Scaffolds Fabricated from Natural Polymers/Composites by Electrospinning for Bone Tissue Regeneration.

Naturally bone is a hierarchical and highly integrative dynamic tissue that is continuously remodeled by osteoblasts and osteoclasts. Deformities in bone due to trauma and/or disease are highly prevalent and mostly need surgical intervention. However, the methods of surgical treatments are associated with donor site morbidity, infection and/or complete rejection. Bone tissue-engineering provides a platform for growth of new bone tissue by fabricating scaffolds along with cells, growth factors and other dynamic forces. The polymeric materials especially natural polymers in their nanofibrous forms have been developed and introduced for bone tissue regeneration. At the nanoscale, natural polymers possess tunable properties and can be surface functionalized or blended with other polymers to provide juncture for cell-seeding, proliferation, differentiation and further resulting in regenerated tissue formation. These scaffolds fabricated from natural polymers and additives by electrospinning are bio-inspired to mimic the natural extracellular matrix resembling the native collagen of bone. This chapter focuses on the fabrication techniques as state of art nanofibrous scaffolds from natural polymers/additives during the recent years by the process of electrospinning for use in bone tissue regeneration. Further on, this chapter highlights the development in the scaffold fabrication from natural polymers like silk fibroin, chitosan, collagen, gelatin, cellulose, starch and, zein. The importance of add-on materials like stem cells, hydroxyapatite, apatite-wollanstonite, growth factors, osteogenic cells, bone morphogenic proteins and osteogenic drugs have been discussed and illustrated by various examples for enhancing the formation of new bone tissue. Furthermore, this chapter explains how these natural polymers influence the several signaling pathways to regulate bone regeneration.

Prospects of Natural Polymeric Scaffolds in Peripheral Nerve Tissue-Regeneration.

Tissue-engineering is emerging field and can be considered as a novel therapeutic intervention in nerve tissue-regeneration. The various pitfalls associated with the use of autografts in nerve-regeneration after injuries have inspired researchers to explore the possibilities using various natural polymers. In this context, the present chapter summarizes the advances of the various types of natural polymeric scaffolds such as fibrous scaffolds, porous scaffolds, and hydrogels in nerve-regeneration and repair process. The functionalization of the scaffolds with wide-range of biomolecules and their biocompatibility analysis by employing various cells (e.g., mesenchymal, neural progenitor stem cells) along with the in vivo regeneration outcomes achieved upon implantation are discussed here. Besides, the various avenues that have been explored so far in nerve tissue-engineering, the use of the extracellular matrix in enhancing the functional polymeric scaffolds and their corresponding outcomes of regeneration are mentioned. We conclude with the present challenges and prospects of efficient exploration of natural polymeric scaffolds in the future to overcome the problems of nerve-regeneration associated with various nerve injuries and neurodegenerative disorders.

Regenerated cellulose nanofibers from cellulose acetate: Incorporating hydroxyapatite (HAp) and silver (Ag) nanoparticles (NPs), as a scaffold for tissue engineering applications.

Cellulose nanofibers, which are troublesome to spin into fibers, can be easily fabricated by post-regeneration of its acetate-derived threads. Cellulose is a natural polymer; it enjoys better biocompatibility, cellular mimicking, and hydrophilic properties than its proportionate analog. Herein, we regenerated acetate-free nanofibers by alkaline de-acetylation of as-spun nanofibers. The resultant cellulose nanofibers previously loaded with hydroxyapatite (HAp) were immobilized using silver (Ag) nanoparticles (NPs) by reduction of adsorbed Ag ions on using sodium borohydride. These amalgamated nanofibers were characterized for SEM, EDX, TEM, FTIR, and hydrophilicity tests revealing the existence of both HAp and Ag NPs in/on the nanofiber scaffolds. The de-acetylation of composite nanofibers resulted in spontaneous hydrophilicity. These nanofibers were cytocompatible, as resolved by MTT assay conducted on chicken embryo fibroblasts. The SEM of the samples after cell culture revealed that these composites allowed a proliferation of the fibroblasts over and within the nanofiber network, and increased concentration of HAp levitated the excessive of apatite formation as well as increased cell growth. The antimicrobial activity of these nanofibers was assessed on E. coli (BL21) and S. aureus, suggesting the potential of de-acetylated nanofibers to restrain bacterial growth. The degradation study for 10, 30, and 60 days indicated degradation of the fibers much is faster in enzymes as compared to degradation in PBS. The results certify that these nanofibers possess enormous potential for soft and hard tissue engineering besides their antimicrobial properties.

Experimental Protocol for Culture and Differentiation of Osteoblasts on 3D Abode Using Nanofiber Scaffolds.

Nanofibrous structures provide a three-dimensional topography in vivo to allow the attachment, migration, proliferation, and differentiation of the cells in an environment which exactly mimics the native tissue. Herein, we report the standard protocols to carry out the cell culture of human osteoblast on nanofiber scaffolds. We also have described protocols for the determination of cell viability, morphology, mineralization, and phenotypic characterization of the osteoblasts.

Experimental Protocol of MSC Differentiation into Neural Lineage for Nerve Tissue Regeneration Using Polymeric Scaffolds.

The treatment of neurodegenerative diseases is still a challenging grindstone in reconstructive surgeries and regenerative medicine. The retention of mesenchymal stem cells (MSCs) to retain remarkable properties of differentiating into motor neuron-like cells and Schwann cells can prove to be effective in repairing disorders. Moreover, the ultrafine electrospun nanofibers provide a favorable and conducive platform for proliferation and differentiation of MSCs. The development of new 3D culture methods with electrospun scaffolds that closely mimic the physiological niche of cells will help us to understand the functional benefits of MSCs in regeneration process. This article highlights the protocols for isolation of MSCs from rat bone marrow and their subsequent culture on nanofiber scaffolds. Furthermore, this chapter summarizes the various procedures including isolation of the MSCs, their seeding on electrospun nanofibrous scaffolds, and their proliferation and differentiation into neural lineage upon appropriate induction. The materials and preparation of various reagents used at different steps of the protocol are also summarized in detail.

Methodology Involved in the Osteogenic Differentiation of Mesenchymal Stem Cells on Chitosan-Collagen Nanofibers Incorporated with Titanium Dioxide Nanoparticles.

Tissue regeneration especially in case of bones is a complex process as a repair involved is often inadequate. The electrospun chitosan nanofibers incorporated with titanium dioxide and collagen due to their ability to enhance biomineralization have been widely explored for bone tissue regeneration. Moreover, the mesenchymal stem cells (MSCs) possessing the properties of both self-renewal and multipotency offer a suitable recourse for cell-based regeneration strategies. This chapter summarizes the fabrication steps involved in the synthesis of titanium dioxide nanoparticles using sol-gel technique and their subsequent loading into chitosan/collagen nanofibers using the electrospinning process. Further on, the protocol involved in isolation of MSCs from bone marrow, seeding on fabricated nanofibers, and differentiation into osteoblasts is reported. The methods and techniques involved such as MTT assay, qRT-PCR, ALP activity, and immunofluorescence staining are also highlighted to investigate the potential of multifunctional nanofibers for the development of bony tissues.

Protocol for Determining the Induction of Human Embryonic Stem Cells into Myogenic Lineage Using Electrospun Nanofibers.

An efficient method for the development of myogenic differentiation using the stem cells can be beneficial in patients with severely compromised mobility, muscular damage, or degenerative diseases. The stem cells can prove to be excellent clinical source of myogenic progenitor cells due to their ability of self-proliferation, renewal, and differentiation into a specific phenotype. They represent an essential component of tissue engineering along with other factors (e.g., 3D scaffolds, ECM mimicking environment, and growth factors). In this chapter, we describe the experimental protocols for isolation of the embryonic stem cells, their proliferation on nanofiber scaffolds, and finally their differentiation into myogenic cells. Furthermore, this chapter elaborates experimental methods to assess the myogenic fate of embryonic stem cells on the nanofiber scaffolds.

Electrospun nanofibers for the delivery of active drugs through nasal, oral and vaginal mucosa: Current status and future perspectives.

Transmucosal surfaces bypass many limitations associated with conventional drug delivery (oral and parenteral routes), such as poor absorption rate, enzymatic activity, acidic environment and first-pass metabolism occurring inside the liver. However, these surfaces have several disadvantages such as poor retention time, narrow absorption window and continuous washout of the drug by the surrounding fluids. Electrospun nanofibers with their unique surface properties and encapsulation efficiency may act as novel drug carriers to overcome the challenges associated with conventional drug delivery routes, so as to achieve desired therapeutic responses. This review article provides detailed information regarding the challenges faced in the mucosal delivery of drugs, and the use of nanofiber systems as an alternative to deliver drugs to the systemic circulation, as well as local drug administration. The physiological and anatomical features of different types of mucosal surfaces and current challenges are systematically discussed. We also address future considerations in the area of transmucosal delivery of some important drugs.

Experimental Protocol for Induction of Transgene Expression in Neural Stem Cells Through Polymeric Nanoparticles.

Therapy based on stem cells utilizes these cells in neurodegeneration, brain/spinal cord injury, and much recently in repairing of severe heart diseases. Owning to their stemness, these cells are the potential source of progenitors that can offer a therapeutic remedy to a variety of diseases and/or disorders. The ability of these cells to regenerate and differentiate into specified phenotypes has great utility in tissue regeneration applications. This chapter provides a detailed account for isolation of neural stem cells from the mice embryo. Furthermore, the fabrication of chitosan-tripolyphosphate/hyaluronic acid-based nanoparticles and evaluating their efficiency in inducing transfection in the isolated neural stem cells as an approach for the treatment of neurodegenerative disorders.

Fabrication of multifunctional cellulose/TiO2 /Ag composite nanofibers scaffold with antibacterial and bioactivity properties for future tissue engineering applications.

In the present work, a novel strategy was explored to fabricate nanofiber scaffolds consisting of cellulose assimilated with titanium dioxide (TiO2 ) and silver (Ag) nanoparticles (NPs). The concentration of the TiO2 NPs in the composite was adjusted to 1.0, 1.5, and 2.0 wt % with respect to polymer concentration used for the electrospinning of colloidal solutions. The fabricated composite scaffolds were dispensed to alkaline deacetylation using 0.05 M NaOH to remove the acetyl groups in order to generate pure cellulose nanofibers containing TiO2 NPs. Moreover, to augment our nanofiber scaffolds with antibacterial activity, the in situ deposition approach of using Ag NPs was utilized with varied molar concentrations of 0.14, 0.42, and 0.71 M. The physicochemical properties of the nanofibers were identified by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) and contact angle meter studies. This demonstrated the presence of both TiO2 and Ag NPs and complete deacetylation of nanofibers. The antibacterial efficiency of the nanofibers was scrutinized against Escherichia coli and Staphylococcus aureus, revealing proper in situ deposition of Ag NPs and confirming the nanofibers are antibacterial in nature. The biocompatibility of the scaffolds was accustomed using chicken embryo fibroblasts, which confirmed their potential role to be used as wound-healing materials. Furthermore, the fabricated scaffolds were subjected to analysis in simulated body fluid at 37°C to induce mineralization for future osseous tissue integration. These results indicate that fabricated composite nanofiber scaffolds with multifunctional characteristics will have a highest potential as a future candidate for promoting new tissues artificially.

Recent trends in peripheral nervous regeneration using 3D biomaterials.

Mesenchymal stem cells (MSCs) owing their multipotency are known as progenitors for the regeneration of adult tissues including that of neuronal tissue. The repair and/or regeneration of traumatic nerves is still a challenging task for neurosurgeons. It is also a well-established fact that the microenvironment plays a primary role in determining the fate of stem cells to a specific lineage. In recent years, with the advent of nanotechnology and its positive influence on designing and fabrication of various 3D biomaterials have progressed to a greater extent. The production of 3D biomaterials such as nanofibers, conduits and hydrogels are providing a suitable environment for mimicking physiological niche of stem cells. These 3D biomaterials in combination with MSCs have been successfully analyzed for their potential in the regeneration of degenerative neurological disorders. This review primarily highlights the combinatorial effect of multipotent MSCs seeded on various 3D polymeric scaffolds in repair and regeneration of nervous tissue. The elaboration of MSCs from distinct sources reported so far in literature are summarized to understand their role in regeneration processes. Furthermore, we accentuate the application of 3D biomaterials especially the nanofibers, polymeric conduits, hydrogels infiltrated with MSCs harvested from distinct sources in the field of peripheral nerve regeneration studies.

Recent Trends in the Fabrication of Starch Nanofibers: Electrospinning and Non-electrospinning Routes and Their Applications in Biotechnology.

Electrospinning a versatile and the most preferred technique for the fabrication of nanofibers has revolutionized by opening unlimited avenues in biomedical fields. Presently, the simultaneous functionalization and/or post-modification of as-spun nanofibers with biomolecules has been explored, to serve the distinct goals in the aforementioned field. Starch is one of the most abundant biopolymers on the earth. Besides, being biocompatible and biodegradable in nature, it has unprecedented properties of gelatinization and retrogradation. Therefore, starch has been used in numerous ways for wide range of applications. Keeping these properties in consideration, the present article summarizes the recent expansion in the fabrication of the pristine/modified starch-based composite scaffolds by electrospinning along with their possible applications. Apart from electrospinning technique, this review will also provide the comprehensive information on various other techniques employed in the fabrication of the starch-based nanofibers. Furthermore, we conclude with the challenges to be overcome in the fabrication of nanofibers by the electrospinning technique and future prospects of starch-based fabricated scaffolds for exploration of its applications.

Novel lavender oil and silver nanoparticles simultaneously loaded onto polyurethane nanofibers for wound-healing applications.

Synthetic polymers, especially those with biocompatible and biodegradable characteristics, may offer effective alternatives for the treatment of severe wounds and burn injuries. Ideally, the scaffold material should induce as little pain as possible, enable quick healing, and direct the growth of defect-free epidermal cells. The best material with this multifunctionality, such as self-healing dressings, should be hydrophilic and have uninterrupted and direct contact with the damaged tissue. In addition, the ideal biomaterial should have some antibacterial properties. In this study, a novel technique was used to fabricate composite electrospun wound-dressing nanofibers composed of polyurethane encasing lavender oil and silver (Ag) nanoparticles (NPs). After electrospinning, the fabricated nanofibers were identified using various techniques, including scanning electron microscopy (SEM) and transmission electron microscopy (TEM). An abundance of Ag NPs in the fibers decreased the diameter of the fibers while increased concentration of the lavender oil increased the diameter. Fourier transform infrared (FTIR) and X-ray diffraction (XRD) studies showed the presence of the lavender oil and Ag NPs in the fiber dressings. The Ag NPs and lavender oil improved the hydrophilicity of the nanofibers and ensured the proliferation of chicken embryo fibroblasts cultured in-vitro on these fiber dressings. The antibacterial efficiency of the nanofiber dressings was investigated using E. coli and S. aureus, which yielded zones of inhibition of 16.2 ±â€¯0.8 and 5.9 ±â€¯0.5 mm, respectively, indicating excellent bactericidal properties of the dressings. The composite nanofiber dressings have great potential to be used as multifunctional wound dressings; offering protection against external agents as well as promoting the regeneration of new tissue.

Reconstructing nanofibers from natural polymers using surface functionalization approaches for applications in tissue engineering, drug delivery and biosensing devices.

Previously, the nanofibers were predominantly fabricated from synthetic polymers due to their excellent mechanical properties. Understanding the different complex processes in fabrication and various process parameters involved have not only allowed the use of natural polymers for fabricating nanofibers but also broadened the scope of applications. To date, many of the natural polymeric composites have been fabricated by different functionalization techniques to increase their applicability. Nanofibers fabricated from natural polymers have been chemically functionalized by a variety of molecules like drugs, enzymes, metal ions etc. by techniques such as plasma treatment, wet chemical method, graft polymerization and co-electrospinning of surface-active molecules. Furthermore, the nanofibers derived from natural polymers have been surface-coated on the synthetic polymers to induce extracellular matrix mirroring properties like cell adhesion, migration, proliferation and differentiation. In this review, we have not only investigated the various novel and facile functionalization approaches but potential properties and applications are discussed as well. The various surface chemistry modifications of the natural polymeric nanofibers and their potential applications in drug delivery, enzymology, catalysis, filtration, biosensing and tissue engineering are discussed. In addition, a brief presentation of an overview of challenges and future scope with the aim of making them a clinical success has been presented.