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Graphene oxide-stimulated myogenic differentiation of C2C12 cells on PLGA/RGD peptide nanofiber matrices
Abstract
During the last decade, much attention has been paid to graphene-based nanomaterials because they are considered as potential candidates for biomedical applications such as scaffolds for tissue engineering and substrates for the differentiation of stem cells. Until now, electrospun matrices composed of various biodegradable copolymers have been extensively developed for tissue engineering and regeneration; however, their use in combination with graphene oxide (GO) is novel and challenging. In this study, nanofiber matrices composed of poly(lactic-co-glycolic acid, PLGA) and M13 phage with RGD peptide displayed on its surface (RGD peptide-M13 phage) were prepared as extracellular matrix (ECM)-mimicking substrates. RGD peptide is a tripeptide (Arg-Gly-Asp) found on ECM proteins that promotes various cellular behaviors. The physicochemical properties of PLGA and RGD peptide-M13 phage (PLGA/RGD peptide) nanofiber matrices were characterized by atomic force microscopy, Fourier-transform infrared spectroscopy and thermogravimetric analysis. In addition, the growth of C2C12 mouse myoblasts on the PLGA/RGD peptide matrices was examined by measuring the metabolic activity. Moreover, the differentiation of C2C12 mouse myoblasts on the matrices when treated with GO was evaluated. The cellular behaviors, including growth and differentiation of C2C12 mouse myoblasts, were substantially enhanced on the PLGA/RGD peptide nanofiber matrices when treated with GO. Overall, these findings suggest that the PLGA/RGD peptide nanofiber matrices can be used in combination with GO as a novel strategy for skeletal tissue regeneration.
Tissue defects are usually caused by trauma, tumors, deformity, and infection. Use of tissue engineering technology to regenerate the defects, especially based on stem cells, has attracted widespread attention in recent years. To achieve faster healing and reconstruction of large scale defects, it is critically important to find scaffolds that are best for attachment and proliferation and can even induce the differentiation of stem cells. Recently, graphene and its derivatives have drawn significant attention due to their special physical and chemical properties, especially their biological properties. In this review, we summarized recent advances in tissue engineering based on graphene-family nanomaterials. For this purpose, a general description was provided to elucidate the biocompatibility, stimulation effects of graphene on cell differentiation, their potential applications in tissue regenerations, and the biodegradation property of the graphene-based materials. Moreover, the limitations and future trends of the applications of graphene in the biomedical field have been presented.
Many Gram-negative bacteria use Type I secretion systems, T1SS, to secrete virulence factors that
contain calcium-binding Repeat-in-ToXin (RTX) motifs. Here, we present structural models of an RTX
protein, RD, in both its intrinsically disordered calcium-free Apo-state and its folded calcium-bound
Holo-state. Apo-RD behaves as a disordered polymer chain comprising several statistical elements
that exhibit local rigidity with residual secondary structure. Holo-RD is a folded multi-domain
protein with an anisometric shape. RTX motifs thus appear remarkably adapted to the structural
and mechanistic constraints of the secretion process. In the low calcium environment of the bacterial
cytosol, Apo-RD is an elongated disordered coil appropriately sized for transport through the narrow
secretion machinery. The progressive folding of Holo-RD in the extracellular calcium-rich environment
as it emerges form the T1SS may then favor its unidirectional export through the secretory channel.
This process is relevant for hundreds of bacterial species producing virulent RTX proteins.
In biomedical applications, there is a need for tissue engineering scaffolds to promote and control cellular behaviors, including adhesion, proliferation and differentiation. In particular, the initial adhesion of cells has a great influence on those cellular behaviors. In this study, we concentrate on developing cell-adhesive substrates applicable for tissue engineering scaffolds. The hybrid nanofiber sheets were prepared by electrospinning poly(lactic-co-glycolic acid) (PLGA) and M13 phage, which was genetically modified to enhance cell adhesion thru expressing RGD peptides on their surface. The RGD peptide is a specific motif of extracellular matrix (ECM) for integrin receptors of cells. RGD peptide-decorated PLGA (RGD-PLGA) nanofiber sheets were characterized by scanning electron microscopy, immunofluorescence staining, contact angle measurement and differential scanning calorimetry. In addition, the initial adhesion and proliferation of four different types of mammalian cells were determined in order to evaluate the potential of RGD-PLGA nanofiber sheets as cell-adhesive substrates. Our results showed that the hybrid nanofiber sheets have a three-dimensional porous structure comparable to the native ECM. Furthermore, the initial adhesion and proliferation of cells were significantly enhanced on RGD-PLGA sheets. These results suggest that biomimetic RGD-PLGA nanofiber sheets can be promising cell-adhesive substrates for application as tissue engineering scaffolds.
The muscle regulators MyoD and Myf-5 control cell cycle withdrawal and induction of differentiation in skeletal muscle cells. By immunofluorescence analysis, we show that MyoD and Myf-5 expression patterns become mutually exclusive when C2 cells are induced to differentiate with Myf-5 staining present in cells which fail to differentiate. Isolation of these undifferentiated cells reveals that upon serum stimulation they reenter the cell cycle, express MyoD and downregulate Myf-5. Similar regulations of MyoD and Myf-5 were observed using cultured primary myoblasts derived from satellite cells. To further analyze these regulations of MyoD and Myf-5 expression, we synchronized proliferating myoblasts. Analysis of MyoD and Myf-5 expression during cell cycle progression revealed distinct and contrasting profiles of expression. MyoD is absent in G0, peaks in mid-G1, falls to its minimum level at G1/S and reaugments from S to M. In contrast, Myf-5 protein is high in G0, decreases during G1 and reappears at the end of G1 to remain stable until mitosis. These data demonstrate that the two myogenic factors MyoD and Myf-5 undergo specific and distinct cell cycle–dependent regulation, thus establishing a correlation between the cell cycle–specific ratios of MyoD and Myf-5 and the capacity of cells to differentiate: (a) in G1, when cells express high levels of MyoD and enter differentiation; (b) in G0, when cells express high levels of Myf-5 and fail to differentiate.
Electrospinning is a simple and effective method for fabricating micro- and nanofiber matrices. Electrospun fibre matrices have numerous advantages for use as tissue engineering scaffolds, such as high surface area-to-volume ratio, mass production capability and structural similarity to the natural extracellular matrix (ECM). Therefore, electrospun matrices, which are composed of biocompatible polymers and various biomaterials, have been developed as biomimetic scaffolds for the tissue engineering applications. In particular, graphene oxide (GO) has recently been considered as a novel biomaterial for skeletal muscle regeneration because it can promote the growth and differentiation of myoblasts. Therefore, the aim of the present study was to fabricate the hybrid fibre matrices that stimulate myoblasts differentiation for skeletal muscle regeneration.
Hybrid fibre matrices composed of poly(lactic-co-glycolic acid, PLGA) and collagen (Col) impregnated with GO (GO-PLGA-Col) were successfully fabricated using an electrospinning process. Our results indicated that the GO-PLGA-Col hybrid matrices were comprised of randomly-oriented continuous fibres with a three-dimensional non-woven porous structure. Compositional analysis showed that GO was dispersed uniformly throughout the GO-PLGA-Col matrices. In addition, the hydrophilicity of the fabricated matrices was significantly increased by blending with a small amount of Col and GO. The attachment and proliferation of the C2C12 skeletal myoblasts were significantly enhanced on the GO-PLGA-Col hybrid matrices. Furthermore, the GO-PLGA-Col matrices stimulated the myogenic differentiation of C2C12 skeletal myoblasts, which was enhanced further under the culture conditions of the differentiation media.
Taking our findings into consideration, it is suggested that the GO-PLGA-Col hybrid fibre matrices can be exploited as potential biomimetic scaffolds for skeletal tissue engineering and regeneration because these GO-impregnated hybrid matrices have potent effects on the induction of spontaneous myogenesis and exhibit superior bioactivity and biocompatibility.
The integration of gene delivery technologies with electrospun nanofibers is a versatile strategy to increase the potential of gene therapy as a key platform technology that can be readily utilized for numerous biomedical applications, including cancer therapy, stem cell therapy, and tissue engineering. As a spatial template for gene delivery, electrospun nanofibers possess highly advantageous characteristics, such as their ease of production, their ECM-analogue nature, the broad range of choices for materials, the feasibility of producing structures with varied physical and chemical properties, and their large surface-to-volume ratios. Thus, electrospun fiber-mediated gene delivery exhibits a great capacity to modulate the spatial and temporal release kinetics of gene vectors and enhance gene delivery efficiency. This review discusses the powerful characteristics of electrospun nanofibers, which can function as spatial interfaces capable of promoting controlled and efficient gene delivery.
3D Printing promises to produce complex biomedical devices according to computer design using patient-specific anatomical data. Since its initial use as pre-surgical visualization models and tooling molds, 3D Printing has slowly evolved to create one-of-a-kind devices, implants, scaffolds for tissue engineering, diagnostic platforms, and drug delivery systems. Fueled by the recent explosion in public interest and access to affordable printers, there is renewed interest to combine stem cells with custom 3D scaffolds for personalized regenerative medicine. Before 3D Printing can be used routinely for the regeneration of complex tissues (e.g. bone, cartilage, muscles, vessels, nerves in the craniomaxillofacial complex), and complex organs with intricate 3D microarchitecture (e.g. liver, lymphoid organs), several technological limitations must be addressed. In this review, the major materials and technology advances within the last five years for each of the common 3D Printing technologies (Three Dimensional Printing, Fused Deposition Modeling, Selective Laser Sintering, Stereolithography, and 3D Plotting/Direct-Write/Bioprinting) are described. Examples are highlighted to illustrate progress of each technology in tissue engineering, and key limitations are identified to motivate future research and advance this fascinating field of advanced manufacturing.
Unlike bone tissue, articular cartilage regeneration has not been very successful and has many challenges ahead. We have previously developed injectable hydrogels using photopolymerizable chitosan (MeGC) that supported growth of chondrocytes. In this study, we demonstrate a biofunctional hydrogel for specific use in cartilage regeneration by conjugating transforming growth factor-β1 (TGF-β1), a well-documented chondrogenic factor, to MeGC hydrogels impregnating type II collagen (Col II), one of the major cartilaginous extracellular matrix (ECM) components.
TGF-β1 was delivered from MeGC hydrogels in a controlled manner with reduced burst release by chemically conjugating the protein to MeGC. The hydrogel system did not compromise viability of encapsulated human synovium-derived mesenchymal stem cells (hSMSCs). Col II impregnation and TGF-β1 delivery significantly enhanced cellular aggregation and deposition of cartilaginous ECM by the encapsulated cells, compared with pure MeGC hydrogels.
This study demonstrates successful engineering of a biofunctional hydrogel with a specific microenvironment tailored to promote chondrogenesis. This hydrogel system can provide promising efficacious therapeutics in the treatment of cartilage defects.
Background
Osteoarthritis (OA) is a degenerative joint disease affecting approximately 27 million Americans, and even more worldwide. OA is characterized by degeneration of subchondral bone and articular cartilage. In this study, a chondrogenic fibrin/hyaluronic acid (HA)-based hydrogel seeded with bone marrow-derived mesenchymal stem cells (BMSCs) was investigated as a method of regenerating these tissues for OA therapy. This chondrogenic hydrogel system can be delivered in a minimally invasive manner through a small gauge needle, forming a three-dimensional (3D) network structure in situ. However, an ongoing problem with fibrin/HA-based biomaterials is poor mechanical strength. This was addressed by modifying HA with methacrylic anhydride (MA) (HA-MA), which reinforces the fibrin gel, thereby improving mechanical properties. In this study, a range of fibrinogen (the fibrin precursor) and HA-MA concentrations were explored to determine optimal conditions for increased mechanical strength, BMSC proliferation, and chondrogenesis potential in vitro.
Results
Increased mechanical strength was achieved by HA-MA reinforcement within fibrin hydrogels, and was directly correlated with increasing HA-MA concentration. Live/dead staining and metabolic assays confirmed that the crosslinked fibrin/HA-MA hydrogels provided a suitable 3D environment for BMSC proliferation. Quantitative polymerase chain reaction (qPCR) of BMSCs incubated in the fibrin/HA-MA hydrogel confirmed decreased expression of collagen type 1 alpha 1 mRNA with an increase in Sox9 mRNA expression especially in the presence of a platelet lysate, suggesting early chondrogenesis.
Conclusion
Fibrin/HA-MA hydrogel may be a suitable delivery method for BMSCs, inducing BMSC differentiation into chondrocytes and potentially aiding in articular cartilage repair for OA therapy.
The purpose of this study was to evaluate the cytotoxicity of human multiple myeloma cells (RPMI-8226) treated with graphene oxide (GO), doxorubicin (DOX), and GO loaded with DOX (GO/DOX). Cell viability was determined using the Cell Counting Kit-8 assay and analyzing the cell cycle and cell apoptosis. Cells treated with GO, GO/DOX, and pure DOX for 24 hours showed a decrease in proliferation. GO/DOX significantly inhibited cell proliferation as compared with pure DOX (P<0.01). When the effects of GO were removed, there was no observed difference between GO/DOX and pure DOX (P>0.05). Flow cytometry analysis of untreated and GO-, DOX-, and GO/DOX-treated cells found no significant differences in the G0/G1 phase (P>0.05), while significant differences were observed in the total apoptotic rates (P<0.05). No significant differences existed in the total apoptotic rates of GO-treated and untreated cells (P>0.05). These findings suggest that GO caused low cytotoxicity and did not induce cell apoptosis or change the cell cycle in multiple myeloma cells. Moreover, GO did not affect the antitumor activity of DOX. In conclusion, GO would be suitable as an anticancer drug nanocarrier and used to treat hematological malignancies.
The main objective of the present paper is to represent a new class of Poly Lactic-co-Glycolic Acid (PLGA) nanofibers containing silver (Ag) nanoparticles (NPs) by electrospinning process. Silver nanoparticles were well loaded without any chemical and structural modifications into PLGA polymer matrix to form an organic-inorganic nanocomposite. Syntheses of silver NPs was carried out by exploiting reduction ability of N, N-dimethylformamide (DMF) solvent which is mainly utilized in decomposition of silver nitrate precursor into silver NPs. Typsically, a sol-gel consisting of different concentration of AgNO3 in PLGA has been electrospun, so, silver NPs have been created in the nanofibers. Observable beads can be noticed with high silver nitrate contents. TEM image of one of the modified nanofiber confirmed that NPs are present in/on the nanofiber. Overall, the size of the nanofibers was within the range of 50 to 100 nm, while the size of silver nanoparticles was within range 5 to 10 nm. The said realized results strongly recommend the use of obtained nanofiber mats as antimicrobial agents in biomaterials or water purifying systems.
Biochemical and topographical features of an artificial extracellular matrix (aECM) can direct stem cell fate. However, it is difficult to vary only the biochemical cues without changing nanotopography to study their unique role. We took advantage of two unique features of M13 phage, a non-toxic nanofiber-like virus, to generate a virus-activated aECM with constant ordered ridge/groove nanotopography but displaying different fibronectin-derived peptides (RGD, its synergy site PHSRN, and a combination of RGD and PHSRN). One feature is the self-assembly of phage into a ridge/groove structure, another is the ease of genetically surface-displaying a peptide. We found that the unique ridge/groove nanotopography and the display of RGD and PHSRN could induce the osteoblastic differentiation of mesenchymal stem cells (MSCs) without any osteogenic supplements. The aECM formed through self-assembly and genetic engineering of phage can be used to understand the role of peptide cues in directing stem cell behavior while keeping nanotopography constant.
Poly(glycolic acid) (PGA) has long been a popular polymer in the tissue engi-neering field. PGA possesses many favorable properties such as biocompati-bility, bioabsorbability, and tensile strength. The traditional fiber formation techniques of melt extrusion and cold-drawing are generally limited to fibers of 10-12 µm in diameter. Electrostatic spinning, or electrospinning, is an attractive approach for the production of much smaller diameter fibers which are of interest as tissue engineering scaffolds. We demonstrate the ability to control the fiber diameter of PGA as a function of solution concentration and fiber orientation, as well as show a correlation between the fiber orientation, elastic modulu, and strain to failure of PGA in a uniaxial model.
Piezoelectric materials can convert mechanical energy into electrical energy, and piezoelectric devices made of a variety of inorganic materials and organic polymers have been demonstrated. However, synthesizing such materials often requires toxic starting compounds, harsh conditions and/or complex procedures. Previously, it was shown that hierarchically organized natural materials such as bones, collagen fibrils and peptide nanotubes can display piezoelectric properties. Here, we demonstrate that the piezoelectric and liquid-crystalline properties of M13 bacteriophage (phage) can be used to generate electrical energy. Using piezoresponse force microscopy, we characterize the structure-dependent piezoelectric properties of the phage at the molecular level. We then show that self-assembled thin films of phage can exhibit piezoelectric strengths of up to 7.8 pm V(-1). We also demonstrate that it is possible to modulate the dipole strength of the phage, hence tuning the piezoelectric response, by genetically engineering the major coat proteins of the phage. Finally, we develop a phage-based piezoelectric generator that produces up to 6 nA of current and 400 mV of potential and use it to operate a liquid-crystal display. Because biotechnology techniques enable large-scale production of genetically modified phages, phage-based piezoelectric materials potentially offer a simple and environmentally friendly approach to piezoelectric energy generation.
Recently, there has been considerable effort to develop suitable scaffolds for tissue engineering applications. Cell adhesion is a prerequisite for cells to survive. In nature, the extracellular matrix (ECM) plays this role. Therefore, an ideal scaffold should be structurally similar to the natural ECM and have biocompatibility and biodegradability. In addition, the scaffold should have biofunctionality, which provides the potent ability to enhance the cellular behaviors, such as adhesion, proliferation and differentiation. This study concentrates on fabricating cell-adhesive matrices composed of RGD peptide-displaying M13 bacteriophage (RGD-M13 phage) and poly(lactic-co-glycolic acid, PLGA) nanofibers. Long rod-shaped M13 bacteriophages are non-toxic and can express many desired proteins on their surface. A genetically engineered M13 phage was constructed to display RGD peptides on its surface. PLGA is a biodegradable polymer with excellent biocompatibility and suitable physicochemical property for adhesive matrices. In this study, RGD-M13 phage/PLGA hybrid nanofiber matrices were fabricated by electrospinning. The physicochemical properties of these matrices were characterized by scanning electron microscopy, atomic force microscopy, Raman spectroscopy, and contact angle measurement. In addition, the cellular behaviors, such as the initial attachment, proliferation and differentiation, were analyzed by a CCK-8 assay and immunofluorescence staining to evaluate the potential application of these matrices to tissue engineering scaffolds. The RGD-M13 phage/PLGA nanofiber matrices could enhance the cellular behaviors and promote the differentiation of C2C12 myoblasts. These results suggest that the RGD-M13 phage/PLGA nanofiber matrices are beneficial to myoblast differentiation and can serve as effective tissue engineering scaffolds.
The preparation of graphitic oxide by methods described in the literature is time consuming and hazardous. A rapid, relatively safe method has been developed for preparing graphitic oxide from graphite in what is essentially an anhydrous mixture of sulfuric acid, sodium nitrate and potassium permanganate.
The cell–cell adhesion molecule N-cadherin, with its associated catenins, is expressed by differentiating skeletal muscle and its precursors. Although N-cadherin's role in later events of skeletal myogenesis such as adhesion during myoblast fusion is well established, less is known about its role in earlier events such as commitment and differentiation. Using an in vitro model system, we have determined that N-cadherin– mediated adhesion enhances skeletal muscle differentiation in three-dimensional cell aggregates. We transfected the cadherin-negative BHK fibroblastlike cell line with N-cadherin. Expression of exogenous N-cadherin upregulated endogenous β-catenin and induced strong cell–cell adhesion. When BHK cells were cultured as three-dimensional aggregates, N-cadherin enhanced withdrawal from the cell cycle and stimulated differentiation into skeletal muscle as measured by increased expression of sarcomeric myosin and the 12/101 antigen. In contrast, N-cadherin did not stimulate differentiation of BHK cells in monolayer cultures. The effect of N-cadherin was not unique since E-cadherin also increased the level of sarcomeric myosin in BHK aggregates. However, a nonfunctional mutant N-cadherin that increased the level of β-catenin failed to promote skeletal muscle differentiation suggesting an adhesion-competent cadherin is required. Our results suggest that cadherin-mediated cell–cell interactions during embryogenesis can dramatically influence skeletal myogenesis.
Degradable nanofiber scaffold is known to provide a suitable, versatile and temporary structure for tissue regeneration. However, synthetic nanofiber scaffold must be properly designed to display appropriate tissue response during the degradation process. In this context, this publication focuses on the design of a finely-tuned poly(lactide-co-ϵ-caprolactone) terpolymer (PLCL) that may be appropriate for vascular biomaterials applications and its comparison with well-known semi-crystalline poly(l-lactide) (PLLA). The degradation mechanism of polymer film and nanofiber scaffold and endothelial cells behavior cultured with degradation products is elucidated. The results highlights benefits of using PLCL terpolymer as vascular biomaterial compared to PLLA.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Although extensively used in the fields of drug-carrier and tissue engineering, the biocompatibility and mechanical properties of polylactide-polyglycolide (PLGA) nanofiber membranes still limit their applications. The objective of this study was to improve their utility by introducing cellulose nanocrystals (CNCs) into PLGA nanofiber membranes. PLGA and PLGA/CNC composite nanofiber membranes were prepared via electrospinning, and the morphology and thermodynamic and mechanical properties of these nanofiber membranes were characterized by scanning electron microscopy (SEM), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and dynamic mechanical analysis (DMA). The cytocompatibility and cellular responses of the nanofiber membranes were also studied by WST-1 assay, SEM, and confocal laser scanning microscopy (CLSM). Incorporation of CNCs (1, 3, 5, and 7wt.%) increased the average fiber diameter of the prepared nanofiber membranes from 100nm (neat PLGA) to ∼400nm (PLGA/7wt.% CNC) and improved the thermal stability of the nanofiber membranes. Among the PLGA/CNC composite nanofiber membranes, those loaded with 7wt.% CNC nanofiber membranes had the best mechanical properties, which were similar to those of human skin. Cell culture results showed that the PLGA/CNC composite nanofiber membranes had better cytocompatibility and facilitated fibroblast adhesion, spreading, and proliferation compared with neat PLGA nanofiber membranes. These preliminary results suggest that PLGA/CNC composite nanofiber membranes are promising new materials for the field of skin tissue engineering.
Copyright © 2015 Elsevier B.V. All rights reserved.
Graphene possesses many unique properties such as two-dimensional planar structure, super conductivity, chemical and mechanical stability, large surface area, and good biocompatibility. In the past few years, graphene-based materials have risen as a shining star on the path of researchers seeking new materials for future regenerative medicine. Herein, the recent research advances made in graphene-based materials mostly utilizing the mechanical and electrical properties of graphene are described. The most exciting findings addressing the impact of graphene-based materials on regenerative medicine are highlighted, with particular emphasis on their applications including nerve, bone, cartilage, skeletal muscle, cardiac, skin, adipose tissue regeneration, and their effects on the induced pluripotent stem cells. Future perspectives and emerging challenges are also addressed in this Review article.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
The infiltration of the cells into the scaffolds is important phenomenon to give them good biocompatibility and even biodegradability. Fluid shear stress is one of the candidates for the infiltration of cells into scaffolds. Here we investigated the directional migration of human mesenchymal stem cells and infiltration into PLGA scaffold by fluid shear stress. The human mesenchymal stem cells showed directional migrations following the direction of the flow (8, 16 dyne/cm(2)). In the scaffold models, the fluid shear stress (8 dyne/cm(2)) enhanced the infiltration of cells but did not influence on the infiltration of Poly(lactic-co-glycolic acid) particles.
Copyright © 2015. Published by Elsevier Inc.
Extracellular matrices (ECMs) are network structures that play an essential role in regulating cellular growth and differentiation. In this study, novel nanofibrous matrices were fabricated by electrospinning M13 bacteriophage and poly(lactic-co-glycolic acid) (PLGA) and were shown to be structurally and functionally similar to natural ECMs. A genetically-engineered M13 bacteriophage was constructed to display Arg-Gly-Asp (RGD) peptides on its surface. The physicochemical properties of RGD peptide-displaying M13 bacteriophage (RGD-M13 phage)/PLGA nanofibers were characterized by using scanning electron microscopy and Fourier-transform infrared spectroscopy. We used immunofluorescence staining to confirm that M13 bacteriophages were homogenously distributed in RGD-M13 phage/PLGA matrices. Furthermore, RGD-M13 phage/PLGA nanofibrous matrices, having excellent biocompatibility, can enhance the behaviors of vascular smooth muscle cells. This result suggests that RGD-M13 phage/PLGA nanofibrous matrices have potentials to serve as tissue engineering scaffolds.
Recently graphene and graphene based composites are emerging as better materials to fabricate scaffolds. Addition of graphene oxide (GO) nanoplatelets (GOnPs) in bioactive polymers was found to enhance its conductivity (σ) and, dielectric permittivity (ϵ) along with biocompatibility. In this paper, human cord blood derived mesenchymal stem cells (CB-hMSCs) were differentiated to skeletal muscle cells (hSkMCs) on spin coated thin GO sheets composed of GOnPs and on electrospun fibrous meshes of GO-PCL (poly-caprolactone) composite. Both substrates exhibited excellent myoblast differentiations and promoted self-alignedmyotubesformation similar to natural orientation. σ, ϵ, microstructural and vibration spectroscopic studies were carried out for the characterizations of GO sheet and the composite scaffolds. Significantly enhanced values of both σ and ϵ of the GO-PCL composite were considered to provide favourable cues for the formation of superior multinucleated myotubes on the electrospun meshes compared to those on thin GO sheets. The present results demonstrated that both substrates might be used as potential candidates for CB-hMSCs differentiation and proliferation for human skeletal muscle tissue regeneration.
Graphene oxide and chitosan are promising materials for tissue regeneration. The present study explores a novel biomimetic mineralization route employing a graphene oxide (GO)-chitosan (CS) conjugate as a template material for the biomineralization of hydroxyapatite (HAP). Structural and morphological studies involving X-ray diffraction, Fourier transform infrared spectroscopy, and electron microscopy indicated that extensive mineralization occurred in the CS-GO conjugate system because of strong electrostatic interactions between the functional groups (carboxyl groups of GO and amino groups of CS) and calcium ions in the simulated body fluid (SBF). The combination of chitosan-graphene oxide conjugate and biomineralization was advantageous in favorably modulating cellular activity (osteoblast functions: cell attachment, proliferation, actin, vinculin and fibronectin expression). It is concluded that biomineralized hydroxyapatite in the HAP-CS-GO system induced homogeneous spatial osteoblastic cell growth and quantitatively (e.g. area) and qualitatively (e.g. mineral-to-matrix ratio) increased mineralization in relation to the HAP-GO system. The data underscore that covalent linkage of HAP to chitosan influences osteoblastic cell differentiation, mineralization, and cell growth. The proposed system and the revelation of fundamental insights merit consideration in tissue engineering.
In this study, hyaluronic acid (HA)/poly(lactic-co-glycolic acid, PLGA) core/shell fiber meshes loaded with epigallocatechin-3-O-gallate (EGCG) (HA/PLGA-E) for application to tissue engineering scaffolds for skin regeneration were prepared via coaxial electrospinning.
Physicochemical properties of HA/PLGA-E core/shell fiber meshes were characterized by SEM, Raman spectroscopy, contact angle, EGCG release profiling and in vitro degradation. Biomechanical properties of HA/PLGA-E meshes were also investigated by a tensile strength test. SEM images showed
that HA/PLGA-E fiber meshes had a three-dimensional interconnected pore structure with an average fiber diameter of about 1270 nm. Raman spectra revealed that EGCG was uniformly dispersed in the PLGA shell of meshes. HA/PLGA-E meshes showed sustained EGCG release patterns by controlled diffusion
and PLGA degradation over 4 weeks. EGCG loading did not adversely affect the tensile strength and elastic modulus of HA/PLGA meshes, while increased their hydrophilicity and surface energy. Attachment of human dermal fibroblasts on HA/PLGA-E meshes was appreciably increased and their proliferation
was steadily retained during the culture period. These results suggest that HA/PLGA-E core/shell fiber meshes can be potentially used as scaffolds supporting skin regeneration.
By using ionic liquid 1-hexylpyridinium hexafluorophosphate based carbon ionic liquid electrode (CILE) as the substrate electrode, graphene (GR) and nickel oxide (NiO) were in situ electrodeposited step by step to get a NiO/GR nanocomposite modified CILE. Myoglobin (Mb) was further immobilized on the surface of NiO/GR/CILE with Nafion film to get the electrochemical sensor denoted as Nafion/Mb/NiO/GR/CILE. Cyclic voltammetric experiments indicated that a pair of well-defined quasi-reversible redox peaks appeared in pH 3.0 phosphate buffer solution with the formal peak potential (E0') located at -0.188 V (vs. SCE), which was the typical characteristics of Mb Fe(III)/Fe(II) redox couples. So the direct electron transfer of Mb was realized and promoted due to the presence of NiO/GR nanocomposite on the electrode. Based on the cyclic voltammetric data, the electrochemical parameters of Mb on the modified electrode were calculated. The Mb modified electrode showed an excellent electrocatalytic activity towards the reduction of different substrates including trichloroacetic acid and H2O2. Therefore a third-generation electrochemical Mb biosensor based on NiO/GR/CILE was constructed with good stability and reproducibility.
Many materials in nature change colours in response to stimuli, making them attractive for use as sensor platform. However, both natural materials and their synthetic analogues lack selectivity towards specific chemicals, and introducing such selectivity remains a challenge. Here we report the self-assembly of genetically engineered viruses (M13 phage) into target-specific, colourimetric biosensors. The sensors are composed of phage-bundle nanostructures and exhibit viewing-angle independent colour, similar to collagen structures in turkey skin. On exposure to various volatile organic chemicals, the structures rapidly swell and undergo distinct colour changes. Furthermore, sensors composed of phage displaying trinitrotoluene (TNT)-binding peptide motifs identified from a phage display selectively distinguish TNT down to 300 p.p.b. over similarly structured chemicals. Our tunable, colourimetric sensors can be useful for the detection of a variety of harmful toxicants and pathogens to protect human health and national security.
This study aims at generating highly aligned functional myotubes using graphene as the underlying scaffold. Graphene not only supports the growth of C2C12 muscle cells but also enhances its differentiation and leads to spontaneous patterning of myotubes.
This study concentrates upon investigating differential cellular responses to carbon nanoparticles (CNPs) such as pristine graphene flakes, single-walled carbon nanotubes (SWCNTs) and multiwalled CNTs (MWCNTs), in human dermal fibroblasts (HDFs) vs. the L-929 fibroblast cell line. Characterizing the surface morphology of each CNP by using scanning electron microscopy, we determined their cytotoxicity profiles by quantifying the mitochondrial activity. Graphene was found to have an irregular flake-like shape with various lengths within the ranges of 50 ∼ 1500 nm. The estimated sizes of the SWCNTs and the MWCNTs were about 400 nm and 1000 nm in diameter, respectively and several µm in length. All the CNPs tested here exerted adverse effects on the viability of HDFs even at 15.6 ppm, through intracellular uptake whereas except MWCNTs, they did not show any cytotoxicity against L-929 cells at 250 ppm. This result suggests that CNPs can have a differential influence on normal fibroblasts vs. immortalized ones.
Chitosan has been a popular material for fabrication of tissue engineering scaffolds and has been reported to be a suitable substrate for the culture and differentiation of mesenchymal stem cells (MSCs). In this study, chitosan scaffolds were conjugated with RGD peptides to further enhance the functions of chitosan scaffolds. Biomimetic chitosan scaffolds were prepared using two types of chitosan derivatives, one containing photoreactive azides for UV-crosslinking and the other tethered with RGD peptides. We first fabricated several different chitosan scaffolds with various azide contents to evaluate the mechanical properties of the chitosan scaffolds. The attachment and proliferation of MSCs were greatly enhanced in RGD-incorporated chitosan scaffolds compared with the control scaffolds without RGD conjugation during 2 weeks of culture without osteoinduction. RGD-conjugation also significantly enhanced mineralization of MSCs after 14 days of osteogenic culture. Taken together, the results indicate that the conjugation of RGD peptides to chitosan scaffolds allows for a more favorable microenvironment for MSCs to grow, differentiate into osteoblasts and produce mineralized matrix compared with unmodified chitosan scaffolds. These findings demonstrate the potential of RGD-incorporated, crosslinked chitosan scaffolds for bone tissue engineering applications.
In this study, a porous PLGA/Ti biphasic scaffold was prepared and its potential to repair osteochondral defects was evaluated. The microstructure and mechanical properties of porous PLGA and porous Ti and their interface were characterized. The as-prepared biphasic scaffolds were implanted into rabbit knees for 1 and 3 months (PLGA/Ti group), and the porous PLGA (PLGA group) or empty defect (Untreated group) was also used. The results showed that both porous PLGA and porous Ti had an interconnected porous structure. The mechanical strength of porous PLGA and porous Ti was similar to that of cartilage and subchondral bone, respectively. The interface analysis revealed that the as-prepared biphasic scaffold had good overall integrity and interface stability. The gross observation and histological evaluation of specimens showed that hyaline-like cartilage filled the defects to a certain extent at 3 months for both PLGA/Ti and PLGA groups while the defects remained in an untreated group. In the PLGA/Ti group, better cartilage and subchondral bone repair was observed, and quantitative macroscopic and histological score evaluations confirmed this result, indicating that the porous biphasic PLGA/Ti scaffold had good biocompatibility and the best osteochondral defect repair ability in this study. The as-prepared porous biphasic scaffold showed the potential to be used in osteochondral tissue engineering for osteochondral defect repair.
Significant improvements in the thermomechanical and surface chemical properties of nanocomposite nanofibers of poly(d, l-lactic-co-glycolic acid) (PLGA) were achieved by adding 2-dimensional nanoscale fillers of graphene oxide (GO) nanosheets to PLGA nanofibers. The significant enhancement of storage and loss moduli of the PLGA/GO (2wt.%.) nanocomposite nanofibers were presumably caused by enhanced chemical bonding between the oxygenated functional groups of the highly dispersible GO nanosheets and the hydroxyl groups of the polymer chains in the PLGA matrix, resulting in strong interfacial interactions between the nanofiller and polymer matrix. Enhanced hydrophilicity of nanocomposite nanofibers caused by embedded GO nanosheets also allowed for good biocompatibility of neuronal cells, resulting in enhanced cell proliferation and viability. Our findings indicate that nanocomposite biopolymer nanofibers embedded with GO nanosheets are attractive candidates for use in biomedical applications such as scaffolds.
Electrospinning technique can be used to produce the three-dimensional nanofibrous scaffold similar to natural extracellular matrix, which satisfies particular requirements of tissue engineering scaffold. Randomly-oriented and aligned poly(lactic-co-glycolic acid) (PLGA) and PLGA/gelatin biocomposite scaffolds were successfully produced by electrospinning in the present study. The resulting nanofibrous scaffolds exhibited smooth surface and high porous structure. Blending PLGA with gelatin enhanced the hydrophilicity but decreased the average fiber diameter and the mechanical properties of the scaffolds under the same electrospinning condition. The cell culture results showed that the elongation of the osteoblast on the aligned nanofibrous scaffold was parallel to the fiber arrangement and the cell number was similar to that of randomly-oriented scaffold, indicating that the aligned nanofibrous scaffold provide a beneficial approach for the bone regeneration.
Porous scaffolds fabricated from biocompatible and biodegradable polymers play vital roles in tissue engineering and regenerative medicine. Among various scaffold matrix materials, poly(lactide-co-glycolide) (PLGA) is a very popular and an important biodegradable polyester owing to its tunable degradation rates, good mechanical properties and processibility, etc. This review highlights the progress on PLGA scaffolds. In the latest decade, some facile fabrication approaches at room temperature were put forward; more appropriate pore structures were designed and achieved; the mechanical properties were investigated both for dry and wet scaffolds; a long time biodegradation of the PLGA scaffold was observed and a three-stage model was established; even the effects of pore size and porosity on in vitro biodegradation were revealed; the PLGA scaffolds have also been implanted into animals, and some tissues have been regenerated in vivo after loading cells including stem cells.
Optical clearing (OC) is a promising method to overcome limitations in biomedical depth-resolved optical studies. Mechanisms of OC in purified bovine Achilles tendon, chicken skin, and chicken tendon were studied using time-lapsed, three-dimensional second harmonic generation (SHG) and two-photon fluorescence microscopic imaging. Quantified nonlinear optical measurements allowed temporal separation of two processes in collagen OC with glycerol. The first one is a fast process of tissue dehydration accompanied with collagen shrinkage and the second relatively slow process is glycerol penetration into the interfibrillar space of collagen alongside with CF swelling. The use of 50% glycerol induced less-expressed OC via partial substitution of water molecules with glycerol molecules. We also found that phosphate-buffered saline- and glycerol-treatments were reversible, and fiber morphology and SHG signal intensity were recovered after the removal of immersion agents. It was shown that tissue OC was a dynamic process and elucidation of its physical mechanisms may help choose optimal diagnostic, treatment, and modification regimes for collagen-based as well as other types of biomaterials.
Graphene has unique mechanical, electronic, and optical properties, which researchers have used to develop novel electronic materials including transparent conductors and ultrafast transistors. Recently, the understanding of various chemical properties of graphene has facilitated its application in high-performance devices that generate and store energy. Graphene is now expanding its territory beyond electronic and chemical applications toward biomedical areas such as precise biosensing through graphene-quenched fluorescence, graphene-enhanced cell differentiation and growth, and graphene-assisted laser desorption/ionization for mass spectrometry. In this Account, we review recent efforts to apply graphene and graphene oxides (GO) to biomedical research and a few different approaches to prepare graphene materials designed for biomedical applications.
A well-engineered scaffold for regenerative medicine, which is suitable to be translated from the bench to the bedside, combines inspired design, technical innovation and precise craftsmanship. Electrospinning and additive manufacturing are separate approaches to manufacturing scaffolds for a variety of tissue engineering applications. A need to accurately control the spatial distribution of pores within scaffolds has recently resulted in combining the two processing methods, to overcome shortfalls in each technology. This review describes where electrospinning and additive manufacturing are used together to generate new porous structures for biological applications.
The utilization of high-surface-area, nanofibrillar, electrospun TiO2 thin films with intact fiber morphology as photoanodes in dye-sensitized solar cells has been demonstrated. Electrochemical impedance spectroscopy measurements indicated that these photoanodes exhibited good charge transport and collection. Devices fabricated from these interconnected nanofibers (3 μm thick) attained efficiencies of 4.2% (N719 dye). For comparison, nanoparticle photoanodes of similar thickness were also prepared. The fabrication steps of the cells which preserve the interconnected morphology of the photoanodes are simple and economical.
Graphene-based nanomaterials have received much attention in biomedical applications for drug/gene delivery, cancer therapy, imaging, and tissue engineering. Despite the capacity of 2D carbon materials as a nontoxic and implantable platform, their effect on myogenic differentiation has been rarely studied. We investigated the myotube formation on graphene-based nanomaterials, particularly graphene oxide (GO) and reduced graphene oxide (rGO). GO sheets were immobilized on amine-modified glass to prepare GO-modified glass, which was further reduced by hydrazine treatment for the synthesis of rGO-modified substrate. We studied the behavior, including adhesion, proliferation, and differentiation, of mouse myoblast C2C12 on unmodified, GO-, and rGO-modified glass substrates. According to our analyses of myogenic protein expression, multinucleate myotube formation, and expression of differentiation-specific genes (MyoD, myogenin, Troponin T, and MHC), myogenic differentiation was remarkably enhanced on GO, which resulted from serum protein adsorption and nanotopographical cues. Our results demonstrate the ability of GO to stimulate myogenic differentiation, showing a potential for skeletal tissue engineering applications.
This work presents the latest results on direct laser writing of polymeric materials for tissue engineering applications. A femtosecond Yb:KGW laser (300 fs, 200 kHz, 515 nm) was used as a light source for non-linear lithography. Fabrication was implemented in various photosensitive polymeric materials, such as: hybrid organic-inorganic sol-gel based on silicon-zirconium oxides, commercial ORMOCER® class photoresins. These materials were structured via multi-photon polymerization technique with submicron resolution. Porous three-dimensional scaffolds for artificial tissue engineering were fabricated with constructed system and were up to several millimeters in overall size with 10 to 100 μm internal pores. Biocompatibility of the used materials was tested in primary rabbit muscle-derived stem cell culture in vitro and using laboratory rats in vivo. This interdisciplinary study suggests that proposed technique and materials are suitable for tissue engineering applications.
Long rod-shaped M13 viruses were used to fabricate one-dimensional (1D) micro- and nanosized diameter fibers by mimicking the spinning process of the silk spider. Liquid crystalline virus suspensions were extruded through the micrometer diameter capillary tubes in a cross-linking solution of glutaraldehyde. Resulting fibers were 10−20 μm in diameter. AFM imaging verified that the molecular long axis of the virus fibers was parallel to the fiber long axis. M13 viruses were suspended in 1,1,1,3,3,3-hexafluoro-2-propanol and were then electrospun into fibers. After blending with a highly water soluble polymer, polyvinyl pyrolidone (PVP), M13 viruses were spun into continuous uniform virus-blended PVP (virus-PVP) nanofibers. Resulting virus-PVP electrospun fibers maintained their ability to infect bacterial hosts after resuspending in buffer solution.
A systematic study of the effects of Mw, flow rate, voltage, and composition on the morphology of electrospun PLGA nanofibers is reported. It is shown that changes of voltage and flow rate do not appreciably affect the morphology. However, the Mw of PLGA predominantly determines the formation of bead structures. Uniform electrospun PLGA nanofibers with controllable diameters can be formed through optimization. Further, multi-walled carbon nanotubes can be incorporated into the PLGA nanofibers, significantly enhancing their tensile strength and elasticity without compromising the uniform morphology. The variable size, porosity, and composition of the nanofibers are essential for their applications in regenerative medicine.
Graphene, with its excellent physical, chemical, and mechanical properties, holds tremendous potential for a wide variety of biomedical applications. As research on graphene-based nanomaterials is still at a nascent stage due to the short time span since its initial report in 2004, a focused review on this topic is timely and necessary. In this feature review, we first summarize the results from toxicity studies of graphene and its derivatives. Although literature reports have mixed findings, we emphasize that the key question is not how toxic graphene itself is, but how to modify and functionalize it and its derivatives so that they do not exhibit acute/chronic toxicity, can be cleared from the body over time, and thereby can be best used for biomedical applications. We then discuss in detail the exploration of graphene-based nanomaterials for tissue engineering, molecular imaging, and drug/gene delivery applications. The future of graphene-based nanomaterials in biomedicine looks brighter than ever, and it is expected that they will find a wide range of biomedical applications with future research effort and interdisciplinary collaboration.
Graphene oxide (GO), has created an unprecedented opportunity for development and application in biology, due to its abundant functional groups and well water solubility. Recently, the potential toxicity of GO in the environment and in humans has garnered more and more attention. In this paper, we systematically studied the cytotoxicity of GO nanosheets via examining the effect of GO on the morphology, viability and differentiation of a human neuroblastoma SH-SY5Y cell line, which was an ideal model used to study neuronal disease in vitro. The results suggested that GO had no obvious cytotoxicity at low concentration (<80 μg mL(-1)) for 96 h, but the viability of cells exhibited dose- and time-dependent decreases at high concentration (≥ 80 μg mL(-1)). Moreover, GO did not induce apoptosis. Very interestingly, GO significantly enhanced the differentiation of SH-SY5Y induced-retinoic acid (RA) by evaluating neurite length and the expression of neuronal marker MAP2. These data provide a promising application for neurodegenerative diseases.
We evaluated the toxicity of graphite nanoplatelets (GNPs) in the model organism Caenorhabditis elegans. The GNPs resulted nontoxic by measuring longevity as well as reproductive capability end points. An imaging technique based on Fourier transform infrared spectroscopy (FT-IR) mapping was also developed to analyze the GNPs spatial distribution inside the nematodes. Conflicting reports on the in vitro antimicrobial properties of graphene-based nanomaterials prompted us to challenge the host-pathogen system C. elegans-Pseudomonas aeruginosa to assess these findings through an in vivo model.
Electrospinning is a promising approach to create nanofiber structures that are capable of supporting adhesion and guiding extension of neurons for nerve regeneration. Concurrently, electrical stimulation of neurons in the absence of topographical features also has been shown to guide axonal extension. Therefore, the goal of this study was to form electrically conductive nanofiber structures and to examine the combined effect of nanofiber structures and electrical stimulation. Conductive meshes were produced by growing polypyrrole (PPy) on random and aligned electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers, as confirmed by scanning electron micrographs and X-ray photon spectroscopy. PPy–PLGA electrospun meshes supported the growth and differentiation of rat pheochromocytoma 12 (PC12) cells and hippocampal neurons comparable to non-coated PLGA control meshes, suggesting that PPy–PLGA may be suitable as conductive nanofibers for neuronal tissue scaffolds. Electrical stimulation studies showed that PC12 cells, stimulated with a potential of 10 mV/cm on PPy–PLGA scaffolds, exhibited 40–50% longer neurites and 40–90% more neurite formation compared to unstimulated cells on the same scaffolds. In addition, stimulation of the cells on aligned PPy–PLGA fibers resulted in longer neurites and more neurite-bearing cells than stimulation on random PPy–PLGA fibers, suggesting a combined effect of electrical stimulation and topographical guidance and the potential use of these scaffolds for neural tissue applications.
In this work, we evaluate the physical properties of nylon 6 nonwoven mats produced from solutions with formic acid. Nonwoven electrospun mats from various solutions with different concentration are examined regarding their morphology, pore size, surface area, and gas transport properties. Each nonwoven mat with average fiber diameters from 90 to 500 nm was prepared under controlled electrospinning process parameters. From the results, it was observed that the fiber diameter was strongly affected by the polymer concentration (polymer viscosity). In additional the results showed that the pore size, Brunauer–Emmett–Teller (BET) surface area, and gas transport property of electrospun nylon 6 nonwoven mats were affected by the fiber diameter.
Electrospinning is a process that produces continuous polymer fibers with diameters in the sub-micron range through the action of an external electric field imposed on a polymer solution or melt. Non-woven textiles composed of electrospun fibers have a large specific surface area and small pore size compared to commercial textiles, making them excellent candidates for use in filtration and membrane applications. While the process of electrospinning has been known for over half a century, current understanding of the process and those parameters, which influence the properties of the fibers produced from it, is very limited. In this work, we have evaluated systematically the effects of two of the most important processing parameters: spinning voltage and solution concentration, on the morphology of the fibers formed. We find that spinning voltage is strongly correlated with the formation of bead defects in the fibers, and that current measurements may be used to signal the onset of the processing voltage at which the bead defect density increases substantially. Solution concentration has been found to most strongly affect fiber size, with fiber diameter increasing with increasing solution concentration according to a power law relationship. In addition, electrospinning from solutions of high concentration has been found to produce a bimodal distribution of fiber sizes, reminiscent of distributions observed in the similar droplet generation process of electrospray. In addition, we find evidence that electrostatic effects influence the macroscale morphology of electrospun textiles, and may result in the formation of heterogeneous or three-dimensional structures.