studied and compared to that from free nanoparticles. The effect of dextran on the in vitro release profile of dexamethasone from the hydrogels was investigated. The obtained results indicate that PLGA nanoparticles are able to release dexamethasone following a diffusion-controlled mechanism. The entrapment of the nanoparticles into the hydrogels affects only slightly this mechanism of drug release. In addition, dextran/PVA hydrogels release a higher amount of drug with respect to pure PVA hydrogels and by increasing dextran content in the hydrogels, the amount of drug released increases.
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Poly(vinyl alcohol) hydrogels prepared by freeze-thawing procedure represent synthetic systems widely investigated as non-biodegradable scaffolds for tissue regeneration. In order to improve the biocompatibility properties of pure poly(vinyl alcohol) (PVA) hydrogels, blends of PVA with different biological macromolecules, such hyaluronic acid, dextran, and gelatin were prepared and used to produce "bioartificial hydrogels". The porosity characteristics of these hydrogels were investigated by scanning electron microscopy and mercury intrusion porosimetry. The morphology of bioartificial hydrogels was evaluated and compared with that of pure PVA hydrogels. In particular the effect exerted by each biological component on pore size and distribution was investigated. The obtained results indicate that when a natural macromolecule is added to PVA the internal structure of the material changes. A small amount of biopolymer induces the structural elements of PVA matrix to take on a well evident lamellar appearance and an apparent preferential orientation. Comparing the results of SEM and mercury intrusion porosimetry it was concluded that hydrogels containing 20% of biological component have the most regular structure and at the same time the lowest total porosity. On the contrary samples with the highest content of natural polymer (40%) show the less regular structure and the highest total porosity.