Fabrication and evaluation of porous and conductive nanofibrous scaffolds for nerve tissue engineering.
Ontology highlight
ABSTRACT: Peripheral nerve repair is still one of the major clinical challenges which has received a great deal of attention. Nerve tissue engineering is a novel treatment approach that provides a permissive environment for neural cells to overcome the constraints of repair. Conductivity and interconnected porosity are two required characteristics for a scaffold to be effective in nerve regeneration. In this study, we aimed to fabricate a conductive scaffold with controlled porosity using polycaprolactone (PCL) and chitosan (Chit), FDA approved materials for the use in implantable medical devices. A novel method of using tetrakis (hydroxymethyl) phosphonium chloride (THPC) and formaldehyde was applied for in situ synthesis of gold nanoparticles (AuNPs) on the scaffolds. In order to achieve desirable porosity, different percentage of polyethylene oxide (PEO) was used as sacrificial fiber. Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FE-SEM) results demonstrated the complete removing of PEO from the scaffolds after washing and construction of interconnected porosities, respectively. Elemental and electrical analysis revealed the successful synthesis of AuNPs with uniform distribution and small average diameter on the PCL/Chit scaffold. Contact angle measurements showed the effect of porosity on hydrophilic properties of the scaffolds, where the porosity of 75-80% remarkably improved surface hydrophilicity. Finally, the effect of conductive nanofibrous scaffold on Schwann cells morphology and vaibility was investigated using FE-SEM and MTT assay, respectively. The results showed that these conductive scaffolds had no cytotoxic effect and support the spindle-shaped morphology of cells with elongated process which are typical of Schwann cell cultures.
Project description:Tissue engineering techniques using a combination of polymeric scaffolds and cells represent a promising approach for nerve regeneration. We fabricated electrospun scaffolds by blending of Poly (3-hydroxybutyrate) (PHB) and Poly (3-hydroxy butyrate-co-3- hydroxyvalerate) (PHBV) in different compositions in order to investigate their potential for the regeneration of the myelinic membrane. The thermal properties of the nanofibrous blends was analyzed by differential scanning calorimetry (DSC), which indicated that the melting and glass temperatures, and crystallization degree of the blends decreased as the PHBV weight ratio increased. Raman spectroscopy also revealed that the full width at half height of the band centered at 1725 cm(-1) can be used to estimate the crystalline degree of the electrospun meshes. Random and aligned nanofibrous scaffolds were also fabricated by electrospinning of PHB and PHBV with or without type I collagen. The influence of blend composition, fiber alignment and collagen incorporation on Schwann cell (SCs) organization and function was investigated. SCs attached and proliferated over all scaffolds formulations up to 14 days. SCs grown on aligned PHB/PHBV/collagen fibers exhibited a bipolar morphology that oriented along the fiber direction, while SCs grown on the randomly oriented fibers had a multipolar morphology. Incorporation of collagen within nanofibers increased SCs proliferation on day 14, GDNF gene expression on day 7 and NGF secretion on day 6. The results of this study demonstrate that aligned PHB/PHBV electrospun nanofibers could find potential use as scaffolds for nerve tissue engineering applications and that the presence of type I collagen in the nanofibers improves cell differentiation.
Project description:Bioresorbable polymers have been studied for several decades as attractive candidates for promoting the advancement of medical science and bio-technology in modern society. In particular, with a well-defined architecture, bioresorbable polymers have prominent advantages over their bulk counterparts for applications in biomedical and implant devices, such as cell delivery, scaffolds for tissue engineering, and hydrogels as well as in the pharmaceutical fields. Biocompatible implant devices based on bioresorbable materials (for instance, bioresorbable polymers that combine the unique advantages of biocompability and easy handling) have emerged as a highly active field due to their promising applications in artificial implant systems and biomedical devices. In this paper, we report an approach to fabricate porous polycaprolactone (PCL) scaffolds using a 3D printing system. And its surface was treated to a hydrophilic surface using plasma treatment. Then, the aspirin and atorvastatin calcium salt mixture was dip coated onto the surface. The drug coating technology was used to deposit the drug material onto the scaffold surface. Our porous PCL scaffold was coated with aspirin and atorvastatin calcium salt to reduce the blood LDL cholesterol and restenosis. These results suggest that our approach may provide a promising scaffold for developing bioresorbable drug-delivery-biomaterials. We further demonstrate that our bioresorbable medical device can be used as vascular scaffolds to provide a wide range of applications for the design of medical devices.
Project description:Microchannel scaffolds accelerate nerve repair by guiding growing neuronal processes across injury sites. Although geometry, materials chemistry, stiffness, and porosity have been shown to influence nerve growth within nerve guidance scaffolds, independent tuning of these properties in a high-throughput manner remains a challenge. Here, fiber drawing is combined with salt leaching to produce microchannels with tunable cross sections and porosity. This technique is applicable to an array of biochemically inert polymers, and it delivers hundreds of meters of porous microchannel fibers. Employing these fibers as filaments during 3D printing enables the production of microchannel scaffolds with geometries matching those of biological nerves, including branched topographies. Applied to sensory neurons, fiber-based porous microchannels enhance growth as compared to non-porous channels with matching materials and geometries. The combinatorial scaffold fabrication approach may advance the studies of neural regeneration and accelerate the development of nerve repair devices.
Project description:In this work, carbon nanofibers were used as doping material to develop a highly conductive chitosan-based composite. Scaffolds based on chitosan only and chitosan/carbon composites were prepared by precipitation. Carbon nanofibers were homogeneously dispersed throughout the chitosan matrix, and the composite scaffold was highly porous with fully interconnected pores. Chitosan/carbon scaffolds had an elastic modulus of 28.1 ± 3.3 KPa, similar to that measured for rat myocardium, and excellent electrical properties, with a conductivity of 0.25 ± 0.09 S/m. The scaffolds were seeded with neonatal rat heart cells and cultured for up to 14 days, without electrical stimulation. After 14 days of culture, the scaffold pores throughout the construct volume were filled with cells. The metabolic activity of cells in chitosan/carbon constructs was significantly higher as compared to cells in chitosan scaffolds. The incorporation of carbon nanofibers also led to increased expression of cardiac-specific genes involved in muscle contraction and electrical coupling. This study demonstrates that the incorporation of carbon nanofibers into porous chitosan scaffolds improved the properties of cardiac tissue constructs, presumably through enhanced transmission of electrical signals between the cells.
Project description:IntroductionIn search for cross-linkers with multifunctional characteristics, the present work investigated the utility of quaternary ammonium organosilane (QOS) as a potential cross-linker for electrospun collagen nanofibers. We hypothesized that the quaternary ammonium ions improve the electrospinnability by reducing the surface tension and confer antimicrobial properties, while the formation of siloxane after alkaline hydrolysis could cross-link collagen and stimulate cell proliferation.Materials and methodsQOS collagen nanofibers were electrospun by incorporating various concentrations of QOS (0.1%-10% w/w) and were cross-linked in situ after exposure to ammonium carbonate. The QOS cross-linked scaffolds were characterized and their biological properties were evaluated in terms of their biocompatibility, cellular adhesion and metabolic activity for primary human dermal fibroblasts and human fetal osteoblasts.Results and discussionThe study revealed that 1) QOS cross-linking increased the flexibility of otherwise rigid collagen nanofibers and improved the thermal stability; 2) QOS cross-linked mats displayed potent antibacterial activity and 3) the biocompatibility of the composite mats depended on the amount of QOS present in dope solution - at low QOS concentrations (0.1% w/w), the mats promoted mammalian cell proliferation and growth, whereas at higher QOS concentrations, cytotoxic effect was observed.ConclusionThis study demonstrates that QOS cross-linked mats possess anti-infective properties and confer niches for cellular growth and proliferation, thus offering a useful approach, which is important for hard and soft tissue engineering and regenerative medicine.
Project description:Tendon and ligament injuries are a persistent orthopedic challenge given their poor innate healing capacity. Nonwoven electrospun nanofibrous scaffolds composed of polyesters have been used to mimic the mechanics and topographical cues of native tendons and ligaments. However, nonwoven nanofibers have several limitations that prevent broader clinical application, including poor cell infiltration, as well as tensile and suture-retention strengths that are inferior to native tissues. In this study, multilayered scaffolds of aligned electrospun nanofibers of two designs-stacked or braided-were fabricated. Mechanical properties, including structural and mechanical properties and suture-retention strength, were determined using acellular scaffolds. Human bone marrow-derived mesenchymal stem cells (MSCs) were seeded on scaffolds for up to 28 days, and assays for tenogenic differentiation, histology, and biochemical composition were performed. Braided scaffolds exhibited improved tensile and suture-retention strengths, but reduced moduli. Both scaffold designs supported expression of tenogenic markers, although the effect was greater on braided scaffolds. Conversely, cell infiltration was superior in stacked constructs, resulting in enhanced cell number, total collagen content, and total sulfated glycosaminoglycan content. However, when normalized against cell number, both designs modulated extracellular matrix protein deposition to a similar degree. Taken together, this study demonstrates that multilayered scaffolds of aligned electrospun nanofibers supported tenogenic differentiation of seeded MSCs, but the macroarchitecture is an important consideration for applications of tendon and ligament tissue engineering.
Project description:Currently, the challenge for bone tissue engineering is to design a scaffold that would mimic the structure and biological functions of the extracellular matrix and would be able to direct the appropriate response of cells through electrochemical signals, thus stimulate faster bone formation. The purpose of the presented research was to perform and evaluate PCL/n-HAp scaffolds locally modified with a conductive polymer-polyaniline. The material was obtained using electrospinning, and a simple ink-jet printing method was applied to receive the conductive polyaniline patterns on the surface of the electrospun materials. The samples of scaffolds were analyzed by scanning electron microscopy (SEM), X-ray diffraction (XRD), thermal analysis (DSC, TGA), and infrared spectroscopy (FTIR) before and after immersion of the material in Simulated Body Fluid. The effect of PANI patterns on changes in the SBF mineralization process and cell morphology was evaluated in order to prove that the presented material enables the growth and proliferation of bone cells.
Project description:Conductivity is a desirable property of an ideal nerve guide conduit (NGC) that is being considered for peripheral nerve regeneration. Most of the conductive polymers reported in use for fabrication of tissue engineering scaffolds such as polypyrrole (PPy), polyaniline, polythiophene, and poly(3,4-ethylenedioxythiophene) are non-biodegradable and possess weak mechanical properties to be fabricated into 3D structures. In this study, a biodegradable and conductive block copolymer of PPy and Polycaprolactone (PPy-b-PCL) was used to fabricate 3D porous NGCs using a novel electrohydrodynamic jet 3D printing process which offers superior control over fiber diameter, pore size, porosity, and fiber alignment. PCL/PPy scaffolds with three different concentrations of PPy-b-PCL (0.5, 1, and 2% v/v) were fabricated as a mesh (pore size 125 ± 15 μm) and the effect of incorporation of PPy-b-PCL on mechanical properties, biodegradability, and conductivity of the NGCs were studied. The mechanical properties of the scaffolds decreased with the addition of PPy-b-PCL which aided the ability to fabricate softer scaffolds that are closer to the properties of the native human peripheral nerve. With increasing concentrations of PPy-b-PCL, the scaffolds displayed a marked increase in conductivity (ranging from 0.28 to 1.15 mS/cm depending on concentration of PPy). Human embryonic stem cell-derived neural crest stem cells (hESC-NCSCs) were used to investigate the impact of PPy-b-PCL based conductive scaffolds on the growth and differentiation to peripheral neuronal cells. The hESC-NCSCs were able to attach and differentiate to peripheral neurons on PCL and PCL/PPy scaffolds, in particular the PCL/PPy (1% v/v) scaffolds supported higher growth of neural cells and a stronger maturation of hESC-NCSCs to peripheral neuronal cells. Overall, these results suggest that PPy-based conductive scaffolds have potential clinical value as cell-free or cell-laden NGCs for peripheral neuronal regeneration.
Project description:The success of tissue engineering will rely on the ability to generate complex, cell seeded three-dimensional (3D) structures. Therefore, methods that can be used to precisely engineer the architecture and topography of scaffolding materials will represent a critical aspect of functional tissue engineering. Previous approaches for 3D scaffold fabrication based on top-down and process driven methods are often not adequate to produce complex structures due to the lack of control on scaffold architecture, porosity, and cellular interactions. The proposed projection stereolithography (PSL) platform can be used to design intricate 3D tissue scaffolds that can be engineered to mimic the microarchitecture of tissues, based on computer aided design (CAD). The PSL system was developed, programmed and optimized to fabricate 3D scaffolds using gelatin methacrylate (GelMA). Variation of the structure and prepolymer concentration enabled tailoring the mechanical properties of the scaffolds. A dynamic cell seeding method was utilized to improve the coverage of the scaffold throughout its thickness. The results demonstrated that the interconnectivity of pores allowed for uniform human umbilical vein endothelial cells (HUVECs) distribution and proliferation in the scaffolds, leading to high cell density and confluency at the end of the culture period. Moreover, immunohistochemistry results showed that cells seeded on the scaffold maintained their endothelial phenotype, demonstrating the biological functionality of the microfabricated GelMA scaffolds.
Project description:Collagen-based scaffolds lack mechanical strength, flexibility, and tunable pore structure, affecting tissue repair outcomes and restricting their wide clinical application. Here, two kinds of scaffolds were prepared by a combination of vacuum homogenization, natural air drying, water soaking, lyophilization, and crosslinking. Compared with the scaffolds made of collagen molecules (Col-M), the scaffolds made of collagen aggregates (Col-A) exhibited higher mechanical strength (ultimate tensile strength: 1.38 ± 0.26 MPa vs 15.46 ± 1.55 MPa), stronger flexibility, advanced cell adhesion, survival, and proliferation. Subcutaneous implantation in rats showed that Col-A scaffolds promoted cell infiltration, macrophage polarization, and vascularization. Furthermore, the Col-A scaffolds inhibited abdominal bulges due to their adequate mechanical support, and they also promoted vascularized muscle regeneration in a rat abdominal hernia defect model. Our study provides a novel strategy for generating high-strength, flexible, porous collagen-based scaffolds, which can be applied to tissue repair with mechanical strength requirements. It broadens their application range in the field of regenerative medicine.