Sing Yian Chew

Current applications and future perspectives of artificial nerve conduits

Artificial nerve guide conduits have the advantage over autografts in terms of their availability and ease of fabrication. However, clinical outcomes associated with the use of artificial nerve conduits are often inferior to that of autografts, particularly over long lesion gaps. There have been significant advances in the designs of artificial nerve conduits over the years. In terms of materials selection and design, a wide variety of new synthetic polymers and biopolymers have been evaluated. The inclusion of nerve conduit lumen fillers has also been demonstrated as essential to enable nerve regeneration across large defect gaps. These lumen filler designs have involved the integration of physical cues for contact guidance and biochemical signals to control cellular function and differentiation. Novel conduit architectural designs using porous and fibrous substrates have also been developed. This review highlights the recent advances in synthetic nerve guide designs for peripheral nerve regeneration, and the in vivo applicability and future prospects of these nerve guide conduits.

The application of nanofibrous scaffolds in neural tissue engineering.

The repairing process in the nervous system is complicated and brings great challenges to researchers. Tissue engineering scaffolds provide an alternative approach for neural regeneration. Sub-micron and nano-scale fibrous scaffolds which mimic the topography of natural extracellular matrix (ECM) can be potential scaffold candidates for neural tissue engineering. Two fiber-fabrication methods have been explored in the field of nerve regeneration: electrospinning and self-assembly. Electrospinning produces fibers with diameters ranging from several micrometers to hundreds of nanometers. The fibrous nerve conduits can be introduced at lesion sites by implantation. Self-assembly fibers have diameters of tens of nanometers and can be injected for central nervous system (CNS) injury repair. Both fibrous scaffolds would enhance neurite extension and axon regrowth. These functional nanofibrous scaffolds can serve as powerful tools for neural tissue engineering.

The Role of Electrospinning in the Emerging Field of Nanomedicine

The fact that in vivo the extracellular matrix (ECM) or substratum with which cells interact often includes topography at the nanoscale underscores the importance of investigating cell-substrate interactions and performing cell culture at the submicron scale. An important and exciting direction of research in nanomedicine would be to gain an understanding and exploit the cellular response to nanostructures. Electrospinning is a simple and versatile technique that can produce a macroporous scaffold comprising randomly oriented or aligned nanofibers. It can also accommodate the incorporation of drug delivery function into the fibrous scaffold. Endowed with both topographical and biochemical signals such electrospun nanofibrous scaffolds may provide an optimal microenvironment for the seeded cells. This review covers the analysis and control of the electrospinning process, and describes the types of electrospun fibers fabricated for biomedical applications such as drug delivery and tissue engineering.

Aligned core–shell nanofibers delivering bioactive proteins

AIMS Continuous nanostructures embedded with proteins may synergistically present topographical and biochemical signals to cells for tissue engineering applications. This study presents the co-axial electrospinning of aligned poly(epsilon-caprolactone) nanofibers encapsulated with bovine serum albumin and platelet-derived growth factor-bb for demonstration of controlled release and bioactivity retention, respectively. MATERIALS & METHODS Controllable release kinetics is achieved by incorporation of poly(ethylene glycol) as a porogen in the shell of the nanofibers. RESULTS & DISCUSSION Poly(ethylene glycol) leaches out in a concentration- and molecular weight-dependent fashion, leading to bovine serum albumin release half-lives that range from 1 to 20 days. Optimized platelet-derived growth factor-bb-encapsulated nanofibers can completely release the protein with near zero-order kinetics and preserved bioactivity. CONCLUSION Co-axial electrospinning is shown to be a versatile technique in achieving the delivery of biochemical signals in a controlled manner for regenerative medicine applications.

Mechanical properties of single electrospun drug-encapsulated nanofibres

The mechanical and structural properties of a surface play an important role in determining the morphology of attached cells, and ultimately their cellular functions. As such, mechanical and structural integrity are important design parameters for a tissue scaffold. Electrospun fibrous meshes are widely used in tissue engineering. When in contact with electrospun scaffolds, cells see the individual micro- or nanofibres as their immediate microenvironment. In this study, tensile testing of single electrospun nanofibres composed of poly(ε-caprolactone) (PCL), and its copolymer, poly(caprolactone-co-ethyl ethylene phosphate) (PCLEEP), revealed a size effect in the Youngs modulus, E, and tensile strength, σ(T). Both strength and stiffness increase as the fibre diameter decreases from bulk (∼5 μm) into the nanometre region (200-300 nm). In particular, E and σ(T) of individual PCL nanofibres were at least two-fold and an order of magnitude higher than that of PCL film, respectively. PCL films were observed to have more pronounced crystallographic texture than the nanofibres; however no difference in crystalline fraction, perfection, or texture was detected among the various fibres. When drugs were encapsulated into single PCLEEP fibres, mechanical properties were enhanced with 1-20 wt% of loaded retinoic acid, but weakened by 10-20 wt% of encapsulated bovine serum albumin. This understanding of the effect of size and drug and protein encapsulation on the mechanical properties of electrospun fibres may help in the optimization of tissue scaffold design that combines biochemical and biomechanical cues for tissue regeneration.

The topographical effect of electrospun nanofibrous scaffolds on the in vivo and in vitro foreign body reaction.

Topographical cues play an important role in influencing cellular behavior and are considered as significant parameters to be controlled in tissue engineering applications. This work investigated the biocompatibility with regard to scaffold architecture and topographical effect of nanofibrous scaffolds on the in vivo and in vitro foreign body reaction. Random and aligned polycaprolactone (PCL) nanofibers were fabricated by electrospinning technique, with diameters of 313 +/- 5 nm and 506 +/- 24 nm, respectively. Primary monocytes isolated from five human donors were cultured on PCL nanofibers, PCL film, and RGD-coated glass in vitro and cell density and morphology was evaluated at time points of day 0 (2 h), day 3, day 7, and day 10. The in vivo study was carried out by implanting PCL nanofibers and film scaffolds subcutaneously in rats to test the biocompatibility and host response at time points of week 1, week 2, and week 4. The in vitro studies revealed that the initial monocyte adhesion on the aligned fiber scaffold was significantly less (p < 0.001) when compared to the random fiber scaffold. The in vivo study showed that the thicknesses of fibrous capsule on fibrous scaffolds were 7.55 +/- 0.54 microm for aligned fibers and 4.13 +/- 0.31 microm for random fibers, which were significantly thinner than that of film implants 37.7 +/- 0.25 microm (p < 0.001). Additionally, cell infiltration was observed in aligned fibrous scaffolds both in vitro and in vivo, while on random fibers and films, distinct fibrous capsule boundaries were found on the surfaces. These results indicate that aligned electrospun nanofibers may serve as a promising scaffold for tissue engineering by minimizing host response, enhancing tissue-scaffold integration, and eliciting a thinner fibrous capsule.

RNA interference by nanofiber-based siRNA delivery system.

SiRNA delivery has found useful applications particularly as therapeutic agents against genetic diseases. Currently, the delivery of siRNA typically takes the form of nanoparticles. In order to expand the applications of these potent but labile molecules for long-term use required by tissue engineering and regenerative medicine, alternative delivery vehicles are required. This work presents a scaffold-mediated approach to siRNA delivery. By encapsulating siRNA within polycaprolactone (PCL) nanofibers (300-400nm in diameter) controlled release of intact siRNA could be achieved for at least 28days under physiological conditions. The successful transfection of HEK 293 cells with GAPDH siRNA released from fibrous scaffolds at day 5, 15 and 30 demonstrated that the encapsulated molecules remained bioactive throughout the period of sustained release, providing silencing efficiency of 61-81% that was comparable to conventional siRNA transfection. Direct seeding of cells on these biofunctional scaffolds, with and without transfection reagent, demonstrated enhanced cellular uptake and efficient GAPDH gene-silencing. This work demonstrates the potential of nanofibrous scaffold-mediated siRNA delivery for long-term gene-silencing applications. The combination of topographical features provided by nanofibrous scaffolds may provide synergistic contact guidance and biochemical signals to mediate and support cellular development in regenerative medicine.

Nanofiber-mediated controlled release of siRNA complexes for long term gene-silencing applications

Nanofiber scaffold-mediated delivery of small-interfering RNA (siRNA) holds great potential in regenerative medicine by providing biomimicking topographical signals and enhanced gene silencing effects to seeded cells. While the delivery of naked siRNA was demonstrated previously using poly (ε-caprolactone) (PCL) nanofibers, the resulting siRNA release kinetics and gene knockdown efficiencies were sub-optimal. In this study, we investigated the feasibility of encapsulating siRNA and transfection reagent (TKO) complexes within nanofibers comprising of a copolymer of caprolactone and ethyl ethylene phosphate (PCLEEP, diameter ∼ 400 nm). Sustained release of bioactive naked siRNA and siRNA/TKO complexes were obtained for at least 28 days. By copolymerizing EEP with caprolactone, siRNA release was significantly enhanced (total siRNA that was released by day 49 was ∼ 89.3-97.2% as compared to previously reported 3% by plain PCL nanofiber delivery). Using GAPDH as the model protein, bioactivity analyses by supernatant transfection revealed the partial retention of bioactivity of naked siRNA and siRNA/TKO complexes for at least 30 days. In particular, GAPDH siRNA/TKO supernatant alone induced significant gene silencing (∼40%), indicating the feasibility of co-encapsulating siRNA and transfection reagent within a single scaffold construct for sustained delivery. Direct culture of cells on siRNA incorporated scaffolds for scaffold-mediated gene transfection revealed significant gene knockdown even in the absence of transfection reagent (21.3% knockdown efficiency by scaffolds incorporating naked siRNA only). By encapsulating siRNA/TKO complexes, more significant gene knockdown was obtained (30.9% knockdown efficiency as compared to previously reported 18% by plain PCL scaffold-mediated transfection). Taken together, the results demonstrated the feasibility of co-encapsulating siRNA-transfection reagent complexes within a single nanofiber construct for sustained siRNA delivery and enhanced gene knockdown efficiency. The study also highlights the potential of PCLEEP as a platform for tailoring siRNA release kinetics for long-term gene silencing applications.

Nanofiber topography and sustained biochemical signaling enhance human mesenchymal stem cell neural commitment.

Stem cells hold great promise in enhancing nerve regeneration. In particular, human mesenchymal stem cells (MSC) represent a clinically viable cell source due in part to their abundance and accessibility. Unfortunately, current methods to direct the fate of stem cells remains largely limited to biochemical-based approaches on two-dimensional substrates with restricted efficacies. Here we have evaluated a scaffold-based approach to directing stem cell differentiation. We demonstrate the combined effects of nanofiber topography and controlled drug release on enhancing MSC neural commitment. By encapsulating up to 0.3 wt.% retinoic acid (RA) within aligned poly(ε-caprolactone) (PCL) nanofibers (average diameter ∼270 nm, AF750), sustained released of RA was obtained for at least 14 days (∼60% released). Compared with tissue culture polystyrene (TCPS), the nanofiber topography arising from plain PCL nanofibers significantly up-regulated the expressions of neural markers, Tuj-1, MAP2, GalC and RIP at the mRNA and protein levels. Combined with sustained drug availability, more significant changes in cell morphology and enhancement of neural marker expression were observed. In particular, scaffold-based controlled delivery of RA enhanced MAP2 and RIP expression compared with bolus delivery despite lower amounts of drug (>8 times lower). The generally higher expression of the mature neuronal marker MAP2 compared with glial markers at the mRNA and protein levels suggested an enhanced potential of MSC neuronal differentiation. In addition, positive staining for synaptophysin was detected only in cells cultured on aligned scaffolds in the presence of RA. Taken together, the results highlight the advantage of the scaffold-based approach in enhancing the potential of MSC neuronal differentiation and demonstrated the importance of the drug delivery approach in directing cell fate. Such biomimicking drug-encapsulating scaffolds may permit subsequent direct cell transplantation and provide guidance cues to control the fate of endogenously recruited stem cells.

Photochemical crosslinked electrospun collagen nanofibers: synthesis, characterization and neural stem cell interactions.

Currently available crosslinking methods for electrospun collagen nanofibers do not preserve the fibrous architecture over prolonged periods of time. In addition, electrospinning of collagen often involves solvents that lead to extensive protein denaturation. In this study, we demonstrate the advantage of acetic acid over 1,1,1,3,3,3 hexafluoroisopropanol (HFP) in preventing collagen denaturation. A novel photochemical crosslinking method using rose bengal as the photoinitiator is also introduced. Using circular dichorism analyses, we demonstrate the fraction of collagen helical structure to be significantly greater in acetic acid-spun fibers than HFP-spun fibers (28.9 +/- 5.9% vs. 12.5 +/- 2.0%, p < 0.05). By introducing 0.1% (w/v) rose bengal into collagen fibers and subjecting these scaffolds to laser irradiation at a wavelength of 514 nm for 100 sec, biodegradable crosslinked scaffolds were obtained. Scaffold degradation as evaluated by soaking crosslinked collagen scaffolds in PBS at 37 degrees C, indicated a mass loss of 47.7 +/- 7.4% and 68.9 +/- 24.7% at day 7 and day 15, respectively. However, these scaffolds retained fibrous architecture for at least 21 days under physiological conditions. Neural stem cell line, C17.2, cultured on crosslinked collagen scaffolds proliferated after 7 days by forming a confluent layer of cells with extensive cellular projections that were indicative of neurite outgrowth. Taken together, these findings support the potential of acetic acid-electrospun photochemical crosslinked collagen nanofibers for neural tissue engineering.