Nanofibers and their applications in tissue engineering

Developing scaffolds that mimic the architecture of tissue at the nanoscale is one of the major challenges in the field of tissue engineering. The development of nanofibers has greatly enhanced the scope for fabricating scaffolds that can potentially meet this challenge. Currently, there are three techniques available for the synthesis of nanofibers: electrospinning, self-assembly, and phase separation. Of these techniques, electrospinning is the most widely studied technique and has also demonstrated the most promising results in terms of tissue engineering applications. The availability of a wide range of natural and synthetic biomaterials has broadened the scope for development of nanofibrous scaffolds, especially using the electrospinning technique. The three dimensional synthetic biodegradable scaffolds designed using nanofibers serve as an excellent framework for cell adhesion, proliferation, and differentiation. Therefore, nanofibers, irrespective of their method of synthesis, have been used as scaffolds for musculoskeletal tissue engineering (including bone, cartilage, ligament, and skeletal muscle), skin tissue engineering, vascular tissue engineering, neural tissue engineering, and as carriers for the controlled delivery of drugs, proteins, and DNA. This review summarizes the currently available techniques for nanofiber synthesis and discusses the use of nanofibers in tissue engineering and drug delivery applications.     

Biochemical and biophysical aspects of collagen nanostructure in the extracellular matrix

ECM is composed of different collagenous and non-collagenous proteins. Collagen nanofibers play a dominant role in maintaining the biological and structural integrity of various tissues and organs, including bone, skin, tendon, blood vessels, and cartilage. Artificial collagen nanofibers are increasingly significant in numerous tissue engineering applications and seem to be ideal scaffolds for cell growth and proliferation. The modern tissue engineering task is to develop three-dimensional scaffolds of appropriate biological and biomechanical properties, at the same time mimicking the natural extracellular matrix and promoting tissue regeneration. Furthermore, it should be biodegradable, bioresorbable and non-inflammatory, should provide sufficient nutrient supply and have appropriate viscoelasticity and strength. Attributed to collagen features mentioned above, collagen fibers represent an obvious appropriate material for tissue engineering scaffolds. The aim of this minireview is, besides encapsulation of the basic biochemical and biophysical properties of collagen, to summarize the most promising modern methods and technologies for production of collagen nanofibers and scaffolds for artificial tissue development.     

Fabrication,Characterization and Cellular Compatibility of Poly(Hydroxy Alkanoate) Composite Nanofibrous Scaffolds for Nerve Tissue Engineering

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.     

Electrospun scaffold tailored for tissue-specific extracellular matrix

The natural extracellular matrix (ECM) is a complex structure that is built to meet the specific requirements of the tissue and organ. Primarily consisting of nanometer diameter fibrils, ECM may contain other vital substances such as proteoglycans, glycosaminoglycan and various minerals. Current research in tissue engineering involves trying to replicate the ECM such that it provides the environment for tissue regeneration. Electrospinning is a versatile process that results in nanofibers by applying a high voltage to electrically charge a liquid. A variety of polymers and other substances have been incorporated into the artificial nanofibrous scaffold. Surface modification and cross-linking of the nanofibers are some ways to improve the biocompatibility and stability of the scaffold. Electrospun scaffolds with oriented nanofibers and other assemblies can be constructed by modifying the electrospinning setup. Using electrospinning, researchers are able to specifically tailor the electrospun scaffold to meet the requirements of the tissue that they seek to regenerate. In vitro and in vivo experiments demonstrate that electrospun scaffolds hold great potential for tissue engineering applications.     

Potential of Magnetic Nanofiber Scaffolds with Mechanical and Biological Properties Applicable for Bone Regeneration

Magnetic nanofibrous scaffolds of poly(caprolactone) (PCL) incorporating magnetic nanoparticles (MNP) were produced, and their effects on physico-chemical, mechanical and biological properties were extensively addressed to find efficacy for bone regeneration purpose. MNPs 12 nm in diameter were citrated and evenly distributed in PCL solutions up to 20% and then were electrospun into nonwoven nanofibrous webs. Incorporation of MNPs greatly improved the hydrophilicity of the nanofibers. Tensile mechanical properties of the nanofibers (tensile strength, yield strength, elastic modulus and elongation) were significantly enhanced with the addition of MNPs up to 15%. In particular, the tensile strength increase was as high as ∼25 MPa at 15% MNPs vs. ∼10 MPa in pure PCL. PCL-MNP nanofibers exhibited magnetic behaviors, with a high saturation point and hysteresis loop area, which increased gradually with MNP content. The incorporation of MNPs substantially increased the degradation of the nanofibers, with a weight loss of ∼20% in pure PCL, ∼45% in 10% MNPs and ∼60% in 20% MNPs. Apatite forming ability of the nanofibers tested in vitro in simulated body fluid confirmed the substantial improvement gained by the addition of MNPs. Osteoblastic cells favored the MNPs-incorporated nanofibers with significantly improved initial cell adhesion and subsequent penetration through the nanofibers, compared to pure PCL. Alkaline phosphatase activity and expression of genes associated with bone (collagen I, osteopontin and bone sialoprotein) were significantly up-regulated in cells cultured on PCL-MNP nanofibers than those on pure PCL. PCL-MNP nanofibers subcutaneously implanted in rats exhibited minimal adverse tissue reactions, while inducing substantial neoblood vessel formation, which however, greatly limited in pure PCL. In vivo study in radial segmental defects also signified the bone regeneration ability of the PCL-MNP nanofibrous scaffolds. The magnetic, bone-bioactive, mechanical, cellular and tissue attributes of MNP-incorporated PCL nanofibers make them promising candidate scaffolds for bone regeneration.     

Heparin mimetic peptide nanofibers promote angiogenesis

New blood vessel formation (angiogenesis) is one of the most important processes required for functional tissue formation. Induction of angiogenesis is usually triggered by growth factors released by cells. Glycosaminoglycans (e.g., heparan sulphates) in the extracellular matrix aid in proper functioning of these growth factors. Therefore, exogeneous heparin or growth factors were required for promoting angiogenesis in previous regenerative medicine studies. Here we report for the first time induction of angiogenesis by a synthetic nanofibrous peptide scaffold without the addition of any exogenous growth factors or heparin. We designed and synthesized a self-assembling peptide amphiphile molecule that is functionalized with biologically active groups to mimic heparin. Like heparin, this molecule has the ability to interact with growth factors and effectively enhance their bioactivity. The nanofibers formed by these molecules were shown to form a 3D network mimicking the structural proteins in the extracellular matrix. Because of heparin mimicking capabilities of the peptide nanofibers, angiogenesis was induced without the addition of exogenous growth factors in vitro. Bioactive interactions between the nanofibers and the growth factors enabled robust vascularization in vivo as well. Heparin mimetic peptide nanofibers presented here provide new opportunities for angiogenesis and tissue regeneration by avoiding the use of heparin and exogenous growth factors. The synthetic peptide nanofiber scaffolds enriched with proper chemical functional groups shown in this study can be used to induce various desired physiological responses for tissue regeneration.     

Electrospun poly (ɛ-caprolactone)/silk fibroin core-sheath nanofibers and their potential applications in tissue engineering and drug release

One of the key tenets of tissue engineering is to develop scaffold materials with favorable biodegradability, surface properties, outstanding mechanical strength and controlled drug release property. In this study, we generated core-sheath nanofibers composed of poly (?-caprolactone) (PCL) and silk fibroin (SF) blends via emulsion electrospinning. Nanofibrous scaffolds were characterized by combined techniques of scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), contact angle and tensile measurements. An in vitro FITC release study was conducted to evaluate sustained release potential of the core-sheath structured nanofibers. We found that the conformation of SF contained in PCL/SF composite nanofibers was transformed from random coil to β-sheet when treated with methanol, leading to improved crystallinity and tensile strength of nanofibrous scaffolds. The hydrophobicity and diameter of nanofibers decreased when we increased the content of SF in PCL/SF composite nanofibers. Furthermore, we evaluated the potential of fabricated PCL/SF composite nanofibers as scaffold in vitro. The results confirmed that fabricated PCL/SF scaffolds improved cell attachment and proliferation. Our results demonstrated the feasibility to generate core-sheath nanofibers composed of PCL and SF using a single-nozzle technique. The produced nanofibrous scaffolds with sustained drug release have potential application in tissue engineering.     

Cell culture in autologous fibrin scaffolds for applications in tissue engineering

In tissue engineering techniques, three-dimensional scaffolds are needed to adjust and guide cell growth and to allow tissue regeneration. The scaffold must be biocompatible, biodegradable and must benefit the interactions between cells and biomaterial. Some natural biomaterials such as fibrin provide a structure similar to the native extracellular matrix containing the cells. Fibrin was first used as a sealant based on pools of commercial fibrinogen. However, the high risk of viral transmission of these pools led to the development of techniques of viral inactivation and elimination and the use of autologous fibrins. In recent decades, fibrin has been used as a release system and three-dimensional scaffold for cell culture. Fibrin scaffolds have been widely used for the culture of different types of cells, and have found several applications in tissue engineering. The structure and development of scaffolds is a key point for cell culture because scaffolds of autologous fibrin offer an important alternative due to their low fibrinogen concentrations, which are more suitable for cell growth.     

Advanced biomaterials and their potential applications in the treatment of periodontal disease

Periodontal disease is considered as a widespread infectious disease and the most common cause of tooth loss in adults. Attempts for developing periodontal disease treatment strategies, including drug delivery and regeneration approaches, provide a useful experimental model for the evaluation of future periodontal therapies. Recently, emerging advanced biomaterials including hydrogels, films, micro/nanofibers and particles, hold great potential to be utilized as cell/drug carriers for local drug delivery and biomimetic scaffolds for future regeneration therapies. In this review, first, we describe the pathogenesis of periodontal disease, including plaque formation, immune response and inflammatory reactions caused by bacteria. Second, periodontal therapy and an overview of current biomaterials in periodontal regenerative medicine have been discussed. Third, the roles of state-of-the-art biomaterials, including hydrogels, films, micro/nanofibers and micro/nanoparticles, developed for periodontal disease treatment and periodontal tissue regeneration, and their fabrication methods, have been presented. Finally, biological properties, including biocompatibility, biodegradability and immunogenicity of the biomaterials, together with their current applications strategies are given. Conclusive remarks and future perspectives for such advanced biomaterials are discussed.     

骨髓间充质干细胞复合支架材料的骨组织工程研究进展

近年来,组织工程技术飞速发展,将种子细胞与支架材料相复合的骨组织工程研究已成为热点,并日趋走向成熟。这一全新的治疗方案将成为解决临床上各种原因造成的骨组织缺损的最有效途径之一。骨组织工程技术包括种子细胞、支架材料和生长因子三个方面。其中,BMSCs因具有多向分化能力、强大的增殖能力以及低免疫源性被认为是最理想的种子细胞,而支架材料的种类有很多种,目前对支架材料的选择也尚有分歧。如何找到理想的支架材料是骨组织工程研究中亟待解决的重要问题。本文就组织工程中与骨髓间充质干细胞(BMSCs)相复合的各类支架材料的研究现状进行综述,这些支架材料的研究将为骨组织工程支架材料的选择提供有效依据。     

High yield of cells committed to the photoreceptor-like cells from conjunctiva mesenchymal stem cells on nanofibrous scaffolds

Transplantation of stem cells using biodegradable and biocompatible nanofibrous scaffolds is a promising therapeutic approach for treating inherited retinal degenerative diseases such as retinitis pigmentosa and age-related macular degeneration. In this study, conjunctiva mesenchymal stem cells (CJMSCs) were seeded onto poly-l-lactic acid (PLLA) nanofibrous scaffolds and were induced to differentiate toward photoreceptor cell lineages. Furthermore, the effects of orientation of scaffold on photoreceptor differentiation were examined. Scanning electron microscopy (SEM) imaging, quantitative real time RT-PCR (qPCR) and immunocytochemistry were used to analyze differentiated cells and their expression of photoreceptor-specific genes. Our observations demonstrated the differentiation of CJMSCs to photoreceptor cells on nanofibrous scaffolds and suggested their potential application in retinal regeneration. SEM imaging showed that CJMSCs were spindle shaped and well oriented on the aligned nanofiber scaffolds. The expression of rod photoreceptor-specific genes was significantly higher in CJMSCs differentiated on randomly-oriented nanofibers compared to those on aligned nanofibers. According to our results we may conclude that the nanofibrous PLLA scaffold reported herein could be used as a potential cell carrier for retinal tissue engineering and a combination of electrospun nanofiber scaffolds and MSC-derived conjunctiva stromal cells may have potential application in retinal regenerative therapy.     

Neural differentiation of mouse embryonic stem cells on conductive nanofiber scaffolds

Nerve tissue engineering requires suitable precursor cells as well as the necessary biochemical and physical cues to guide neurite extension and tissue development. An ideal scaffold for neural regeneration would be both fibrous and electrically conductive. We have contrasted the growth and neural differentiation of mouse embryonic stem cells on three different aligned nanofiber scaffolds composed of poly L: -lactic acid supplemented with either single- or multi-walled carbon-nanotubes. The addition of the nanotubes conferred conductivity to the nanofibers and promoted mESC neural differentiation as evidenced by an increased mature neuronal markers expression. We propose that the conductive scaffold could be a useful tool for the generation of neural tissue mimics in vitro and potentially as a scaffold for the repair of neural defects in vivo.     

Electrospun poly(epsilon-caprolactone) microfiber and multilayer nanofiber/microfiber scaffolds: characterization of scaffolds and measurement of cellular infiltration

The physical and spatial architectural geometries of electrospun scaffolds are important to their application in tissue engineering strategies. In this work, poly(epsilon-caprolactone) microfiber scaffolds with average fiber diameters ranging from 2 to 10 microm were individually electrospun to determine the parameters required for reproducibly fabricating scaffolds. As fiber diameter increased, the average pore size of the scaffolds, as measured by mercury porosimetry, increased (values ranging from 20 to 45 microm), while a constant porosity was observed. To capitalize on both the larger pore sizes of the microfiber layers and the nanoscale dimensions of the nanofiber layers, layered scaffolds were fabricated by sequential electrospinning. These scaffolds consisted of alternating layers of poly(epsilon-caprolactone) microfibers and poly(epsilon-caprolactone) nanofibers. By electrospinning the nanofiber layers for different lengths of time, the thickness of the nanofiber layers could be modulated. Bilayered constructs consisting of microfiber scaffolds with varying thicknesses of nanofibers on top were generated and evaluated for their potential to affect rat marrow stromal cell attachment, spreading, and infiltration. Cell attachment after 24 h did not increase with increasing number of nanofibers, but the presence of nanofibers enhanced cell spreading as evidenced by stronger F-actin staining. Additionally, increasing the thickness of the nanofiber layer reduced the infiltration of cells into the scaffolds under both static and flow perfusion culture for the specific conditions tested. The scaffold design presented in this study allows for cellular infiltration into the scaffolds while at the same time providing nanofibers as a physical mimicry of extracellular matrix.     

Incorporated-bFGF polycaprolactone/polyvinylidene fluoride nanocomposite scaffold promotes human induced pluripotent stem cells osteogenic differentiation

Bioactive scaffolds that can increase transplanted cell survival time at the defect site have a great promising potential to use clinically since tissue regeneration or secretions crucially depend on the transplanted cell survival. In this study embedded basic fibroblast growth factor (bFGF)-polycaprolactone-polyvinylidene fluoride (PCL-PVDF) hybrid was designed and fabricated by electrospinning as a bio-functional nanofibrous scaffold for bone tissue engineering. After morphological characterization of the PCL-PVDF (bFGF) scaffold, nanofibers biocompatibility was investigated by culturing of the human induced pluripotent stem cells (iPSCs). Then, the bone differentiation capacity of the iPSCs was evaluated when grown on the PCL-PVDF and PCL-PVDF (bFGF) scaffolds in comparison with culture plate as a control using evaluating of the common osteogenic markers. The viability assay displayed a significant increase in iPSCs survival rate when grown on the bFGF content scaffold. The highest alkaline phosphatase activity and mineralization were detected in the iPSCs while grown on the PCL-PVDF (bFGF) scaffolds. Obtained results from gene and protein expression were also demonstrated the higher osteoinductive property of the bFGF content scaffold compared with the scaffold without it. According to the results, the release of bFGF from PCL-PVDF nanofibers increased survival and proliferation rate of the iPSCs, which followed by an increase in its osteogenic differentiation potential. Taking together, PCL-PVDF (bFGF) nanofibrous scaffold demonstrated that can be noted as a promising candidate for treating the bone lesions by tissue engineering products.     

Three-dimensional hierarchical composite scaffolds consisting of polycaprolactone, β-tricalcium phosphate, and collagen nanofibers: fabrication, physical properties, and in vitro cell activity for bone tissue regeneration

β-Tricalcium phosphate (β-TCP) and collagen have been widely used to regenerate various hard tissues, but although Bioceramics and collagen have various biological advantages with respect to cellular activity, their usage has been limited due to β-TCP's inherent brittleness and low mechanical properties, along with the low shape-ability of the three-dimensional collagen. To overcome these material deficiencies, we fabricated a new hierarchical scaffold that consisted of a melt-plotted polycaprolactone (PCL)/β-TCP composite and embedded collagen nanofibers. The fabrication process was combined with general melt-plotting methods and electrospinning. To evaluate the capability of this hierarchical scaffold to act as a biomaterial for bone tissue regeneration, physical and biological assessments were performed. Scanning electron microscope (SEM) micrographs of the fabricated scaffolds indicated that the β-TCP particles were uniformly embedded in PCL struts and that electrospun collagen nanofibers (diameter = 160 nm) were well layered between the composite struts. By accommodating the β-TCP and collagen nanofibers, the hierarchical composite scaffolds showed dramatic water-absorption ability (100% increase), increased hydrophilic properties (20%), and good mechanical properties similar to PCL/β-TCP composite. MTT assay and SEM images of cell-seeded scaffolds showed that the initial attachment of osteoblast-like cells (MG63) in the hierarchical scaffold was 2.2 times higher than that on the PCL/β-TCP composite scaffold. Additionally, the proliferation rate of the cells was about two times higher than that of the composite scaffold after 7 days of cell culture. Based on these results, we conclude that the collagen nanofibers and β-TCP particles in the scaffold provide good synergistic effects for cell activity.     

Composite scaffolds for cartilage tissue engineering

Tissue engineering remains a promising therapeutic strategy for the repair or regeneration of diseased or damaged tissues. Previous approaches have typically focused on combining cells and bioactive molecules (e.g., growth factors, cytokines and DNA fragments) with a biomaterial scaffold that functions as a template to control the geometry of the newly formed tissue, while facilitating the attachment, proliferation, and differentiation of embedded cells. Biomaterial scaffolds also play a crucial role in determining the functional properties of engineered tissues, including biomechanical characteristics such as inhomogeneity, anisotropy, nonlinearity or viscoelasticity. While single-phase, homogeneous materials have been used extensively to create numerous types of tissue constructs, there continue to be significant challenges in the development of scaffolds that can provide the functional properties of load-bearing tissues such as articular cartilage. In an attempt to create more complex scaffolds that promote the regeneration of functional engineered tissues, composite scaffolds comprising two or more distinct materials have been developed. This paper reviews various studies on the development and testing of composite scaffolds for the tissue engineering of articular cartilage, using techniques such as embedded fibers and textiles for reinforcement, embedded solid structures, multi-layered designs, or three-dimensionally woven composite materials. In many cases, the use of composite scaffolds can provide unique biomechanical and biological properties for the development of functional tissue engineering scaffolds.     

Increased FGF-2 secretion and ability to support neurite outgrowth by astrocytes cultured on polyamide nanofibrillar matrices

An electrospun nonwoven matrix of polyamide nanofibers was employed as a new model for the capillary basement membrane at the blood-brain barrier (BBB). The basement membrane separates astrocytes from endothelial cells and is associated with growth factors, such as fibroblast growth factor-2 (FGF-2). FGF-2 is produced by astrocytes and induces specialized functions in endothelial cells, but also has actions on astrocytes. To investigate potential autocrine actions of FGF-2 at the BBB, astrocytes were cultured on unmodified nanofibers or nanofibers covalently modified with FGF-2. The former assumed an in vivo-like stellate morphology that was enhanced in the presence of cross-linked FGF-2. Furthermore, astrocyte monolayers established on unmodified nanofibers were more permissive for neurite outgrowth when cultured with an overlay of neurons than similar monolayers established on standard tissue culture surfaces, while astrocytes cultured on FGF-2-modifed nanofibers were yet more permissive. The observed differences were due in part to progressively increasing amounts of FGF-2 secreted by the astrocytes into the medium; hence FGF-2 increases its own expression in astrocytes to modulate astrocyte–neuron interactions. Soluble FGF-2 was unable to replicate the effects of cross-linked FGF-2. Nanofibers alone up-regulated FGF-2, albeit to a lesser extent than nanofibers covalently modified with FGF-2. These results underscore the importance of both surface topography and growth factor presentation on cellular function. Moreover, these results indicate that FGF-2-modified nanofibrillar scaffolds may demonstrate utility in tissue engineering applications for replacement and regeneration of lost tissue following central nervous system (CNS) injury or disease.     

Novel chitin and chitosan nanofibers in biomedical applications

Chitin and its deacetylated derivative, chitosan, are non-toxic, antibacterial, biodegradable and biocompatible biopolymers. Due to these properties, they are widely used for biomedical applications such as tissue engineering scaffolds, drug delivery, wound dressings, separation membranes and antibacterial coatings, stent coatings, and sensors. In the recent years, electrospinning has been found to be a novel technique to produce chitin and chitosan nanofibers. These nanofibers find novel applications in biomedical fields due to their high surface area and porosity. This article reviews the recent reports on the preparation, properties and biomedical applications of chitin and chitosan based nanofibers in detail.     

Bone tissue engineering using marrow stromal cells

Bone tissue defects cause a significant socioeconomic problem, and bone is the most frequently transplanted tissue beside blood. Autografting is considered the gold standard treatment for bone defects, but its utility is limited due to donor site morbidity. Hence much research has focused on bone tissue engineering as a promising alternative method for repair of bone defects. Marrow stromal cells (MSCs) are considered to be potential cell sources for bone tissue engineering. In bone tissue engineering using MSCs, bone is formed through intramembranous and endochondral ossification in response to osteogenic inducers. Angiogenesis is a complex process mediated by multiple growth factors and is crucial for bone regeneration. Vascular endothelial growth factor plays important roles in bone tissue regeneration by promoting the migration and differentiation of osteoblasts, and by inducing angiogenesis. Scaffold materials used for bone tissue engineering include natural components of bone, such as calcium phosphate and collagen I, and biodegradable polymers such as poly(lactide-coglycolide) However, ideal scaffolds for bone tissue engineering have yet to be found. Bone tissue engineering has been successfully used to treat bone defects in several human clinical trials to regenerate bone defects. Through investigation of MSC biology and the development of novel scaffolds, we will be able to develop advanced bone tissue engineering techniques in the future.