Metin Uz

Electrical Differentiation of Mesenchymal Stem Cells into Schwann-Cell-Like Phenotypes Using Inkjet-Printed Graphene Circuits

Graphene-based materials (GBMs) have displayed tremendous promise for use as neurointerfacial substrates as they enable favorable adhesion, growth, proliferation, spreading, and migration of immobilized cells. This study reports the first case of the differentiation of mesenchymal stem cells (MSCs) into Schwann cell (SC)-like phenotypes through the application of electrical stimuli from a graphene-based electrode. Electrical differentiation of MSCs into SC-like phenotypes is carried out on a flexible, inkjet-printed graphene interdigitated electrode (IDE) circuit that is made highly conductive (sheet resistance < 1 kΩ/sq) via a postprint pulse-laser annealing process. MSCs immobilized on the graphene printed IDEs and electrically stimulated/treated (etMSCs) display significant enhanced cellular differentiation and paracrine activity above conventional chemical treatment strategies [≈85% of the etMSCs differentiated into SC-like phenotypes with ≈80 ng mL-1 of nerve growth factor (NGF) secretion vs. 75% and ≈55 ng mL-1 for chemically treated MSCs (ctMSCs)]. These results help pave the way for in vivo peripheral nerve regeneration where the flexible graphene electrodes could conform to the injury site and provide intimate electrical simulation for nerve cell regrowth.

Effect of PEG Grafting Density and Hydrodynamic Volume on Gold Nanoparticle–Cell Interactions: An Investigation on Cell Cycle, Apoptosis, and DNA Damage

In this study, interactions of polyethylene glycol (PEG)-coated gold nanoparticles (AuNPs) with cells were investigated with particular focus on the relationship between the PEG layer properties (conformation, grafting density, and hydrodynamic volume) and cell cycle arrest, apoptosis, and DNA damage. Steric hindrance and PEG hydrodynamic volume controlled the protein adsorption, whereas the AuNP core size and PEG hydrodynamic volume were primary factors for cell uptake and viability. At all PEG grafting densities, the particles caused significant cell cycle arrest and DNA damage against CaCo2 and PC3 cells without apoptosis. However, at a particular PEG grafting density (∼0.65 chains/nm(2)), none of these severe damages were observed on 3T3 cells indicating discriminating behavior of the healthy (3T3) and cancer (PC3 and CaCo2) cells. It was concluded that the PEG grafting density and hydrodynamic volume, tuned with the PEG concentration and AuNP size, played an important role in particle-cell interactions.

Development of multifunctional films for peripheral nerve regeneration.

In this study, a poly(lactic acid) (PLLA) porous film with longitudinal surface micropatterns was fabricated by a dry phase inversion technique to be used as potential conduit material for peripheral nerve regeneration applications. The presence of a nerve growth factor (NGF) gradient on the patterned film surface and protein loaded, surface-eroding, biodegradable, and amphiphilic polyanhydride (PA) microparticles within the film matrix, enabled co-delivery of neurotrophic factors with controlled release properties and enhanced neurite outgrowth from PC12 cells. The protein loading capacity of PA particles was increased up to 80% using the spray drying technique, while the surface loading of NGF reached 300ng/cm2 through ester-amine interactions. The NGF surface gradient provided initial fast release from the film surface and facilitated directional neurite outgrowth along with the longitudinal micropatterns. Furthermore, the variable backbone chemistry and surface eroding nature of protein-loaded PA microparticles within the film matrix ensured protein stability and enabled controlled protein release. This novel co-delivery strategy yielded tunable diffusion coefficients varying between 6×10-14 and 1.67×10-10cm2/min and dissolution constants ranging from 1×10-4 to 1×10-3min-1 with released amounts of ∼100-300ng/mL. This strategy promoted guided neurite extension from PC12 cells of up to 10μm total neurite length per cell in 2days. Overall, this unique strategy can potentially be extended for individually programmed delivery of multiple growth factors through the use of PA microparticle cocktails and can further be investigated for in vivo performance as potential conduit material for peripheral nerve regeneration applications. STATEMENT OF SIGNIFICANCE This manuscript focuses on the development of multifunctional degradable polymer films that provide topographic cues for guided growth, surface gradients of growth factors as well as nanoparticles in the films for tunable release of growth factors to enable peripheral nerve regeneration. The combination of cues was designed to overcome limitations of current strategies to facilitate peripheral nerve regeneration. These multifunctional films successfully provided high protein loading capacities while persevering activity, protein gradients on the surface, and tunable release of bioactive nerve growth factor that promoted directional and guided neurite extension of PC12 cells of up to 10μm in 2days. These multifunctional films can be made into conduits for peripheral nerve regeneration.

Gelatin-based 3D conduits for transdifferentiation of mesenchymal stem cells into Schwann cell-like phenotypes

In this study, gelatin-based 3D conduits with three different microstructures (nanofibrous, macroporous and ladder-like) were fabricated for the first time via combined molding and thermally induced phase separation (TIPS) technique for peripheral nerve regeneration. The effects of conduit microstructure and mechanical properties on the transdifferentiation of bone marrow-derived mesenchymal stem cells (MSCs) into Schwann cell (SC) like phenotypes were examined to help facilitate neuroregeneration and understand material-cell interfaces. Results indicated that 3D macroporous and ladder-like structures enhanced MSC attachment, proliferation and spreading, creating interconnected cellular networks with large numbers of viable cells compared to nanofibrous and 2D-tissue culture plate counterparts. 3D-ladder-like conduit structure with complex modulus of ∼0.4×106Pa and pore size of ∼150μm provided the most favorable microenvironment for MSC transdifferentiation leading to ∼85% immunolabeling of all SC markers. On the other hand, the macroporous conduits with complex modulus of ∼4×106Pa and pore size of ∼100μm showed slightly lower (∼65% for p75, ∼75% for S100 and ∼85% for S100β markers) immunolabeling. Transdifferentiated MSCs within 3D-ladder-like conduits secreted significant amounts (∼2.5pg/mL NGF and ∼0.7pg/mL GDNF per cell) of neurotrophic factors, while MSCs in macroporous conduits released slightly lower (∼1.5pg/mL NGF and 0.7pg/mL GDNF per cell) levels. PC12 cells displayed enhanced neurite outgrowth in media conditioned by conduits with transdifferentiated MSCs. Overall, conduits with macroporous and ladder-like 3D structures are promising platforms in transdifferentiation of MSCs for neuroregeneration and should be further tested in vivo. STATEMENT OF SIGNIFICANCE This manuscript focuses on the effect of microstructure and mechanical properties of gelatin-based 3D conduits on the transdifferentiation of mesenchymal stem cells to Schwann cell-like phenotypes. This work builds on our recently accepted manuscript in Acta Biomaterialia focused on multifunctional 2D films, and focuses on 3D microstructured conduits designed to overcome limitations of current strategies to facilitate peripheral nerve regeneration. The comparison between conduits fabricated with nanofibrous, macroporous and ladder-like microstructures showed that the ladder-like conduits showed the most favorable environment for MSC transdifferentiation to Schwann-cell like phenotypes, as seen by both immunolabeling as well as secretion of neurotrophic factors. This work demonstrates the importance of controlling the 3D microstructure to facilitate tissue engineering strategies involving stem cells that can serve as promising approaches for peripheral nerve regeneration.

Stem Cells, Bioengineering, and 3-D Scaffolds for Nervous System Repair and Regeneration

Abstract:A fundamental issue in biology concerns how cells establish and maintain their identity during early embryogenesis. Gaining a better understanding of these rules is key to future development of experimental therapeutics and is an important foundation of tissue engineering and regenerative medicine. With the successful isolation of embryonic stem cells and the emergence of induced pluripotent stem cell technologies, it has become achievable to recapitulate developmental processes of early development. Furthermore, the advent of cellular reprogramming and transdifferentiation technologies has made it possible to implement rational strategies to generate specific cell types in order to model neurodegenerative diseases and develop cell-based therapies for nervous system disorders. Moreover, with advances in biomaterials and in 3-D scaffold fabrication techniques, it is becoming possible to mimic the neural stem cell niche. In this chapter, we provide an overview of approaches merging stem cells, polymeric scaffolds, drug delivery systems, gene therapy, cellular engineering, and biomaterials to develop experimental strategies for neural tissue engineering. Combined, these enabling technologies are likely to be beneficial for development of therapeutic interventions for translation to the clinic. A summary of a number of current clinical trials is also presented at the end to illustrate how combination of these technologies is helping nervous system rescue and repair.

Responsive pentablock copolymers for siRNA delivery

In this study, temperature and pH responsive cationic and amphiphilic pentablock copolymers, which consist of the temperature responsive triblock Pluronic F127 sandwiched between pH responsive PDEAEM (poly(2-diethylaminoethyl methacrylate)) end blocks, were used for the first time in the development of polyplex and gold nanoparticle (AuNP) based multicomponent siRNA delivery systems (MCSs). Copolymers in both systems protected siRNA from external effects, provided cell entry and endosomal escape. The thermoreversible micellization of the hydrophobic PPO block facilitated the cellular entry while the PDEAEM blocks enhanced the endosomal escape through protonated tertiary amine groups by pH buffering. The synergistic advantages of the different blocks showed an enhanced effect in the MCSs due to attachment and surface configuration reasons. The siRNA transfection efficiency of MCSs against luciferase expressing SKOV3 cells was 15% higher than both the polyplexes alone and the commercial siRNA transfection agent Lipofectamine RNAiMax at the same applied dose, without any toxicity. The results indicated that the multicomponent systems based on the responsive cationic pentablock copolymers and gold nanoparticles have promising potential as an efficient siRNA delivery vector for future applications.

Proteomic analysis of mesenchymal to Schwann cell transdifferentiation

While transplantation of Schwann cells facilitates axon regeneration, remyelination and repair after peripheral nerve injury clinical use is limited by cell bioavailability. We posit that such limitation in cell access can be overcome by the use of autologous bone-marrow derived mesenchymal stem cells (MSCs). As MSCs can transdifferentiate to Schwann cell-phenotypes and accelerate nerve regeneration we undertook proteomic evaluation of the cells to uncover the protein contents that affects Schwann cell formulation. Transdifferentiated MSCs secrete significant amounts of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in cell-conditioned media that facilitated neurite outgrowth. MSC proteins significantly regulated during Schwann cell transdifferentiation included, but were not limited to, GNAI2, MYL9, ACTN4, ACTN1, ACTB, CAV-1, HSPB1, PHB2, TBB4B, CTGF, TGFI1, ARF6, EZR, GELS, VIM, WNT5A, RTN4, EFNB1. These support axonal guidance, myelination, neural development and neural growth and differentiation. The results unravel the molecular events that underlie cell transdifferentiation that ultimately serve to facilitate nerve regeneration and repair in support of cell transplantation. SIGNIFICANCE STATEMENT While Schwann cells facilitate axon regeneration, remyelination and repair after peripheral nerve injury clinical use is limited by cell bioavailability. We posit that such limitation in cell access can be overcome by the use of bone-marrow derived mesenchymal stem cells (MSCs) transdifferentiated to Schwann cell-phenotypes. In the present study, we undertook the first proteomic evaluation of these transdifferentiated cells to uncover the protein contents that affects Schwann cell formulation. Furthermore, these transdifferentiated MSCs secrete significant amounts of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in cell-conditioned media that facilitated neurite outgrowth. Our results demonstrate that a number of MSC proteins were significantly regulated following transdifferentiation of the MSCs supporting roles in axonal guidance, myelination, neural development and differentiation. The conclusions of the present work unravel the molecular events that underlie cell transdifferentiation that ultimately serve to facilitate nerve regeneration and repair in support of cell transplantation. Our study was the first proteomic comparison demonstrating the transdifferentiation of MSCs and these reported results can affect a wide field of stem cell biology, tissue engineering, and proteomics.

CHAPTER 14:Smart Materials for Nerve Regeneration and Neural Tissue Engineering

Stimuli-responsive smart-biomaterial-based approaches have been identified as a promising tool for nerve regeneration and neural tissue engineering. Understanding the stimuli-responsive behavior of the smart materials, along with the fundamentals of cellular interactions, is the key to future strategies for neural tissue engineering. Advances in the development and application of smart biomaterials and 3-D scaffold fabrication techniques as well as cellular reprogramming and transdifferentiation technologies make it possible to combine stem cells, cellular engineering, drug/gene delivery systems, nanotechnology and biomaterial-based therapies to develop experimental and clinical strategies for neural tissue engineering. The application of smart biomaterials in these technologies is likely to contribute synergistically to the improvement of therapeutic strategies for clinical translation. This review chapter focuses on the use of strategies combining stimuli-responsive smart biomaterials with other technologies in neural tissue engineering. A specific emphasis on temperature, pH, enzyme, photo-triggered, self-assembling and electrical stimuli-sensitive mono or multi-responsive smart biomaterials in neural tissue engineering is presented. A summary of the clinical potential and applications of smart materials in neural tissue engineering is also presented at the end to illustrate how smart materials can be effective in combination with these technologies to enhance neural regeneration.