Paul Wieringa

Nanotopography induced contact guidance of the F11 cell line during neuronal differentiation: a neuronal model cell line for tissue scaffold development

The F11 hybridoma, a dorsal root ganglion-derived cell line, was used to investigate the response of nociceptive sensory neurons to nanotopographical guidance cues. This established this cell line as a model of peripheral sensory neuron growth for tissue scaffold design. Cells were seeded on substrates of cyclic olefin copolymer (COC) films imprinted via nanoimprint lithography (NIL) with a grating pattern of nano-scale grooves and ridges. Different ridge widths were employed to alter the focal adhesion formation, thereby changing the cell/substrate interaction. Differentiation was stimulated with forskolin in culture medium consisting of either 1 or 10% fetal bovine serum (FBS). Per medium condition, similar neurite alignment was achieved over the four day period, with the 1% serum condition exhibiting longer, more aligned neurites. Immunostaining for focal adhesions found the 1% FBS condition to also have fewer, less developed focal adhesions. The robust response of the F11 to guidance cues further builds on the utility of this cell line as a sensory neuron model, representing a useful tool to explore the design of regenerative guidance tissue scaffolds.

Peptide functionalized polyhydroxyalkanoate nanofibrous scaffolds enhance Schwann cells activity

Interactions between Schwann cells (SCs) and scaffolds are important for tissue development during nerve regeneration, because SCs physiologically assist in directing the growth of regenerating axons. In this study, we prepared electrospun scaffolds combining poly (3-hydroxybutyrate) (PHB) and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) functionalized with either collagen I, H-Gly-Arg-Gly-Asp-Ser-OH (GRGDS), H-Tyr-Ile-Gly-Ser-Arg-NH2 (YIGSR), or H-Arg-Asn-Ile-Ala-Glu-Ile-Ile-Lys-Asp-Ile-OH (p20) neuromimetic peptides to mimic naturally occurring ECM motifs for nerve regeneration. Cells cultured on fibrous mats presenting these biomolecules showed a significant increase in metabolic activity and proliferation while exhibiting unidirectional orientation along the orientation of the fibers. Real-time PCR showed cells cultured on peptide-modified scaffolds had a significantly higher neurotrophin expression compared to those on untreated nanofibers. Our study suggests that biofunctionalized aligned PHB/PHBV nanofibrous scaffolds may elicit essential cues for SCs activity and could serve as a potential scaffold for nerve regeneration. From the clinical editor: Nanotechnology-based functionalized scaffolds represent one of the most promising approaches in peripheral nerve recovery, as well as spinal cord recovery. In this study, bio-functionalized and aligned PHB/PHBV nanofibrous scaffolds were found to elicit essential cues for Schwann cell activity, therefore could serve as a potential scaffold for nerve regeneration.

Bifurcating microchannels as a scaffold to induce separation of regenerating neurites

Many neural interfacing strategies, such as the sieve electrode and the cultured probe, rely on neurite growth to establish neural contact. But this growth is subject to natural fasciculation, compromising the effectiveness of these interfacing strategies by reducing potential selectivity. This in vitro study shows that the fasciculation mechanism can be manipulated by providing an appropriate microchannel scaffold to guide and influence growth, thereby achieving a high degree of selectivity. The microchannels employed have a bifurcation from a primary channel into two secondary channels. This bifurcating microstructure was able to support and promote fasciculated growth over 70% of the time for microchannels widths of 2.5, 5, 10 and 20 microm. Fasciculation is shown to be a strong force during ingrowth, with the initiation of neurite separation related to random spatial exploration. Narrower microchannels initiate separated growth better. Once separated growth starts fasciculation results in an even distribution of neurite growth across the bifurcation. The reduction from 20 microm to 10 microm wide channels also resulted in a 3-fold decrease in ingrowing neurites performing 180 degrees turns to exit the microchannel via the entrance. No neurite turning was observed for both the 5 and 2.5 microm wide channels.

In vitro Verification of a 3-D Regenerative Neural Interface Design: Examination of Neurite Growth and Electrical Properties Within a Bifurcating Microchannel Structure

Toward the development of neuroprosthesis, we propose a 3-D regenerative neural interface design for connecting with the peripheral nervous system. This approach relies on bifurcating microstructures to achieve defasciculated ingrowth patterns and, consequently, high selectivity. In vitro studies were performed to validate this design by showing that fasciculation during nerve regeneration can be influenced by providing a scaffold to guide growth appropriately. With this approach, neurites can be separated from one another and guided toward specific electrode sites to create a highly selective interface. The neurite separation characteristics were examined for smaller microchannel structures (2.5 and 5 ¿m wide) and larger microchannels (10 and 20 ¿m wide), with smaller microchannels shown to be statistically more effective at initiating separation. Electrodes incorporated at different locations within the microchannels allowed for the recording and tracking of action potential propagation. Microchannel size was also found to play an important role in this regard, with smaller microchannels amplifying the recordable extracellular signal; a twofold increase in the signal to noise ratio was found for 5 ¿m wide microchannels.

Influence of Solution Properties and Process Parameters on the Formation and Morphology of YSZ and NiO Ceramic Nanofibers by Electrospinning

The fabrication process of ceramic yttria-stabilized zirconia (YSZ) and nickel oxide nanofibers by electrospinning is reported. The preparation of hollow YSZ nanofibers and aligned nanofiber arrays is also demonstrated. The influence of the process parameters of the electrospinning process, the physicochemical properties of the spinning solutions, and the thermal treatment procedure on spinnability and final microstructure of the ceramic fibers was determined. The fiber diameter can be varied from hundreds of nanometers to more than a micrometer by controlling the solution properties of the electrospinning process, while the grain size and surface roughness of the resulting fibers are mainly controlled via the final thermal annealing process. Although most observed phenomena are in qualitative agreement with previous studies on the electrospinning of polymeric nanofibers, one of the main differences is the high ionic strength of ceramic precursor solutions, which may hamper the spinnability. A strategy to control the effective ionic strength of precursor solutions is also presented.

Peot/pbt Guides Enhance Nerve Regeneration in Long Gap Defects

Development of new nerve guides is required for replacing autologous nerve grafts for the repair of long gap defects after nerve injury. A nerve guide comprised only of electrospun fibers able to bridge a critical (15 mm) nerve gap in a rat animal model is reported for the first time. The nerve conduits are made of poly(ethylene oxide terephthalate) and poly(butylene terephthalate) (PEOT/PBT), a biocompatible copolymer composed of alternating amorphous, hydrophilic poly(ethylene oxide terephthalate), and crystalline, hydrophobic poly(butylene terephthalate) segments. These guides show suitable mechanical properties, high porosity, and fibers aligned in the longitudinal axis of the guide. In vitro studies show that both neurites and Schwann cells exhibit growth alignment with PA fibers. In vivo studies reveal that, after rat sciatic nerve transection and repair with PEOT/PBT guides, axons grow occupying a larger area compared to silicone tubes. Moreover, after repair of limiting (10 mm) and critical (15 mm) nerve gaps, PEOT/PBT guides significantly increase the percentage of regenerated nerves, the number of regenerated myelinated axons, and improve motor, sensory, and autonomic reinnervation in both gaps. This nerve conduit design combines the properties of PEOT/PBT with electrospun structure, demonstrating that nerve regeneration through long gaps can be achieved through the design of instructive biomaterial constructs.

A combinatorial approach towards the design of nanofibrous scaffolds for chondrogenesis

The extracellular matrix (ECM) is a three-dimensional (3D) structure composed of proteinaceous fibres that provide physical and biological cues to direct cell behaviour. Here, we build a library of hybrid collagen-polymer fibrous scaffolds with nanoscale dimensions and screen them for their ability to grow chondrocytes for cartilage repair. Poly(lactic acid) and poly (lactic-co-glycolic acid) at two different monomer ratios (85:15 and 50:50) were incrementally blended with collagen. Physical properties (wettability and stiffness) of the scaffolds were characterized and related to biological performance (proliferation, ECM production, and gene expression) and structure-function relationships were developed. We found that soft scaffolds with an intermediate wettability composed of the highly biodegradable PLGA50:50 and collagen, in two ratios (40:60 and 60:40), were optimal for chondrogenic differentiation of ATDC5 cells as determined by increased ECM production and enhanced cartilage specific gene expression. Long-term cultures indicated a stable phenotype with minimal de-differentiation or hypertrophy. The combinatorial methodology applied herein is a promising approach for the design and development of scaffolds for regenerative medicine.

Direct Writing Electrospinning of Scaffolds with Multidimensional Fiber Architecture for Hierarchical Tissue Engineering

Nanofibrous structures have long been used as scaffolds for tissue engineering (TE) applications, due to their favorable characteristics, such as high porosity, flexibility, high cell attachment and enhanced proliferation, and overall resemblance to native extracellular matrix (ECM). Such scaffolds can be easily produced at a low cost via electrospinning (ESP), but generally cannot be fabricated with a regular and/or complex geometry, characterized by macropores and uniform thickness. We present here a novel technique for direct writing (DW) with solution ESP to produce complex three-dimensional (3D) multiscale and ultrathin (∼1 μm) fibrous scaffolds with desirable patterns and geometries. This technique was simply achieved via manipulating technological conditions, such as spinning solution, ambient conditions, and processing parameters. Three different regimes in fiber morphologies were observed, including bundle with dispersed fibers, bundle with a core of aligned fibers, and single fibers. The transition between these regimes depended on tip to collector distance (Wd) and applied voltage (V), which could be simplified as the ratio V/Wd. Using this technique, a scaffold mimicking the zonal organization of articular cartilage was further fabricated as a proof of concept, demonstrating the ability to better mimic native tissue organization. The DW scaffolds directed tissue organization and fibril matrix orientation in a zone-dependent way. Comparative expression of chondrogenic markers revealed a substantial upregulation of Sox9 and aggrecan (ACAN) on these structures compared to conventional electrospun meshes. Our novel method provides a simple way to produce customized 3D ultrathin fibrous scaffolds, with great potential for TE applications, in particular those for which anisotropy is of importance.

Cell-instructive high-resolution micropatterned polylactic acid surfaces

Micro and nanoscale topographical structuring of biomaterial surfaces has been a valuable tool for influencing cell behavior, including cell attachment, proliferation and differentiation. However, most fabrication techniques for surface patterning of implantable biomaterials suffer from a limited resolution, not allowing controlled generation of sub-cellular three-dimensional features. Here, a direct laser lithography technique based on two-photon absorption was used to construct several patterns varying in size between 500 nm and 15 μm. Through replication via an intermediate mold, the patterns were transferred into polylactic acid (PLA), a widely used biomedical polymer, while retaining the original geometry. An osteoblast-like cell line, MG-63 was used for characterizing the morphological response to the topographical patterns. The results indicated that semi-continuous (dashed) lines, with a height of 1 μm were able to induce cell elongation in the direction of the lines. However, when dashes with a height of 0.5 μm were combined with perpendicularly crossing continuous lines (rails) with a height of 8 μm, the contact guidance effect of the dashes was lost and elongation of the cells was observed in the direction of the larger features. A second pattern, consisting of different arrays of pillars showed that, depending on the pillar height, the cells were either able to spread over the pattern or were confined between the pattern features. These differences in the ability of cells to spread further resulted in the formation of tension forces through stress fibers and displacement of vimentin. The method for high-resolution micropatterning of PLA as presented here can also be applied to other biomedical polymers, making it useful both for fundamental studies and for designing new biomaterials with improved functionality.

Surface Modification of Polyimide Thin Films for Peripheral Invasive Neural Interfaces

Despite recognized as one key component for establishing a functional electrical connection with nerves, neural invasive peripheral interfaces are still not optimal for long-term applications in humans. An improvement in the field of biocompatible and nontoxic materials is necessary to overcome the issues of interface/tissue mismatch and physiological reactions. The present work aimed to study, implement and characterize a novel approach to modify the surface of neural mi-crolectrodes basedon polyimide thin films. The purpose was to improve biocompatibility and to promote neuronal migration, growth and differentiation by increasing the surface roughness and endowing the surface with structure-reactivity for thiol-containing amino acids or peptides. L-Cysteine-Rhodamine B, used as a model biomolecule, was successfully grafted on samples surface via the introduction of cross-linkable vinyl groups on polyimide foils. Preliminary in vitro biological analysis allowed to evaluate the tendency of PC12 cells to adhere and to proliferate.