Aligned Electrospun Scaffolds Research Articles

Fabrication and characterisation of random and aligned electrospun scaffolds to investigate hypothalamic stem/progenitor cell behaviour

Tanycytes are stem/progenitor cells that reside in the hypothalamus of the adult vertebrate brain. Tanycytes can be cultured as free-floating neurospheres in vitro but tend to spontaneously differentiate over time. Here we asked whether morphological cues provided by engineered polymer scaffolds can modify spontaneous differentiation. Tanycyte-derived neurospheres were cultured on electrospun scaffolds, prepared with either random or aligned fiber morphologies. Cells dispersed widely on the scaffolds, and - on aligned scaffolds - were highly organized, orientated parallel to the fibers. Immunocytochemical analysis showed that cells cultured on aligned scaffolds showed significantly greater expression of the neural stem/progenitor cell marker, NrCAM and reduced expression of differentiated cell markers in comparison to those cultured on random scaffolds. Together this shows that tanycytes respond to local engineered cues, and that a morphologically constrained environment can better maintain tanycytes as stem cells. The aligned scaffold culture system provides a powerful tool to better investigate this novel stem/progenitor cell population.

Bioinspired electrospun decellularized extracellular matrix scaffolds promote muscle regeneration in a rat skeletal muscle defect model.

Volumetric muscle loss is a debilitating injury that can leave patients with long-lasting or permanent structural and functional deficits. With clinical treatments failing to address these shortcomings, there is a great need for tissue-engineered therapies to promote skeletal muscle regeneration. In this study, we aim to assess the potential for electrospun decellularized skeletal muscle extracellular matrix (dECM) to promote skeletal muscle regeneration in a rat partial thickness tibialis anterior defect model. Aligned electrospun scaffolds with varying degrees of crosslinking density were implanted into the defect site and compared to an empty defect control. After 8 weeks, muscles were harvested, weighed, and cellular and morphological analyses were performed via histology and immunohistochemistry. Cell infiltration, angiogenesis, and myogenesis were observed in the defect site in both dECM groups. However, favorable mechanical properties and slower degradation kinetics resulted in greater support of tissue remodeling in the more crosslinked scaffolds and preservation of existing myofiber area in both dECM groups compared to the empty defect control. More sustained release of pro-regenerative degradation products also promoted greater myofiber formation in the defect site. This study allowed for a greater understanding of how electrospun skeletal muscle scaffolds interact with existing skeletal muscle and can inform their potential as a therapy in a wide variety of soft tissue applications.

Architected fibrous scaffolds for engineering anisotropic tissues

Mimicking the native three-dimensional microenvironment is of crucial importance when biofabricating a new healthcare material. One aspect of the native tissue that is often omitted when designing a suitable scaffold is its anisotropy. Not only is matching native mechanical properties important when designing implantable scaffolds or healthcare materials, but matching physiological structure is also important as many cell populations respond differently to fiber orientation. Therefore, novel aligned electrospun scaffolds with varying fiber angles and spacing of bundles were created and mechanically characterized. Through controlling the angle between the fibers in each layer of the scaffold, a range of different physiological anisotropic mechanical properties were achieved that encompasses values found in native tissues. Extrapolation of this mechanical data allowed for any native tissue’s anisotropic Young’s modulus to be mimicked by electrospinning fibers at a particular angle. These electrospun scaffolds were then incorporated with cell-laden hydrogels to create hybrid structures that contain the benefits of both scaffolding techniques with the ability to encapsulate cells in the hydrogel. To conclude, this study develops a novel bundled fiber scaffold that was architected to yield anisotropic properties matching native tissues.

Adipose-Derived Stem Cells Growth and Proliferation Enhancement Using Poly(Lactic-co-Glycolic Acid) (PLGA)/Fibrin Nanofiber Mats

Introduction: A synthetic biomaterial, such as poly(lactic-co-glycolic acid) (PLGA) with superior mechanical properties, along with a natural polymer such as fibrin, which facilitates cell attachment and enhances biocompatibility, can be used in the production of novel composite tissue engineering scaffolds. In this study, we fabricated a PLGA/Fibrin scaffold which was evaluated to the enhancement of adipose-derived stem cells' growth and proliferation. Materials and Methods: 10% polymer solutions with different ratios of PLGA:Fibrin, including 10:0, 9:1, 8:2, and 7:3, were prepared and used in the production of aligned and unaligned electrospun nanofiber scaffolds. Human adipose-derived stem cells (h-ADSCs) were cultured on the scaffolds and they were characterized using FTIR, EDX, SEM, mechanical, hydrophilic, degradation, water absorption, and biocompatibility tests. Results: The obtained scaffolds consisted of homogeneous fibers, without any beads and water droplets. The percentage of porosities and internal correlation of the cavities are not significantly different between aligned and unaligned electrospun scaffolds (P>0.05) and by adding fibrin, these properties are improved, while tensile strength and elasticity are decreased. All the scaffolds are hydrophobic and the highest and lowest swelling rates belong to PLGA/30%Fibrin scaffolds and pure PLGA scaffolds (more than 90% and less than 45%, respectively). There is a significant difference in degradation rates between fibrin-contained scaffolds and pure PLGA scaffolds. Moreover, compared with aligned electrospun scaffolds, a higher degradation rate of unaligned ones was observed. Conclusions: Considering the results of SEM and bio-compatibility experiments, the aligned electrospun PLGA/10%Fibrin scaffold with numerous spindle shape h-ADSCs and unaligned electrospun PLGA/20%Fibrin scaffold with lots of spindle shape cells together with sort of round shape cells are introduced as the optimal options.

Investigation of the batch-to-batch inconsistencies of Collagen in PCL-Collagen nanofibers

The application of tissue engineered constructs is an approach in regenerative therapies to support regeneration of damaged muscular tissue. Therefore the development of highly aligned electrospun scaffolds based on polycaprolactone (PCL) and collagen enables a versatile development towards tailor made applications. However, the application of natural polymers like collagen brings the risk of batch-to-batch inconsistencies, which influence the reproducibility of the electrospinning process. Aligned PCL-Collagen nanofibers were fabricated via electrospinning using benign solvents. The spinnability of different collagen batches and polymer concentrations in diluted acetic acid as solvent was investigated. Furthermore spinning parameters and fiber morphology were investigated in order to determine the most stable spinning conditions and analyze the batch-to-batch variations. Finally the effect of the solution temperature and the time of pure collagen in solution were investigated, to complete the analysis of the influences on the spinning behavior.

Enhanced Osteogenesis of BMP2-Transfected Human Periodontal Ligament Stem Cells by Aligned Electrospun Scaffolds for Bone Tissue Engineering

Enhanced Osteogenesis of BMP2-Transfected Human Periodontal Ligament Stem Cells by Aligned Electrospun Scaffolds for Bone Tissue Engineering

Enhancing the Biomechanical Performance of Anisotropic Nanofibrous Scaffolds in Tendon Tissue Engineering: Reinforcement with Cellulose Nanocrystals.

Anisotropically aligned electrospun nanofibrous scaffolds based on natural/synthetic polymer blends have been established as a reasonable compromise between biological and biomechanical performance for tendon tissue engineering (TE) strategies. However, the limited tensile properties of these biomaterials restrict their application in this field due to the load-bearing nature of tendon/ligament tissues. Herein, the use of cellulose nanocrystals (CNCs) as reinforcing nanofillers in aligned electrospun scaffolds based on a natural/synthetic polymer blend matrix, poly-ε-caprolactone/chitosan (PCL/CHT) is reported. The incorporation of small amounts of CNCs (up to 3 wt%) into tendon mimetic nanofiber bundles has a remarkable biomaterial-toughing effect (85% ± 5%, p < 0.0002) and raises the scaffolds mechanical properties to tendon/ligament relevant range (σ = 39.3 ± 1.9 MPa and E = 540.5 ± 83.7 MPa, p < 0.0001). Aligned PCL/CHT/CNC nanocomposite fibrous scaffolds meet not only the mechanical requirements for tendon TE applications but also provide tendon mimetic extracellular matrix (ECM) topographic cues, a key feature for maintaining tendon cell's morphology and behavior. The strategy proposed here may be extended to other anisotropic aligned nanofibrous scaffolds based on natural/synthetic polymer blends and enable the full exploitation of the advantages provided by their tendon mimetic fibrous structures in tendon TE.

Electrospun fiber constructs for vocal fold tissue engineering: Effects of alignment and elastomeric polypeptide coating

Vocal fold lamina propria extracellular matrix (ECM) is highly aligned and when injured, becomes disorganized with loss of the tissue’s critical biomechanical properties. This study examines the effects of electrospun fiber scaffold architecture and elastin-like polypeptide (ELP4) coating on human vocal fold fibroblast (HVFF) behavior for applications toward tissue engineering the vocal fold lamina propria. Electrospun Tecoflex™ scaffolds were made with aligned and unaligned fibers, and were characterized using scanning electron microscopy and uniaxial tensile testing. ELP4 was successfully adsorbed onto the scaffolds; HVFFs were seeded and their viability, proliferation, morphology and gene expression were characterized. Aligned and unaligned scaffolds had initial elastic moduli of ∼14MPa, ∼5MPa and ∼0.3MPa, ∼0.6MPa in the preferred and cross-preferred directions, respectively. Scaffold topography had an effect on the orientation of the cells, with HVFFs seeded on aligned scaffolds having a significantly different (p<0.001) angle of orientation than HVFFs cultured on unaligned scaffolds. This same effect and significant difference (p<0.001) was seen on aligned and unaligned scaffolds coated with ELP4. Scaffold alignment and ELP4 coating impacted ECM gene expression. ELP4 coating, and aligned scaffolds upregulated elastin synthesis when tested on day 7 without a concomitant upregulation of collagen III synthesis. Collectively, results indicate that aligned electrospun scaffolds and ELP4 coating are promising candidates in the development of biodegradeable vocal fold lamina propria constructs.

Random and aligned PLLA : PRGF electrospun scaffolds for regenerative medicine

ABSTRACTRandom and aligned electrospun scaffolds were prepared combining poly(l‐lactic acid) (PLLA) and activated platelet‐rich plasma (PRGF) at various proportions, with the aim of elucidating the role of nanofibers orientation and growth factors on cell attachment and proliferation. PRGF is released from scaffolds in a sustained way for at least 3 weeks, without an initial burst effect. Mesenchymal stem cells (MSCs) seeded on the random scaffolds present a polygonal and random orientation in any direction of the scaffold. On the other hand, aligned scaffolds are able to promote cell attachment and proliferation in the direction of the nanofibers. The incorporation of PRGF in the scaffolds enhances cell proliferation for at least 2 weeks. Overall, aligned electrospun PLLA : PRGF scaffolds can encapsulate growth factors at relatively large proportions and sustain their release to enhance cell attachment and proliferation as well as eliciting cell alignment. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 41372.

Human airway smooth muscle maintain in situ cell orientation and phenotype when cultured on aligned electrospun scaffolds.

Human airway smooth muscle (HASM) contraction plays a central role in regulating airway resistance in both healthy and asthmatic bronchioles. In vitro studies that investigate the intricate mechanisms that regulate this contractile process are predominantly conducted on tissue culture plastic, a rigid, 2D geometry, unlike the 3D microenvironment smooth muscle cells are exposed to in situ. It is increasingly apparent that cellular characteristics and responses are altered between cells cultured on 2D substrates compared with 3D topographies. Electrospinning is an attractive method to produce 3D topographies for cell culturing as the fibers produced have dimensions within the nanometer range, similar to cells' natural environment. We have developed an electrospun scaffold using the nondegradable, nontoxic, polymer polyethylene terephthalate (PET) composed of uniaxially orientated nanofibers and have evaluated this topography's effect on HASM cell adhesion, alignment, and morphology. The fibers orientation provided contact guidance enabling the formation of fully aligned sheets of smooth muscle. Moreover, smooth muscle cells cultured on the scaffold present an elongated cell phenotype with altered contractile protein levels and distribution. HASM cells cultured on this scaffold responded to the bronchoconstrictor bradykinin. The platform presented provides a novel in vitro model that promotes airway smooth muscle cell development toward a more in vivo-like phenotype while providing topological cues to ensure full cell alignment.

The influence of electrospun scaffold topography on endothelial cell morphology, alignment, and adhesion in response to fluid flow

Bioengineered vascular grafts provide a promising alternative to autografts for replacing diseased or damaged arteries, but necessitate scaffold designs capable of supporting a confluent endothelium that resists endothelial cell (EC) detachment under fluid flow. To this end, we investigated whether tuning electrospun topography (i.e., fiber diameter and orientation) could impact EC morphology, alignment, and structural protein organization with the goal of forming a confluent and well-adhered endothelium under fluid flow. To test this, a composite polymer blend of poly(ε-caprolactone) (PCL) and type I collagen was electrospun to form scaffolds with controlled fiber diameters ranging from approximately 100-1,200 nm and with varying degrees of fiber alignment. ECs were seeded onto scaffolds, and cell morphology and degree of alignment were quantified using image analysis of fluorescently stained cells. Our results show that ECs form confluent monolayers on electrospun scaffolds, with cell alignment systematically increasing with a larger degree of fiber orientation. Additionally, cells on aligned electrospun scaffolds display thick F-actin bundles parallel to the direction of fiber alignment and strong VE-cadherin expression at cell-cell junctions. Under fluid flow, ECs on highly aligned scaffolds had greater resistance to detachment compared to cells cultured on randomly oriented and semi-aligned scaffolds. These results indicate that scaffolds with aligned topographies may be useful in forming a confluent endothelium with enhanced EC adhesion for vascular tissue engineering applications.

Biaxial mechanics and inter‐lamellar shearing of stem‐cell seeded electrospun angle‐ply laminates for annulus fibrosus tissue engineering

The annulus fibrosus (AF) of the intervertebral disk plays a critical role in vertebral load transmission that is heavily dependent on the microscale structure and composition of the tissue. With degeneration, both structure and composition are compromised, resulting in a loss of AF mechanical function. Numerous tissue engineering strategies have addressed the issue of AF degeneration, but few have focused on recapitulation of AF microstructure and function. One approach that allows for generation of engineered AF with appropriate (+/-)30° lamellar microstructure is the use of aligned electrospun scaffolds seeded with mesenchymal stem cells (MSCs) and assembled into angle-ply laminates (APL). Previous work indicates that opposing lamellar orientation is necessary for development of near native uniaxial tensile properties. However, most native AF tensile loads are applied biaxially, as the disk is subjected to multi-axial loads and is constrained by its attachments to the vertebral bodies. Thus, the objective of this study was to evaluate the biaxial mechanical response of engineered AF bilayers, and to determine the importance of opposing lamellar structure under this loading regime. Opposing bilayers, which replicate native AF structure, showed a significantly higher modulus in both testing directions compared to parallel bilayers, and reached ∼60% of native AF biaxial properties. Associated with this increase in biaxial properties, significantly less shear, and significantly higher stretch in the fiber direction, was observed. These results provide additional insight into native tissue structure-function relationships, as well as new benchmarks for engineering functional AF tissue constructs.

Aligned electrospun scaffolds and elastogenic factors for vascular cell-mediated elastic matrix assembly

Strategies to enhance the production of organized elastic matrix by smooth muscle cells (SMCs) are critical in engineering functional vascular conduits. Therefore, the goal of this study was to determine the effect of different surfaces, i.e. random and aligned electrospun poly(ε-caprolactone) meshes and two-dimensional (2D) controls, and exogenous elastogenic factors on the cultured rat aortic SMC phenotype and production of extracellular matrix. This study demonstrated that aligned electrospun fibres guide cell alignment, induce a more elongated cell morphology and promote a more synthetic phenotype. Importantly, these cells produced greater amounts of elastin-rich matrix per cell on the electrospun scaffolds. In addition, exogenous elastogenic factors severely limited rat aortic smooth muscle cells (RASMCs) proliferation and promoted a more synthetic SMC phenotype on electrospun meshes, but they had less effect on 2D controls. Finally, the elastogenic factors induced the SMCs to generate more matrix collagen and elastin on a per cell basis. Together, these results demonstrate the elastogenic benefits of electrospun meshes.

The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers

Aligned electrospun scaffolds are promising tools for engineering fibrous musculoskeletal tissues, as they reproduce the mechanical anisotropy of these tissues and can direct ordered neo-tissue formation. However, these scaffolds suffer from a slow cellular infiltration rate, likely due in part to their dense fiber packing. We hypothesized that cell ingress could be expedited in scaffolds by increasing porosity, while at the same time preserving overall scaffold anisotropy. To test this hypothesis, poly(ɛ-caprolactone) (a slow-degrading polyester) and poly(ethylene oxide) (a water-soluble polymer) were co-electrospun from two separate spinnerets to form dual-polymer composite fiber-aligned scaffolds. Adjusting fabrication parameters produced aligned scaffolds with a full range of sacrificial (PEO) fiber contents. Tensile properties of scaffolds were functions of the ratio of PCL to PEO in the composite scaffolds, and were altered in a predictable fashion with removal of the PEO component. When seeded with mesenchymal stem cells (MSCs), increases in the starting sacrificial fraction (and porosity) improved cell infiltration and distribution after three weeks in culture. In pure PCL scaffolds, cells lined the scaffold periphery, while scaffolds containing >50% sacrificial PEO content had cells present throughout the scaffold. These findings indicate that cell infiltration can be expedited in dense fibrous assemblies with the removal of sacrificial fibers. This strategy may enhance in vitro and in vivo formation and maturation of functional constructs for fibrous tissue engineering.