RESUMEN
The ability to expand and direct both precursor and stem cells towards a differential fate is considered extremely advantageous in tissue engineering. Platelet-rich plasma (PRP) possesses a milieu of growth factors and cytokines, which have the potential to have either a differentiative or proliferative influence on the cell type tested. Here, we investigated the effect of PRP on C2C12 myoblasts. A range of PRP concentrations in differentiation media was used to determine whether a concentration dependence existed, while PRP embedded in fibres of aligned electrospun polydioxanone and polycaprolactone was used to determine whether this presence of fibres would cause any differences in response. In both cases, it was found that late myogenic markers were suppressed after 7 days in culture. However, an early differentiation marker, MyoD, was upregulated during this same time period. The results from this study represent the ability of PRP to have an influence over both myogenic proliferation and differentiation, a factor which could prove useful in future studies involved with skeletal muscle tissue engineering.
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Diferenciación Celular , Desarrollo de Músculos , Plasma Rico en Plaquetas/metabolismo , Animales , Western Blotting , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Análisis de Fourier , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Desarrollo de Músculos/efectos de los fármacos , Factores Reguladores Miogénicos/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Smad2/metabolismo , Andamios del Tejido/químicaRESUMEN
Spinal cord injury results in tissue necrosis in and around the lesion site, commonly leading to the formation of a fluid-filled cyst. This pathological end point represents a physical gap that impedes axonal regeneration. To overcome the obstacle of the cavity, we have explored the extent to which axonal substrates can be bioengineered through electrospinning, a process that uses an electrical field to produce fine fibres of synthetic or biological molecules. Recently, we demonstrated the potential of electrospinning to generate an aligned matrix that can influence the directionality and growth of axons. Here, we show that this matrix can be supplemented with nerve growth factor and chondroitinase ABC to provide trophic support and neutralize glial-derived inhibitory proteins. Moreover, we show how air-gap electrospinning can be used to generate a cylindrical matrix that matches the shape of the cord. Upon implantation in a completely transected rat spinal cord, matrices supplemented with NGF and chondroitinase ABC promote significant functional recovery. An examination of these matrices post-implantation shows that electrospun aligned monofilaments induce a more robust cellular infiltration than unaligned monofilaments. Further, a vascular network is generated in these matrices, with some endothelial cells using the electrospun fibres as a growth substrate. The presence of axons within these implanted matrices demonstrates that they facilitate axon regeneration following spinal cord injury. Collectively, these results demonstrate the potential of electrospinning to generate an aligned substrate that can provide trophic support, directional guidance cues and regeneration-inhibitory neutralizing compounds to regenerating axons following spinal cord injury. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Axones/metabolismo , Condroitina ABC Liasa , Factor de Crecimiento Nervioso , Traumatismos de la Médula Espinal/terapia , Regeneración de la Medula Espinal/efectos de los fármacos , Andamios del Tejido/química , Animales , Axones/patología , Condroitina ABC Liasa/química , Condroitina ABC Liasa/farmacología , Factor de Crecimiento Nervioso/química , Factor de Crecimiento Nervioso/farmacología , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patologíaRESUMEN
BACKGROUND AND PURPOSE: Current technology for endovascular thrombectomy in ischemic stroke utilizes static loading and is successful in approximately 85% of cases. Existing technology uses either static suction (applied via a continuous pump or syringe) or flow arrest with a proximal balloon. In this paper we evaluate the potential of cyclic loading in aspiration thrombectomy. METHODS: In order to evaluate the efficacy of cyclic aspiration, a model was created using a Penumbra aspiration system, three-way valve and Penumbra 5Max catheter. Synthetic clots were aspirated at different frequencies and using different aspiration mediums. Success or failure of clot removal and time were recorded. All statistical analyses were based on either a one-way or two-way analysis of variance, Holm-Sidak pairwise multiple comparison procedure (α=0.05). RESULTS: Cyclic aspiration outperformed static aspiration in overall clot removal and removal speed (p<0.001). Within cyclic aspiration, Max Hz frequencies (â¼6.3â Hz) cleared clots faster than 1â Hz (p<0.001) and 2â Hz (p=0.024). Loading cycle dynamics (specific pressure waveforms) affected speed and overall clearance (p<0.001). Water as the aspiration medium was more effective at clearing clots than air (p=0.019). CONCLUSIONS: Cyclic aspiration significantly outperformed static aspiration in speed and overall clearance of synthetic clots in our experimental model. Within cyclic aspiration, efficacy is improved by increasing cycle frequency, utilizing specific pressure cycle waveforms and using water rather than air as the aspiration medium. These findings provide a starting point for altering existing thrombectomy technology or perhaps the development of new technologies with higher recanalization rates.
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Isquemia Encefálica/cirugía , Procedimientos Endovasculares/métodos , Accidente Cerebrovascular/diagnóstico por imagen , Succión/métodos , Trombectomía/instrumentación , Trombectomía/métodos , Aire , Humanos , Modelos Neurológicos , Succión/efectos adversos , Ultrasonografía , AguaRESUMEN
We characterize layered, delamination resistant, tissue engineering scaffolds produced by gradient electrospinning using computational fluid dynamics, measurements of fiber diameter with respect to dynamic changes in polymer concentration, SEM analysis, and materials testing. Gradient electrospinning delivers a continuously variable concentration of polymer to the electrospinning jet, resulting in scaffolds that exhibit controlled transitions in fiber diameter across the Z-axis. This makes it possible to produce scaffolds that exhibit very different fiber sizes and material properties on opposing surfaces while eliminating the boundary layers that lead to delamination failures. In materials testing bi-layered laminated electrospun scaffolds (layer 1 = <250 nm, layer 2 = 1000 nm diameter polycaprolactone fibers) exhibit ductile properties and undergo multiphasic failure. In contrast, scaffolds, produced by gradient electrospinning fabricated with fibers of this type on opposing surfaces fracture and fail as unified, and mechanically integrated, structures. Gradient electrospinning also eliminates the anisotropic strain properties observed in scaffolds composed of highly aligned fibers. In burst testing, scaffolds composed of aligned fibers produced using gradient electrospinning exhibit superior material properties with respect to scaffolds composed of random or aligned fibers produced from a single polymer concentration or as bi-layered, laminated structures.
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Poliésteres/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Simulación por Computador , Hidrodinámica , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Estrés Mecánico , Resistencia a la Tracción , Factores de TiempoRESUMEN
The purpose of this study was to create seamless, acellular, small diameter bioresorbable arterial grafts that attempt to mimic the extracellular matrix and mechanical properties of native artery using synthetic and natural polymers. Silk fibroin, collagen, elastin, and polycaprolactone (PCL) were electrospun to create a tri-layered structure for evaluation. Dynamic compliance testing of the electrospun grafts ranged from 0.4-2.5%/100 mmHg, where saphenous vein (1.5%/100 mmHg) falls within this range. Increasing PCL content caused a gradual decrease in medial layer compliance, while changes in PCL, elastin, and silk content in the adventitial layer had varying affects. Mathematical modeling was used to further characterize these results. Burst strength results ranged from 1614-3500 mmHg, where some exceeded the capacity of the pressure regulator. Four week degradation studies demonstrated no significant changes in compliance or burst strength, indicating that these grafts could withstand the initial physiological conditions without risk of degradation. Overall, we were able to manufacture a multi-layered graft that architecturally mimics the native vascular wall and mechanically matches the gold standard of vessel replacement, saphenous vein.
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Materiales Biomiméticos/química , Prótesis Vascular , Colágeno/química , Elastina/química , Poliésteres/química , Seda/química , Andamios del Tejido , Animales , Arterias/fisiología , Fenómenos Biomecánicos , Materiales Biomiméticos/metabolismo , Ensayo de Materiales , Modelos Biológicos , Estrés MecánicoRESUMEN
Electrospun non-woven structures have the potential to form bioresorbable vascular grafts that promote tissue regeneration in situ as they degrade and are replaced by autologous tissue. Current bioresorbable grafts lack appropriate regeneration potential since they do not have optimal architecture, and their fabrication must be altered by the manipulation of process parameters, especially enhancing porosity. We describe here an air-impedance process where the solid mandrel is replaced with a porous mandrel that has pressurized air exiting the pores to impede fiber deposition. The mandrel design, in terms of air-flow rate, pore size, and pore distribution, allows for control over fiber deposition and scaffold porosity, giving greater cell penetration without a detrimental loss of mechanical properties or structural integrity.
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Electroquímica/métodos , Poliésteres/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Línea Celular , Fibroblastos/citología , Humanos , Microscopía Electrónica de Rastreo , PorosidadRESUMEN
Platelet-rich plasma (PRP) therapy has seen a recent spike in clinical interest due to the potential that the highly concentrated platelet solutions hold for stimulating tissue repair and regeneration. The aim of this study was to incorporate PRP into a number of electrospun materials to determine how growth factors are eluted from the structures, and what effect the presence of these factors has on enhancing electrospun scaffold bioactivity. PRP underwent a freeze-thaw-freeze process to lyse platelets, followed by lyophilization to create a powdered preparation rich in growth factors (PRGF), which was subsequently added to the electrospinning process. Release of protein from scaffolds over time was quantified, along with the quantification of human macrophage and adipose-derived stem cell (ADSC) chemotaxis and proliferation. Protein assays demonstrated a sustained release of protein from PRGF-containing scaffolds at up to 35 days in culture. Scaffold bioactivity was enhanced as ADSCs demonstrated increased proliferation in the presence of PRGF, whereas macrophages demonstrated increased chemotaxis to PRGF. In conclusion, the work performed in this study demonstrated that the incorporation of PRGF into electrospun structures has a significant positive influence on the bioactivity of the scaffolds, and may prove beneficial in a number of tissue engineering applications.
Asunto(s)
Plasma Rico en Plaquetas/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Proliferación Celular , Células Cultivadas , Electroquímica/métodos , HumanosRESUMEN
Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery under pulsatile deformations. The goal of this study was to mimic the structure of native artery by fabricating a multi-layered electrospun conduit composed of poly(caprolactone) (PCL) with the addition of elastin and collagen with blends of 45-45-10, 55-35-10, and 65-25-10 PCL-ELAS-COL to demonstrate mechanical properties indicative of native arterial tissue, while remaining conducive to tissue regeneration. Whole grafts and individual layers were analyzed using uniaxial tensile testing, dynamic compliance, suture retention, and burst strength. Compliance results revealed that changes to the middle/medial layer changed overall graft behavior with whole graft compliance values ranging from 0.8 - 2.8 % / 100 mmHg, while uniaxial results demonstrated an average modulus range of 2.0 - 11.8 MPa. Both modulus and compliance data displayed values within the range of native artery. Mathematical modeling was implemented to show how changes in layer stiffness affect the overall circumferential wall stress, and as a design aid to achieve the best mechanical combination of materials. Overall, the results indicated that a graft can be designed to mimic a tri-layered structure by altering layer properties.
Asunto(s)
Materiales Biomiméticos/química , Prótesis Vascular , Colágeno/química , Elastina/química , Técnicas Electroquímicas/métodos , Poliésteres/química , Animales , Bovinos , Modelos BiológicosRESUMEN
We describe the structural and functional properties of three-dimensional (3D) nerve guides fabricated from poly-ε-caprolactone (PCL) using the air gap electrospinning process. This process makes it possible to deposit nano-to-micron diameter fibers into linear bundles that are aligned in parallel with the long axis of a cylindrical construct. By varying starting electrospinning conditions it is possible to modulate scaffold material properties and void space volume. The architecture of these constructs provides thousands of potential channels to direct axon growth. In cell culture functional assays, scaffolds composed of individual PCL fibers ranging from 400 to 1500 nm supported the penetration and growth of axons from rat dorsal root ganglion. To test the efficacy of our guide design we reconstructed 10mm lesions in the rodent sciatic nerve with scaffolds that had fibers 1 µm in average diameter and void volumes >90%. Seven weeks post implantation, microscopic examination of the regenerating tissue revealed dense, parallel arrays of myelinated and non-myelinated axons. Functional blood vessels were scattered throughout the implant. We speculate that end organ targeting might be improved in nerve injuries if axons can be directed to regenerate along specific tissue planes by a guide composed of 3D fiber arrays.
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Aire , Regeneración Tisular Dirigida/métodos , Regeneración Nerviosa/fisiología , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Células Cultivadas , Análisis de Fourier , Implantes Experimentales , Ensayo de Materiales , Nervios Periféricos/fisiología , Nervios Periféricos/ultraestructura , Ratas , SolucionesRESUMEN
Engineered scaffolds function to supplement or replace injured, missing, or compromised tissue or organs. The current direction in this research area is to create scaffolds that mimic the structure and function of the native extracellular matrix (ECM). It is believed that the fabrication of a scaffold that has both structural integrity and allows for normal cellular function and interaction will bring scaffolds closer to clinical relevance. Nanotechnology innovations have aided in the development of techniques for the production of nanofiber scaffolds. The three major processing techniques, self-assembly, phase separation, and electrospinning, produce fibers that rival the size of those found in the native ECM. However, the simplicity, versatility, and scalability of electrospinning make it an attractive processing method that can be used to reproduce aspects of the complexity that characterizes the native ECM. Novel electrospinning strategies include alterations of scaffold composition and architecture, along with the addition and encapsulation of cells, pharmaceuticals and growth factors within the scaffold. This article reviews the major nanofiber fabrication technologies as well as delves into recent significant contributions to the conception of a meaningful and practical electrospun scaffold.
Asunto(s)
Nanomedicina/métodos , Ingeniería de Tejidos/métodos , Andamios del Tejido , HumanosRESUMEN
Throughout native artery, collagen, and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery under pulsatile deformations. The goal of this study was to mimic the structure of native artery by fabricating a multi-layered electrospun conduit composed of poly(caprolactone) (PCL) with the addition of elastin and collagen with blends of 45-45-10, 55-35-10, and 65-25-10 PCL-ELAS-COL to demonstrate mechanical properties indicative of native arterial tissue, while remaining conducive to tissue regeneration. Whole grafts and individual layers were analyzed using uniaxial tensile testing, dynamic compliance, suture retention, and burst strength. Compliance results revealed that changes to the middle/medial layer changed overall graft behavior with whole graft compliance values ranging from 0.8 to 2.8%/100 mm Hg, while uniaxial results demonstrated an average modulus range of 2.0-11.8 MPa. Both modulus and compliance data displayed values within the range of native artery. Mathematical modeling was implemented to show how changes in layer stiffness affect the overall circumferential wall stress, and as a design aid to achieve the best mechanical combination of materials. Overall, the results indicated that a graft can be designed to mimic a tri-layered structure by altering layer properties.
Asunto(s)
Arterias/ultraestructura , Colágeno Tipo I/química , Elastina/química , Poliésteres/química , Animales , Adaptabilidad , Microscopía Electrónica de Rastreo , Modelos Teóricos , Porcinos , Resistencia a la TracciónRESUMEN
One of the major limitations in scaffold-based bone tissue engineering has been the inability to increase the loading of biologically active inorganic mineral. The present study introduces a novel two step strategy to increase overall mineral content of electrospun scaffolds and employs multiple factor interaction as a statistic to identify the combination of factors that yields maximal scaffold mineralization. Different amounts of nHA (0, 10, 25 and 50% by wt. of polymer) were electrospun in combination with polydioxanone (PDO) or poly(glycolide: lactide) to generate composite scaffolds. Successful incorporation of nHA within, on and in between nanofibers was confirmed by transmission and scanning electron microscopy. These scaffolds were immersed in different types (conventional, revised, ionic and modified) of simulated body fluid (SBF), prepared at 1x and 4x concentrations and the incubation was carried out either in static or dynamic setting at biomimetic conditions. At 2 weeks, the total amount of mineral within the scaffold was quantified using a modified Alizarin Red-based assay. Each of the five independent factors was analyzed independently and tested for interaction using random effects ANOVA. Statistics revealed significant higher order interactions among factors and the combination of PDO containing 50% nHA incubated in 1x revised SBF resulted in maximum mineralization.
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Materiales Biomiméticos/química , Sustitutos de Huesos/química , Calcificación Fisiológica , Durapatita/química , Electroquímica/métodos , Nanoestructuras/química , Ingeniería de Tejidos/métodos , Ensayo de Materiales , Nanoestructuras/ultraestructura , RotaciónRESUMEN
Native extracellular matrix (ECM) provides structural support to the multicellular organism on a macroscopic scale and establishes a unique microenvironment (niche) to tissue- and organ-specific cell types. Both these functions are critical for optimal function of the organism. These natural ECMs comprise predominantly fibrillar proteins, collagen and elastin and are synthesized as monomers but undergo hierarchical organization into well-defined nanoscaled structural units. The interaction between the cells and ECM is dynamic, reciprocal and essential for tissue development, maintenance of function, repair and regeneration processes. Tissue-engineering scaffolds are synthetic, biomimetic ECM analogues that have great promise in regenerative medicine. Ongoing efforts in mimicking the native ECM in terms of composition and dimension have resulted in three strategies that permit the generation of scaffolds in nanometer dimensions. Although excellent reviews regarding the applications of these strategies in tissue engineering are available, a comprehensive review of the science behind these fabrication techniques does not exist. This review intends to fill this critical gap in the existing knowledge in the fast-expanding field of nanofibrous scaffolds. A thorough understanding of the fabrication processes would enable us to better exploit available technologies to produce superior tissue-engineering scaffolds.
Asunto(s)
Ingeniería de Tejidos/métodos , Andamios del Tejido , Matriz Extracelular/químicaRESUMEN
We cross-linked scaffolds of electrospun collagen to varying degrees with glutaraldehyde using an ethanol-based solvent system and subsequently defined how the percentage of cross-linking impacts bulk and microscale material properties and fiber structure. At hydration, electrospun fibers underwent coiling; the extent of coiling was proportional to the percentage of cross-linking introduced into the samples and was largely suppressed as cross-linking approached saturation. These data suggest that electrospun collagen fibers are not deposited in a minimal energy state; fiber coiling may reflect a molecular reorganization. This result has functional/structural implications for protein-based electrospun scaffolds. Changes in fiber topology that develop during post-electrospinning processing may alter monomer organization, mask or unmask receptor binding sites, and/or change the biological properties of these nanomaterials. Hydrated scaffolds were mounted into a custom stretching device installed on a microscope stage and photographed after incremental changes in strain. Changes in fiber alignment were measured using the two-dimensional fast Fourier transform method. Fibers in all scaffolds underwent alignment in response to strain; however, the rate and extent of alignment that could be achieved varied as a function of cross-linking. We propose four distinct modes of scaffold response to strain: fiber uncoiling, fiber reorientation, fiber elongation and interfiber sliding. We conclude that bulk material properties and local microscale architecture must be simultaneously considered to optimize the performance of electrospun scaffolds.
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Materiales Biocompatibles/química , Ingeniería de Tejidos/métodos , Animales , Anisotropía , Bovinos , Colágeno/química , Reactivos de Enlaces Cruzados/química , Electroquímica/métodos , Análisis de Fourier , Ensayo de Materiales , Conformación Molecular , Nanoestructuras/química , Nanotecnología , Propiedades de Superficie , TemperaturaRESUMEN
The purpose of this study was to enhance the mechanical properties and slow the degradation of an electrospun fibrinogen scaffold, while maintaining the scaffold's high level of bioactivity. Three different cross-linkers were used to achieve this goal: glutaraldehyde vapour, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in ethanol and genipin in ethanol. Scaffolds with a fibrinogen concentration of 120 mg ml(-1) were electrospun and cross-linked with one of the aforementioned cross-linkers. Mechanical properties were determined through uniaxial tensile testing performed on scaffolds incubated under standard culture conditions for 1 day, 7 days and 14 days. Cross-linked scaffolds were seeded with human foreskin fibroblasts (BJ-GFP-hTERT) and cultured for 7, 14 and 21 days, with histology and scanning electron microscopy performed upon completion of the time course. Mechanical testing revealed significantly increased peak stress and modulus values for the EDC and genipin cross-linked scaffolds, with significantly slowed degradation. However, cross-linking with EDC and genipin was shown to have some negative effect on the bioactivity of the scaffolds as cell migration throughout the thickness of the scaffold was slowed.
Asunto(s)
Reactivos de Enlaces Cruzados/farmacología , Ingeniería de Tejidos/instrumentación , Animales , Materiales Biocompatibles/química , Carbodiimidas/química , Bovinos , Movimiento Celular , Reactivos de Enlaces Cruzados/química , Electroquímica/métodos , Fibrinógeno/química , Fibroblastos/metabolismo , Glutaral/química , Humanos , Glicósidos Iridoides , Iridoides/química , Microscopía Electrónica de Rastreo/métodos , Resistencia a la Tracción , Ingeniería de Tejidos/métodosRESUMEN
In this study we describe how to use a two-dimensional fast Fourier transform (2D FFT) approach to measure fiber alignment in electrospun materials. This image processing function can be coupled with a variety of imaging modalities to assign an objective numerical value to scaffold anisotropy. A data image of an electrospun scaffold is composed of pixels that depict the spatial organization of the constituent fibers. The 2D FFT function converts this spatial information into a mathematically defined frequency domain that maps the rate at which pixel intensities change across the original data image. This output image also contains quantitative information concerning the orientation of objects in a data image. We discuss the theory and practice of using the frequency plot of the 2D FFT function to measure relative scaffold anisotropy and identify the principal axis of fiber orientation. We note that specific degrees of scaffold anisotropy may represent a critical design feature in the fabrication of tissues that will be subjected to well-defined uniaxial mechanical loads. This structural property may also represent a source of guidance cues that can be exploited to regulate cell phenotype.
Asunto(s)
Materiales Biocompatibles/química , Análisis de Fourier , Ingeniería de Tejidos/métodos , Anisotropía , Microscopía Electrónica de RastreoRESUMEN
This study characterizes the cross-linking of electrospun elastin and the mechanical properties of suture-reinforced 1.5mm internal diameter electrospun tubes composed of blended polydioxanone (PDO) and soluble elastin. Several tube configurations were tested to assess the effects of reinforcement on tube mechanical properties. Between the electrospun layers of each double-layered prosthetic, zero, one or two 6-0 sutures were wound, maintaining 1mm spacing with a pitch of 9 degrees . Single-layered tubes without suture were also examined. Samples were cross-linked and tested for compliance and burst strength. Compliance decreased significantly (p <0.05) and burst strength significantly increased (p <0.01) with reinforcement. Uncross-linked tubes were also tested to determine the effects of cross-linking. Results demonstrated that cross-linking significantly decreases burst strength (p <0.01), while decreases in compliance for cross-linked tubes were not significant. Cross-linked suture-reinforced PDO-elastin tubes had burst pressures more than 10 times greater than normal systolic pressures and exhibited a range of compliance values, including those matching native artery. These tubes display many characteristics of the "ideal" small-diameter graft, having mechanical properties that can be tailored to match those desired in vascular replacement applications.
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Materiales Biocompatibles/química , Prótesis Vascular , Elastina/química , Polidioxanona/química , Suturas , Ingeniería de Tejidos , Fenómenos Biomecánicos , Estudios de Factibilidad , Ensayo de Materiales , Microscopía Electrónica de RastreoRESUMEN
Tissue engineering is an interdisciplinary field that has attempted to utilize a variety of processing methods with synthetic and natural polymers to fabricate scaffolds for the regeneration of tissues and organs. The study of structure-function relationships in both normal and pathological tissues has been coupled with the development of biologically active substitutes or engineered materials. The fibrillar collagens, types I, II, and III, are the most abundant natural polymers in the body and are found throughout the interstitial spaces where they function to impart overall structural integrity and strength to tissues. The collagen structures, referred to as extracellular matrix (ECM), provide the cells with the appropriate biological environment for embryologic development, organogenesis, cell growth, and wound repair. In the native tissues, the structural ECM proteins range in diameter from 50 to 500 nm. In order to create scaffolds or ECM analogues, which are truly biomimicking at this scale, one must employ nanotechnology. Recent advances in nanotechnology have led to a variety of approaches for the development of engineered ECM analogues. To date, three processing techniques (self-assembly, phase separation, and electrospinning) have evolved to allow the fabrication of nanofibrous scaffolds. With these advances, the long-awaited and much anticipated construction of a truly "biomimicking" or "ideal" tissue engineered environment, or scaffold, for a variety of tissues is now highly feasible. This review will discuss the three primary technologies (with a focus on electrospinning) available to create tissue engineering scaffolds that are capable of mimicking native tissue, as well as explore the wide array of materials investigated for use in scaffolds.
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Nanoestructuras , Ingeniería de Tejidos/métodos , Colágeno/química , Polímeros/química , Proteínas/químicaRESUMEN
Electrospinning can be used to selectively process a variety of natural and synthetic polymers into highly porous scaffolds composed of nano-to-m diameter fibers. This process shows great potential as a gateway to the development of physiologically relevant tissue engineering scaffolds. In this study, we examine how incremental changes in fiber alignment modulate the material properties of a model scaffold. We prepared electrospun scaffolds of gelatin composed of varying fiber diameters and degrees of anisotropy. The scaffolds were cut into a series of "dog-bone" shaped samples in the longitudinal, perpendicular and transverse orientations and the relative degree of fiber alignment, as measured by the fast Fourier transform (FFT) method, was determined for each sample. We measured peak stress, peak strain and the modulus of elasticity as a function of fiber diameter and scaffold anisotropy. Fiber alignment was the variable most closely associated with the regulation of peak stress, peak strain and modulus of elasticity. Incremental changes, as judged by the FFT method, in the proportion of fibers that were aligned along a specific axis induced incremental changes in peak stress in the model scaffolds. These results underscore the critical role that scaffold anisotropy plays in establishing the material properties of an electrospun tissue engineering scaffold and the native extracellular matrix.
Asunto(s)
Materiales Biocompatibles/química , Matriz Extracelular/química , Gelatina/química , Ingeniería de Tejidos/métodos , Anisotropía , Elasticidad , Electroquímica/métodos , Ensayo de Materiales , Rotación , Resistencia a la Tracción , ViscosidadRESUMEN
In trying to assess the structural integrity of electrospun type II collagen scaffolds, a modified but new technique for cross-linking collagen has been developed. Carbodiimides have been previously used to cross-link collagen in gels and in lyophilized native tissue specimens but had not been used for electrospun mats until recently. This cross-linking agent, and in particular 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), is of extreme interest, especially for tissue-engineered scaffolds composed specifically of native polymers (e.g., collagen), because it is a zero-length cross-linking agent that has not been shown to cause any cytotoxic reactions. The unique aspect of the cross-linking protocol in this study involves the use of ethanol as the solvent for the cross-linking agent, because the pure collagen electrospun mats immediately disintegrate when placed in an aqueous solution. This study examines 2 concentrations of EDC with and without the addition of N-hydroxysuccinimide to the reaction (which has been shown to result in higher cross-linking yields in aqueous solutions) to test the hypothesis that the use of EDC in a nonaqueous solution will cross-link electrospun type II collagen fibrous matrices in a comparable manner to typical glutaraldehyde fixation protocols. The use of EDC is compared with the cross-linking effects of glutaraldehyde via mechanical testing (uniaxial tensile testing) and biochemical testing (analysis of the percentage of free amino groups). The stress-strain curves of the cross-linked samples demonstrated uniaxial tensile behavior more characteristic of native tissue than do the dry, untreated samples. The heated, 50% glutaraldehyde cross-linking protocol resulted in a mean peak stress of 0.76 MPa, a mean strain at break of 127.30%, and a mean tangential modulus of 0.89 MPa; mean values for the samples treated with the EDC protocols ranged from 0.35 to 0.60 MPa for peak stress, from 111.83 to 159.23% for strain at break, and from 0.57 to 0.92 MPa for tangential modulus. Low and high concentrations (20 mM and 200 mM, respectively) of EDC alone were comparable in extent of cross-linking (29% and 29%, respectively) to the heated 50% glutaraldehyde cross-linking protocol (30% cross-linked).