Recent Progress and Potential Biomedical Applications of Electrospun Nanofibers in Regeneration of Tissues and Organs

Electrospun techniques are promising and flexible technologies to fabricate ultrafine fiber/nanofiber materials from diverse materials with unique characteristics under optimum conditions. These fabricated fibers/nanofibers via electrospinning can be easily assembled into several shapes of three-dimensional (3D) structures and can be combined with other nanomaterials. Therefore, electrospun nanofibers, with their structural and functional advantages, have gained considerable attention from scientific communities as suitable candidates in biomedical fields, such as the regeneration of tissues and organs, where they can mimic the network structure of collagen fiber in its natural extracellular matrix(es). Due to these special features, electrospinning has been revolutionized as a successful technique to fabricate such nanomaterials from polymer media. Therefore, this review reports on recent progress in electrospun nanofibers and their applications in various biomedical fields, such as bone cell proliferation, nerve regeneration, and vascular tissue, and skin tissue, engineering. The functionalization of the fabricated electrospun nanofibers with different materials furnishes them with promising properties to enhance their employment in various fields of biomedical applications. Finally, we highlight the challenges and outlooks to improve and enhance the application of electrospun nanofibers in these applications

Polymer- and Hybrid-Based Biomaterials for Interstitial, Connective, Vascular, Nerve, Visceral and Musculoskeletal Tissue Engineering

In this review, materials based on polymers and hybrids possessing both organic and inorganic contents for repairing or facilitating cell growth in tissue engineering are discussed. Pure polymer based biomaterials are predominantly used to target soft tissues. Stipulated by possibilities of tuning the composition and concentration of their inorganic content, hybrid materials allow to mimic properties of various types of harder tissues. That leads to the concept of “one-matches-all” referring to materials possessing the same polymeric base, but different inorganic content to enable tissue growth and repair, proliferation of cells, and the formation of the ECM (extra cellular matrix). Furthermore, adding drug delivery carriers to coatings and scaffolds designed with such materials brings additional functionality by encapsulating active molecules, antibacterial agents, and growth factors. We discuss here materials and methods of their assembly from a general perspective together with their applications in various tissue engineering sub-areas: interstitial, connective, vascular, nervous, visceral and musculoskeletal tissues. The overall aims of this review are two-fold: (a) to describe the needs and opportunities in the field of bio-medicine, which should be useful for material scientists, and (b) to present capabilities and resources available in the area of materials, which should be of interest for biologists and medical doctors.</jats:p

CELL MECHANICS IN CARDIOVASCULAR DISEASE AND ELECTROSPUN SCAFFOLD FOR VASCULAR TISSUE ENGINEERING

Cardiovascular disease (CVD) is the leading cause of death worldwide. Atherosclerosis, one of the primary CVDs, is characterized as a chronic inflammatory disease. In the initial stages of atherosclerosis, there is a buildup of cholesterol and lipoproteins that triggers monocytes to enter the arterial wall and begin accumulating lipids. Vascular smooth muscle cells (VSMCs) begin to detach and migrate from the media toward the intima in a process known as phenotypic switching. Phenotypic switching transitions VSMCs from a contractile to synthetic phenotype and they gain the capacity for migration, proliferation, and secretion of extracellular matrix (ECM) proteins. Synthetic VSMCs experience a variety of microenvironments of differing stiffness and composition within the atherosclerotic plaque which elicit different biomechanical responses. The growth of atherosclerotic plaques can cause stenosis and reduced blood flow. One treatment for this is revascularization surgery using vascular grafts to bypass blockages. In this dissertation, we examine how VSMC biomechanics change in response to substrate stiffness and composition. Then we developed an electrospun polycaprolactone (PCL)-silk fibroin (SF) electrospun scaffold for use in vascular tissue engineering. In specific aim 1, the effect of substrate stiffness and collagen or fibronectin coatings on VSMC migration and cytoskeletal organization was analyzed. Protein coatings and substrate stiffness were found to synergistically regulate migration and cortical actin organization in the opposite manner. In specific aim 2, the differences in biomechanics of atherosclerotic ApoE-/- and wild type (WT) VSMCs was analyzed. ApoE-/- VSMCs were found to have lower adhesion forces but increased migration capacity, cytoskeletal alignment, and stiffness, with the latter two being enhanced by increasing substrate stiffness. In specific aim 3, an exploration into the use of electrospun PCL-SF scaffolds as vascular grafts was conducted. The addition of SF improved the mechanical properties of the graft, making them more similar to those of native arteries, as well as increasing the diameter of the nanofibers. Furthermore, the PCL-SF scaffolds supported the differentiation of mesenchymal stem cells into VSMC-like cells. Therefore, this dissertation provides further insights in the alteration of VSMC biomechanics during atherosclerosis and a promising material for the development a tissue engineered vascular graft

Controlling the fate of stem cells through two-and three-dimensional scaffolds based on bioresorbable polymers and graphenen derivatives: a study towards nerve tissue regeneration.

184 p.Neurological disorders are the major cause of long-term impairment and the second largest cause of death worldwide. Hence, there is an urgent need for new treatments that allow the functional recovery of damaged tissue. Among the experimental treatments, bioresorbable polyesters are showing great results in preclinical and clinical trials due to their biocompatibility, tunable degradability, versatility and physicochemical and mechanical properties. In this PhD thesis nanostructured scaffolds based on bioresorbable polymers and graphene oxide were developed to study the attachment, aligned growth and migration of both murine and human stem cells, avoiding the use of extracellular matrix-like compounds coatings. The use of murine neural stem cells allowed to study the differentiation pattern of the cells over the nanostructuredscaffolds, focusing on the achievement of a balanced neuronal and glial support, for a long-term survival of the cultures in vitro. The use of a relatively new source of stem cells, now considered clinical waste, like the dental pulp stem cells, allowed to minimize the ethical concerns and provide an actual alternative for personalized medicine in future therapies. To test this alternative, the regeneration capabilities of the nanostructured scaffolds were studied after the impairment of the rostral migratory stream in a rodent model in vivo. And with the aim of addressing the enhanced restoration capabilities of the personalized advanced medical products combining polymeric materials and human stem cells, the regeneration of the rostral migratory stream was compared when grafting the dental pulp stem cells, alone or in combination with our nanostructured scaffolds.Finally, to better resemble the neural niche in vitro graphene derivatives-based three-dimensional scaffolds with tunable geometry, mechanical and electrical conductive features were fabricated and their effect studied on cell survival and differentiation. Afterward, cerium oxide nanoparticles were incorporated to provide enhanced antioxidant and neuroprotective features and their effect on the establishment of balanced neuronal and glial co-cultures studied.Overall, this thesis gives new insights into the design of polymeric materials based on graphene derivatives for future personalized advanced medical products in combination with human stem cells for the restoration of the nervous syste

Coaxially electrospun heparin-eluting scaffolds for vascular graft application

The use of electrospun scaffolds for small diameter vascular grafts ( 0.9 MPa, maximum strain > 100 %, suture retention > 2.4 N) or within groups between the longitudinal and circumferential tensile properties. After 6 weeks of in vitro degradation, all groups exhibited similar mechanical losses of approximately 40 % in ultimate tensile stress and 80 % in maximum elongation in circumferential and longitudinal directions. The smaller vascular grafts had burst pressures superior to native vasculature and compliances approximating those of healthy arteries. Thermal analyses (DSC) of the different groups showed similar thermograms with little intergroup variation and indicated that the electrospinning process did not unduly affect the thermal properties or crystallinity, of DP30. There was also no major variations in thermograms of degraded samples. Blend electrospun scaffolds showed the expected initial burst release of HepTBA (47.7 %, 3 days) followed by a sustained release (56.1 %, 6 weeks). Coaxially incorporated HepNa+ also exhibited initial burst release (67.5-69.7 %, 3 days) for both the low and high heparin content groups followed by improved sustained release (81.9 - 97.7%, 6 weeks). Coaxial incorporation had a 2× higher heparin encapsulation efficiency than blend incorporation (approaching 100 %). Heparin, post-TBA-modification, did not fully retain its antithrombotic properties (54.9 % reduction), which was further reduced after incorporation and release (24.2 % reduction). HepNa+ , however, retained its full antithrombotic activity post coaxial incorporation and elution. Coaxial electrospinning of heparin in DP30 shows potential for producing small diameter vascular grafts with mechanical properties comparable to small blood vessels. Although some initial burst release occurred, the sustained release over 6 weeks, incorporation of heparin without the need for modification at improved efficiency, and the retained activity of the heparin after electrospinning incorporation and elution; holds promise for vascular graft applications. Future work should aim for the production of continuous cores within fibre morphology and evaluating graft performance in an in vivo model to determine whether an appropriate and sufficient amount of heparin has been included to affect the desired response

Development of biofunctionalized tubular scaffolds for vascular tissue engineering applications

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Biomateriais, Reabilitação e Biomecânica)One of the major problems related to small-diameter blood vessels replacement is the lack of vascular grafts with suitable mechanical and biological properties. Although there are synthetic vascular grafts in clinical use, these substitutes present thrombogenic behaviour and are too stiff compared to native vessels. Rapid endothelialization and matched mechanical properties are important functional requirements that vascular grafts should accomplish. Herein, an electrospun tubular fibrous (eTF) scaffold was fabricated and functionalized to immobilize tropoelastin at the luminal surface, providing a biomimetic environment to enhance endothelialization. The morphology was assessed by scanning electron microscopy, the effectiveness of surface functionalization by NH2 groups quantification and surface charge measurements, and the mechanical properties by uniaxial tensile tests. Tropoelastin was immobilized at 20 μg/mL by its -NH2 functional groups on activated scaffolds, as well as by its -COOH functional groups on aminolysed scaffolds, in an attempt to expose different conformations of tropoelastin for cell binding. The amount of immobilized tropoelastin on both substrates was quantified by microBCA assay. These constructs were cultured with a cell line of human umbilical vein endothelial cells (HUVECs) for 7 days, to study the endothelialization of eTF scaffolds by evaluating their metabolic activity, proliferation, total protein synthesis, VEGF secretion, as well as cell morphology and phenotype maintenance. Our experimental characterization demonstrated that the eTF scaffolds have a thickness of 240.85 ± 46.91 μm and their luminal surface was 33.55 % porous mix of micro to submicro fibers diameters, pore sizes less than 23 μm and pore areas up to 70 μm2. The eTF scaffolds were successfully functionalized by the insertion of 0.5 ± 0.04 nmol/mg of NH2 groups at their surface and confirmed by the differences observed in surface charge. Untreated, activated and aminolysed scaffolds supported higher stresses and strains in axial direction rather than in radial direction. These values are compatible to those of native blood vessels. The exposure of tropoelastin -COOH groups promoted endothelial cells metabolic activity and growth, whereas when exposed its -NH2 groups a significant influence on protein synthesis was observed. Additionally, eTF scaffolds promoted phenotype maintenance and endothelial cell coverage just after 7 days of culture. Altogether, the results confirm that biofunctional eTF scaffolds are suitable for vascular application since they presented adequate mechanical properties and a rapid endothelialization.Um dos maiores problemas associados à substituição de vasos sanguíneos de pequeno diâmetro é a insuficiência de enxertos vasculares com propriedades mecânicas e biológicas adequadas. Embora existam enxertos vasculares sintéticos na prática clínica, estes substitutos apresentam trombogenicidade e são demasiado rígidos comparativamente aos vasos sanguíneos nativos. Uma rápida endotelização e propriedades mecânicas semelhantes aos vasos sanguíneos humanos são requisitos essenciais que um excerto vascular deve possuir. Neste trabalho, estruturas tubulares fibrosas foram produzidas por electrospinning (eTF scaffolds) e funcionalizadas para imobilizar tropoelastina na superfície interna, proporcionando um ambiente biomimético para promover a endotelização. A morfologia foi analisada por microscopia eletrónica de varrimento (SEM), a eficiência da funcionalização da superfície pela quantificação dos grupos amina (-NH2) e pela carga de superfície, e as propriedades mecânicas foram analisadas por testes uniaxiais à tração. A tropoelastina foi imobilizada a uma concentração de 20 μg/mL através dos seus grupos -NH2 nos eTF scaffolds activados, bem como pelos seus grupos carboxílicos (-COOH) nos scaffolds aminolisados, de forma a expor diferentes conformações para a ligação com as células. A quantidade de tropoelastina imobilizada em ambos os substratos foi quantificada através do método microBCA. Por último, os eTF scaffolds foram semeados com uma linha celular de células endoteliais da veia umbilical humana durante 7 dias para estudar a endotelização. Desta forma, a atividade metabólica, a proliferação celular, a síntese proteica e de VEGF, bem como a morfologia celular e a manutenção do fenótipo dos eTF scaffolds foram investigadas. Os resultados experimentais demonstraram que os eTF scaffolds possuem uma espessura de 240.85 ± 46.91 μm e uma superfície interna 33.55% porosa com diâmetros de fibras na ordem do micro ao submicro, tamanhos de poros inferiores a 23 μm e áreas de poros até 70 μm2. Os eTF scaffolds foram efetivamente funcionalizados através da inserção de 0.5 ± 0.04 nmol de grupos NH2 na superfície e pelas diferenças observadas na carga de superfície. Os eTF scaffolds não tratados, activados e aminolisados suportaram tensões e elongamentos mais elevados na direção axial do que na radial. Estes resultados obtidos são compatíveis com os valores reportados para os vasos sanguíneos nativos. A exposição dos grupos -COOH da tropoelastina induziu um aumento da atividade metabólica e crescimento das células endoteliais. Quando expostos os grupos -NH2, uma influência significativa na síntese proteica foi observada. Além disso, os eTF scaffolds promoveram a manutenção do fenótipo e a formação de uma monocamada de células endoteliais na superfície após 7 dias de cultura. De um modo geral, estes resultados confirmam que estes eTF scaffolds biofuncionais são adequados para aplicação vascular, uma vez que apresentam propriedades mecânicas adequadas e uma rápida endotelização

Nanofibrous Scaffolds for Skin Tissue Engineering and Wound Healing Based on Synthetic Polymers

Nanofibrous scaffolds are popular materials in all areas of tissue engineering, because they mimic the fibrous component of the natural extracellular matrix. In this chapter, we focused on the application of nanofibers in skin tissue engineering and wound healing, because the skin is an organ with several vitally important functions, particularly barrier, thermoregulatory, and sensory functions. Nanofibrous meshes not only serve as carriers for skin cells but also can prevent the penetration of microbes into wounds and can keep appropriate moisture in the damaged skin. The nanofibrous meshes have been prepared from a wide range of synthetic and nature-derived polymers. This review is concentrated on synthetic non-degradable and degradable polymers, which have been explored for skin tissue engineering and wound healing. These synthetic polymers were often combined with natural polymers of the protein or polysaccharide nature, which improved their attractiveness for cell colonization. The nanofibrous scaffolds can also be loaded with various bioactive molecules, such as growth factors, hormones, vitamins, antioxidants, antimicrobial, and antitumor agents. In advanced tissue engineering approaches, the cells on the nanofibrous scaffolds are cultured in dynamic bioreactors enabling appropriate mechanical stimulation of cells and at air-liquid interface. This chapter summarizes recent results achieved in the field of nanofiber-based skin tissue engineering, including results of our research group

Recent Advances in Electrospun Sustainable Composites for Biomedical, Environmental, Energy, and Packaging Applications.

Electrospinning has gained constant enthusiasm and wide interest as a novel sustainable material processing technique due to its ease of operation and wide adaptability for fabricating eco-friendly fibers on a nanoscale. In addition, the device working parameters, spinning solution properties, and the environmental factors can have a significant effect on the fibers\u27 morphology during electrospinning. This review summarizes the newly developed principles and influence factors for electrospinning technology in the past five years, including these factors\u27 interactions with the electrospinning mechanism as well as its most recent applications of electrospun natural or sustainable composite materials in biology, environmental protection, energy, and food packaging materials

Electrospun Nanofibre Scaffolds for Tissue Engineered Small-Diameter Vascular Grafts

Ph.DDOCTOR OF PHILOSOPH