3D Fibrous Scaffolds Research Articles

Electrohydrodynamic jet process for pore-structure-controlled 3D fibrous architecture as a tissue regenerative material: fabrication and cellular activities.

In this study, we propose a new scaffold fabrication method, "direct electro-hydrodynamic jet process," using the initial jet of an electrospinning process and ethanol media as a target. The fabricated three-dimensional (3D) fibrous structure was configured with multilayered microsized struts consisting of randomly entangled micro/nanofibrous architecture, similar to that of native extracellular matrixes. The fabrication of the structure was highly dependent on various processing parameters, such as the surface tension of the target media, and the flow rate and weight fraction of the polymer solution. As a tissue regenerative material, the 3D fibrous scaffold was cultured with preosteoblasts to observe the initial cellular activities in comparison with a solid-freeform fabricated 3D scaffold sharing a similar structural geometry. The cell-culture results showed that the newly developed scaffold provided outstanding microcellular environmental conditions to the seeded cells (about 3.5-fold better initial cell attachment and 2.1-fold better cell proliferation).

Advanced micro- and nanofabrication technologies for tissue engineering

The ability of the living body to heal and regenerate itself following trauma is astonishing. Numerous events of repair and regeneration occur during our lifetime, most of which we are never aware of. Unfortunately, in some cases, the injury or defect cannot be adequately repaired solely by nature and medical intervention is required.

One-stop microfiber spinning and fabrication of a fibrous cell-encapsulated scaffold on a single microfluidic platform

This paper provides a method for microscale fiber spinning and the in situ construction of a 3D fibrous scaffold on a single microfluidic platform. This platform was also used to fabricate a variety of fibrous scaffolds with diverse compositions without the use of complicated devices. We explored the potential utility of the fibrous scaffolds for tissue engineering applications by constructing a fibrous scaffold encapsulating primary hepatocytes. The cells in scaffold were cultured over seven days and maintained higher viability comparing with 3D alginate non-fibrous block. The main advantage of this platform is that the fibrous structure used to form a scaffold can be generated without damaging the mechanically weak alginate fibers or encapsulated cells because all procedures are performed in a single platform without the intervention of the operator. In addition, the proposed fibrous scaffold permitted high diffusion capability of molecules, which enabled better viability of encapsulated cells than non-fibrous scaffold even in massive cell culture.

Scaffold Architecture Controls Insulinoma Clustering, Viability, and Insulin Production

Recently, in vitro diagnostic tools have shifted focus toward personalized medicine by incorporating patient cells into traditional test beds. These cell-based platforms commonly utilize two-dimensional substrates that lack the ability to support three-dimensional cell structures seen in vivo. As monolayer cell cultures have previously been shown to function differently than cells in vivo, the results of such in vitro tests may not accurately reflect cell response in vivo. It is therefore of interest to determine the relationships between substrate architecture, cell structure, and cell function in 3D cell-based platforms. To investigate the effect of substrate architecture on insulinoma organization and function, insulinomas were seeded onto 2D gelatin substrates and 3D fibrous gelatin scaffolds with three distinct fiber diameters and fiber densities. Cell viability and clustering was assessed at culture days 3, 5, and 7 with baseline insulin secretion and glucose-stimulated insulin production measured at day 7. Small, closely spaced gelatin fibers promoted the formation of large, rounded insulinoma clusters, whereas monolayer organization and large fibers prevented cell clustering and reduced glucose-stimulated insulin production. Taken together, these data show that scaffold properties can be used to control the organization and function of insulin-producing cells and may be useful as a 3D test bed for diabetes drug development.

Intrinsically water-stable electrospun three-dimensional ultrafine fibrous soy protein scaffolds for soft tissue engineering using adipose derived mesenchymal stem cells

Soy protein, the plant protein from soybean, was electrospun into intrinsically water-stable scaffolds with large volume and ultrafine fibers oriented randomly and evenly in three dimensions (3D) to simulate native extracellular matrices of soft tissues. The 3D ultrafine fibrous scaffolds from proteins could be favored in soft tissue engineering. However, protein-based biomaterials usually suffered from poor water stability, while the highly crosslinked proteins which had water stability were usually difficult to be fabricated into fibers. Soy protein was a typical protein with intrinsic water stability, attributed to its 1.2% cysteine content. Soy protein has been developed into 3D non-fibrous structures, coarse fibers and films for tissue engineering applications, but not ultrafine fibrous structures. In this research, the disulfide crosslinks in soy protein were cleaved to facilitate its dissolution in an aqueous solvent system. The obtained solution was electrospun into bulky scaffolds composed of ultrafine fibers oriented randomly in three dimensions. Without external crosslinking, the fibrous soy protein scaffolds demonstrated long-term water stability, and maintained their fibrous structures after incubated in PBS for up to 28 days. In vitro study showed that the 3D soy protein scaffolds well supported uniform distribution and adipogenic differentiation of adipose derived mesenchymal stem cells. In summary, the 3D ultrafine fibrous soy protein structures could be good candidates as scaffolds in soft tissue engineering.

Layer-by-layer assembly of silica nanoparticles on 3D fibrous scaffolds: Enhancement of osteoblast cell adhesion, proliferation, and differentiation

Silica nanoparticles were applied onto the fiber surface of an interbonded three-dimensional polycaprolactone fibrous tissue scaffold by an electrostatic layer-by-layer self-assembly technique. The nanoparticle layer was found to improve the fiber wettability and surface roughness. Osteoblast cells were cultured on the fibrous scaffolds to evaluate the biological compatibility. The silica nanoparticle coated scaffold showed enhanced cell attachment, proliferation, and alkaline phosphatase activities. The overall results suggested that interbonded fibrous scaffold with silica nanoparticulate coating could be a promising scaffolding candidate for various applications in bone repair and regeneration.

Highly porous 3D nanofibrous scaffolds processed with an electrospinning/laser process

Electrospinning has been widely used to produce micro/nanosized fibres. Although the method is very simple, easy, and effective for obtaining nanosized material, the fabrication of three dimensional (3D) shapes comprised of micro/nanofibres has been a major obstacle for use in tissue engineering. In this study, a new electrospinning method to fabricate controllable 3D micro/nanofibrous structure (with thickness over 3 mm) is suggested. The fabricated 3D fibrous structure was fully porous and successfully consisted of submicron-sized fibres. However, the pores in the 3D fibrous structure were too small (5–10 μm), so we used a femtosecond laser process to achieve enough cell infiltration and proliferation in the thickness direction of the 3D structure. By controlling appropriate processing conditions, we can successfully fabricate a highly porous 3D micro/nanofibrous structure with various pore sizes ranging from 189 ± 28 μm to 380 ± 21 μm. The fabricated 3D fibrous scaffolds were assessed for in vitro biological capabilities by culturing osteoblast like cells (MG63). Compared with the rapid-prototyped PCL scaffold, the 3D fibrous scaffold exhibited significantly higher biological activities (initial cell attachment and cell proliferation) due to the topographical structure of micro/nanofibres.

A 3D Fibrous Scaffold Inducing Tumoroids: A Platform for Anticancer Drug Development

The development of a suitable three dimensional (3D) culture system for anticancer drug development remains an unmet need. Despite progress, a simple, rapid, scalable and inexpensive 3D-tumor model that recapitulates in vivo tumorigenesis is lacking. Herein, we report on the development and characterization of a 3D nanofibrous scaffold produced by electrospinning a mixture of poly(lactic-co-glycolic acid) (PLGA) and a block copolymer of polylactic acid (PLA) and mono-methoxypolyethylene glycol (mPEG) designated as 3P. Cancer cells cultured on the 3P scaffold formed tight irregular aggregates similar to in vivo tumors, referred to as tumoroids that depended on the topography and net charge of the scaffold. 3P scaffolds induced tumor cells to undergo the epithelial-to-mesenchymal transition (EMT) as demonstrated by up-regulation of vimentin and loss of E-cadherin expression. 3P tumoroids showed higher resistance to anticancer drugs than the same tumor cells grown as monolayers. Inhibition of ERK and PI3K signal pathways prevented EMT and reduced tumoroid formation, diameter and number. Fine needle aspirates, collected from tumor cells implanted in mice when cultured on 3P scaffolds formed tumoroids, but showed decreased sensitivity to anticancer drugs, compared to tumoroids formed by direct seeding. These results show that 3P scaffolds provide an excellent platform for producing tumoroids from tumor cell lines and from biopsies and that the platform can be used to culture patient biopsies, test for anticancer compounds and tailor a personalized cancer treatment.

Surface modification of poly-l-lactic acid fibrous scaffolds by a molecular-designed multi-walled carbon nanotube multilayer for enhancing cell interactions

We presented a molecular-designed multi-walled carbon nanotube (MWCNT) layer-by-layer (LbL) multilayer on poly-l-lactic acid (PLLA) electrospun fibers for engineering cell/CNT interfaces. A stable, positively charged monolayer was created on the fiber surface by the aminolysis reaction of poly(ethylene imine) (PEI) with PLLA, followed by alternate deposition in negatively charged MWCNT and positively charged chitosan (CS). Thermogravimetric analysis indicated a sustained growth of the MWCNT during the self-assembly process. The interactions between MWCNT and polycation crucially affected the specific structure and properties of the MWCNT multilayer. MWCNT/PEI electrostatic interactions reduced the gap between MWCNTs and improved the π→π∗ transitions. However, the CS chains tended to be more serpentine than the chains of PEI molecules, which might have hindered the π→π∗ transitions. On the other hand, the electrostatic interactions might have enhanced the disorder grade of the MWCNT structure, as indicated by Raman analysis. The scaffolds maintained their fibrous and porous structure after MWCNT multilayer modification and supported fibroblast growth. The MWCNT multilayer induced cell migration toward the interior of the scaffolds. Therefore, we created a simple yet efficient method of building a CNT multilayer on three-dimensional (3D) fibrous scaffolds for enhancing cell-matrix interactions.

A novel technique for the fabrication of 3D nanofibrous scaffolds using simultaneous positive voltage electrospinning and negative voltage electrospinning

Electrospinning is a very attractive technology for fabricating nanofibrous structures. However, for potential tissue engineering applications, the conventional electrospinning technique can only produce 2D nanofibrous membranes with limited thickness. In this investigation, a novel technique, which involved simultaneous positive voltage electrospinning (PVES) and negative voltage electrospinning (NVES), was developed for constructing 3D nanofibrous scaffolds with greatly increased thickness. Using a specially designed electrospinning device, the problem of attraction and agglomeration of oppositely charged fibers in mid-air during simultaneous PVES and NVES could be avoided. As a demonstration and for comparison, two types of polymers, polyvinyl alcohol and poly(d,l-lactic acid), were processed into fibrous scaffolds using conventional and novel electrospinning techniques, respectively. For each polymer, the novel technique rendered the formation of 3D fibrous scaffolds that could not be achieved via conventional electrospinning. The polymer type, applied voltage and scaffold fabrication technique were critical factors affecting the thickness of nanofibrous scaffolds. The mechanism for 3D scaffold formation was proposed.

Layer‐by‐layer assembly of antibacterial coating on interbonded 3D fibrous scaffolds and its cytocompatibility assessment

Bonded fibrous matrices have shown great potential in tissue engineering because of their unique 3D structures and pore characteristics. For some applications, bacterial infections must be taken into account, and antibacterial function is highly desired. In this study, an antibacterial polymer, polyhexamethylene biguanide (PHMB), was applied onto the fiber surface of a bonded poly(ε-caprolactone) (PCL) fibrous matrix with the objective to achieve both strong antibacterial effect and good cell compatibility. The coatings were prepared by using an electrostatic layer-by-layer (LbL) assembly technique, which allowed the control of PHMB loading and coating uniformity on the fiber surface. The PHMB coating provided antibacterial activities, but had no toxicity on mammalian cells. This bonded PCL fibrous matrix with electrostatically self-assembled PHMB may provide a new antiinfective tissue scaffold for various biomedical applications.

Novel fabrication technique for three‐dimensional micropatterned electrospun poly(DL‐lactide‐co‐glycolide) acid

AbstractCurrent tissue engineering scaffolds created by electrospinning techniques are mostly limited to two‐dimensional membranes. In this article, a novel method for fabricating three‐dimensional (3D) fibrous scaffolds is presented. The fabrication technique uses the saturation of carbon dioxide in poly(lactide‐co‐glycolide) acid to lower the glass‐transition temperature of the polymer to allow for the sintering of multiple layers of electrospun films. The effect of gas saturation on the films was first examined by variation of the saturation pressure and time. Fiber sintering was observed as the saturation pressure and time were increased. Next, the adhesion strength between two layers of scaffolds that were sintered by gas saturation was examined, and a higher pressure of 400 psi was found to better adhere the layers of the membranes together. From the first two parts of this study, the optimal combination of gas saturation parameters was determined to a pressure of 400 psi for 3 min. With this set of parameters, 3D structures were fabricated by the sintering of 30 layers of electrospun scaffolds. Also, by patterning individual layers using microfeatured plates, we improved the open porosity. This demonstration of the ability to fabricate 3D scaffolds improves current electrospinning techniques and maintains a desirable fibrous structure for tissue engineering. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

A 3D Electroactive Polypyrrole-Collagen Fibrous Scaffold for Tissue Engineering

Fibers that can provide topographical, biochemical and electrical cues would be attractive for directing the differentiation of stem cells into electro-responsive cells such as neuronal or muscular cells. Here we report on the fabrication of polypyrrole-incorporated collagen-based fibers via interfacial polyelectrolyte complexation (IPC). The mean ultimate tensile strength of the fibers is 304.0 ± 61.0 MPa and the Young’s Modulus is 10.4 ± 4.3 GPa. Human bone marrow-derived mesenchymal stem cells (hMSCs) are cultured on the fibers in a proliferating medium and stimulated with an external electrical pulse generator for 5 and 10 days. The effects of polypyrrole in the fiber system can be observed, with hMSCs adopting a neuronal-like morphology at day 10, and through the upregulation of neural markers, such as noggin, MAP2, neurofilament, β tubulin III and nestin. This study demonstrates the potential of this fiber system as an attractive 3D scaffold for tissue engineering, where collagen is present on the fiber surface for cellular adhesion, and polypyrrole is encapsulated within the fiber for enhanced electrical communication in cell-substrate and cell-cell interactions.

Characterizations of Chondrocyte Attachment and Proliferation on Electrospun Biodegradable Scaffolds of PLLA and PBSA for Use in Cartilage Tissue Engineering

The influence of physical characteristics of electrospun three-dimensional (3D) fibrous scaffolds based on polybutylene succinate-co-adipate (PBSA) and poly l-lactic acid (PLLA) on the culture of primary human chondrocytes (PHCs) in terms of cell attachment, proliferation, and re-differentiation was investigated. Physical characteristics assessed for two polymers electrospun at two different delivery rates (PBSA-3, PBSA-16, PLLA-3, and PLLA-16) including average fiber diameter, average pore diameter, porosity, and contact angle. Results demonstrated that 3D fibrous scaffolds are better for PHCs' attachment than two-dimensional (2D) casting films made of the same polymeric materials. It was also found that 3D fibrous scaffolds are appropriate architecture for the proliferation of PHCs than 2D casting films and dependent upon the polymer used. Histological analysis revealed that a significant amount of PHC was found to be growing only within layers of PLLA fibrous scaffolds. The mitochondrial ribonucleic acid (mRNA) expression of both aggrecan and type II collagen by PHCs cultured in tissue culture polystyrene for 28 days decreased significantly. The mRNA expression of both aggrecan and type II collagen by PHCs cultured in PBSA scaffolds increased from 14 to 28 days, whereas only mRNA expression of aggrecan cultured in both PLLA scaffolds increased from 14 to 28 days.

A novel route for the production of chitosan/poly(lactide-co-glycolide) graft copolymers for electrospinning

Both chitosan and polylactide/polyglycolide have good biocompatibility and can be used to produce tissue engineering scaffolds for cultured cells. However the synthetic scaffolds lack groups that would facilitate their modification, whereas chitosan has extensive active amide and hydroxyl groups which would allow it to be subsequently modified for the attachment of peptides, proteins and drugs. Also chitosan is very hydrophilic, whereas PLGA is relatively hydrophobic. Accordingly there are many situations where it would be ideal to have a copolymer of both, especially one that could be electrospun to provide a versatile range of scaffolds for tissue engineering. Our aim was to develop a novel route of chitosan-g-PLGA preparation and evaluate the copolymers in terms of their chemical characterization, their performance on electrospinning and their ability to support the culture of fibroblasts as an initial biological evaluation of these scaffolds. Chitosan was first modified with trimethylsilyl chloride, and catalyzed by dimethylamino pyridine. PLGA-grafted chitosan copolymers were prepared by reaction with end-carboxyl PLGA (PLGA–COOH). FT-IR and1H-NMR characterized the copolymer molecular structure as being substantially different to that of the chitosan or PLGA on their own. Elemental analysis showed an average 18 pyranose unit intervals when PLGA–COOH was grafted into the chitosan molecular chain. Differential scanning calorimetry results showed that the copolymers had different thermal properties from PLGA and chitosan respectively. Contact angle measurements demonstrated that copolymers became more hydrophilic than PLGA. The chitosan-g-PLGA copolymers were electrospun to produce either nano- or microfibers as desired. A 3D fibrous scaffold of the copolymers gave good fibroblast adhesion and proliferation which did not differ significantly from the performance of the cells on the chitosan or PLGA electrospun scaffolds. In summary this work presents a methodology for making a hybrid material of natural and synthetic polymers which can be electrospun and reacts well as a substrate for cell culture.

Myogenic differentiation of human bone marrow mesenchymal stem cells on a 3D nano fibrous scaffold for bladder tissue engineering

Current strategies for engineering bladder tissues include a bladder biopsy for in vitro cell expansion for use in reconstructive procedures. However, this approach cannot be used in patients with bladder cancer who need a complete bladder replacement. Bone marrow mesenchymal stem cells (BMSC) might be an alternative cell source to better meet this need. We investigated the effects of soluble growth factors, bladder extracellular matrix (ECM), and 3D dynamic culture on cell proliferation and differentiation of human BMSC into smooth muscle cells (SMC). Myogenic growth factors (PDGF-BB and TGF-β1) alone, or combined either with bladder ECM or dynamic cultures, induced BMSC to express smooth muscle-specific genes and proteins. Either ECM or the dynamic culture alone promoted cell proliferation but did not induce myogenic differentiation of BMSC. A highly porous poly- l-lactic acid (PLLA) scaffold provided a 3D structure for maximizing the cell-matrix penetration, maintained myogenic differentiation of the induced BMSC, and promoted tissue remolding with rich capillary formation in vivo. Our results demonstrate that myogenic-differentiated BMSC seeded on a nano fibrous PLLA scaffold can be potentially used for cell-based tissue engineering for bladder cancer patients requiring cystoplasty.

Design of Biphasic Polymeric 3-Dimensional Fiber Deposited Scaffolds for Cartilage Tissue Engineering Applications

This report describes a novel system to create rapid prototyped 3-dimensional (3D) fibrous scaffolds with a shell-core fiber architecture in which the core polymer supplies the mechanical properties and the shell polymer acts as a coating providing the desired physicochemical surface properties. Poly[(ethylene oxide) terephthalate-co-poly(butylene) terephthalate] (PEOT/PBT) 3D fiber deposited (3DF) scaffolds were fabricated and examined for articular cartilage tissue regeneration. The shell polymer contained a higher molecular weight of the initial poly(ethylene glycol) (PEG) segments used in the copolymerization and a higher weight percentage of the PEOT domains compared with the core polymer. The 3DF scaffolds entirely produced with the shell or with the core polymers were also considered. After 3 weeks of culture, scaffolds were homogeneously filled with cartilage tissue, as assessed by scanning electron microscopy. Although comparable amounts of entrapped chondrocytes and of extracellular matrix formation were found for all analyzed scaffolds, chondrocytes maintained their rounded shape and aggregated during the culture period on shell-core 3DF scaffolds, suggesting a proper cell differentiation into articular cartilage. This finding was also observed in the 3DF scaffolds fabricated with the shell composition only. In contrast, cells spread and attached on scaffolds made simply with the core polymer, implying a lower degree of differentiation into articular cartilaginous tissue. Furthermore, the shell-core scaffolds displayed an improved dynamic stiffness as a result of a "prestress" action of the shell polymer on the core one. In addition, the dynamic stiffness of the constructs increased compared with the stiffness of the bare scaffolds before culture. These findings suggest that shell-core 3DF PEOT/PBT scaffolds with desired mechanical and surface properties are a promising solution for improved cartilage tissue engineering.

Runx2/Cbfa1-genetically engineered skeletal myoblasts mineralize collagen scaffolds in vitro.

Genetic engineering of progenitor and stem cells is an attractive approach to address cell sourcing limitations associated with tissue engineering applications. Bone tissue engineering represents a promising strategy to repair large bone defects, but has been limited in part by the availability of a sustained, mineralizing cell source. This study examined the in vitro mineralization potential of primary skeletal myoblasts genetically engineered to overexpress Runx2/Cbfa1, an osteoblastic transcriptional regulator essential to bone formation. These cells were viable at the periphery of 3D fibrous collagen scaffolds for 6 weeks of static culture. Exogenous Runx2 expression induced osteogenic differentiation and repressed myogenesis in these constructs relative to controls. Runx2-modified cells deposited significant amounts of mineralized matrix and hydroxyapatite, as determined by microcomputed tomography, histological analysis, and Fourier transform infrared spectroscopy, whereas scaffolds seeded with control cells exhibited no mineralized regions. Although mineralization by Runx2-engineered cells was confined to the periphery of the construct, colocalizing with cell viability, it was sufficient to increase the compressive modulus of constructs 30-fold relative to controls. This work demonstrates that Runx2 overexpression in skeletal myoblasts may address current obstacles of bone tissue engineering by providing a potent cell source for in vitro mineralization and construct maturation. Additionally, the use of genetic engineering methods to express downstream control factors and transcriptional regulators, in contrast to soluble signaling molecules, represents a robust strategy to enhance cellular activities for tissue engineering applications.