Co-immobilization of semaphorin3A and nerve growth factor to guide and pattern axons.

Immobilization of axon guidance cues offers a powerful tissue regenerative strategy to control the presentation and spatial location of these biomolecules. We use our previously developed immobilization strategy to specifically tether recombinant biotinylated nerve growth factor (bNGF) and biotinylated semaphorin3A (bSema3A) to chitosan films as an outgrowth and guidance platform. DRG neurite length and number for a range of single cues of immobilized bNGF or bSema3A were examined to determine a concentration response. Next single and dual cues of bNGF and bSema3A were immobilized and DRG guidance was assessed in response to a step concentration change from zero. Overall, immobilized groups caused axon extension, retraction and turning depending on the ratio of bNGF and bSema3A immobilized in the encountered region. This response indicated the exquisite sensitivity of DRG axons to both attractive and repulsive tethered cues. bSema3A concentrations of 0.10 and 0.49 ng/mm(2), when co-immobilized with bNGF (at 0.86 and 0.43 ng/mm(2) respectively), caused axons to turn away from the co-immobilized region. Immunocytochemical analysis showed that at these bSema3A concentrations, axons inside the co-immobilized region display microtubule degradation and breakdown of actin filaments. At the lowest bSema3A concentration (0.01 ng/mm(2)) co-immobilized with a higher bNGF concentration (2.16 ng/mm(2)), neurite lengths are shorter in the immobilized area, but bNGF dominates the guidance mechanism as neurites are directed toward the immobilized region. Future applications can pattern these cues in various geometries and gradients in order to better modulate axon guidance in terms of polarity, extension and branching. STATEMENT OF SIGNIFICANCE: Nervous system formation and regeneration requires key molecules for guiding the growth cone and nervous system patterning. In vivo these molecules work in conjunction with one another to modulate axon guidance, and often they are tethered to limit spatial distribution. The novelty of this research is that we provide a specific attachment method to immobilize an attractive signal, nerve growth factor, along with an inhibitory cue, semaphorin3A, to a substrate in order to analyze the interplay of these proteins on axon guidance responses. The scientific impact of this manuscript is that we show that dual-cued platforms are necessary in order to finetune and tailor specific axon responses for varying neuronal regenerative purposes.

Micropatterned coumarin polyester thin films direct neurite orientation.

Guidance and migration of cells in the nervous system is imperative for proper development, maturation, and regeneration. In the peripheral nervous system (PNS), it is challenging for axons to bridge critical-sized injury defects to achieve repair and the central nervous system (CNS) has a very limited ability to regenerate after injury because of its innate injury response. The photoreactivity of the coumarin polyester used in this study enables efficient micropatterning using a custom digital micromirror device (DMD) and has been previously shown to be biodegradable, making these thin films ideal for cell guidance substrates with potential for future in vivo applications. With DMD, we fabricated coumarin polyester thin films into 10×20 µm and 15×50 µm micropatterns with depths ranging from 15 to 20 nm to enhance nervous system cell alignment. Adult primary neurons, oligodendrocytes, and astrocytes were isolated from rat brain tissue and seeded onto the polymer surfaces. After 24 h, cell type and neurite alignment were analyzed using phase contrast and fluorescence imaging. There was a significant difference (p<0.0001) in cell process distribution for both emergence angle (from the body of the cell) and orientation angle (at the tip of the growth cone) confirming alignment on patterned surfaces compared to control substrates (unpatterned polymer and glass surfaces). The expected frequency distribution for parallel alignment (≤15°) is 14% and the two micropatterned groups ranged from 42 to 49% alignment for emergence and orientation angle measurements, where the control groups range from 12 to 22% for parallel alignment. Despite depths being 15 to 20 nm, cell processes could sense these topographical changes and preferred to align to certain features of the micropatterns like the plateau/channel interface. As a result this initial study in utilizing these new DMD micropatterned coumarin polyester thin films has proven beneficial as an axon guidance platform for future nervous system regenerative strategies.

Short duration electrical stimulation to enhance neurite outgrowth and maturation of adult neural stem progenitor cells.

New therapies are desperately needed for human central nervous system (CNS) regeneration to circumvent the lack of innate regenerative ability following traumatic injuries. Previously attempted therapies have been stymied by barriers to CNS regeneration largely because of protective mechanisms such as the blood brain barrier, inhibitory molecules, and glial scar formation. The application of electric stimulation (ES) has shown promise for enhancing peripheral nervous system regeneration, but is in its infancy in CNS regeneration. The objective of this study is to better understand how short duration ES can be harnessed to direct adult neural stem progenitor cell (NSPC) neurogenesis, neurite extension, and maturation. Herein, NSPCs were exposed to physiological levels of electrical stimulation of 0.53 or 1.83 V/m (applied power supply setting of 1.2 and 2.5 V) of direct current (DC) for 10 min/days for 2 days with a total differentiation time of 3 days. Culturing conditions consisted of either mitogenic growth factors or the neuronal differentiation factor interferon-γ (IFN-γ). Stimulated NSPCs showed lengths that were over five times longer than unstimulated controls (112.0 ± 88.8 µm at 0.53 V/m vs. 21.3 ± 8.5 µm for 0 V/m with IFN-γ) with the longest neurites reaching up to 600 µm. Additionally, ES resulted in mature neuronal morphologies and signs of differentiation through positive ßIII tubulin, neuronal nuclei (NeuN), and better organized filamentous-actin (f-actin) staining with growth cone formation. Additionally, the neurites and soma of stimulated NSPCs showed increases in intracellular Ca(2+) during stimulation, signifying the presence of functional neurons capable of electrical conductance and communication with other cells. Our study demonstrates that short stimulation times (10 min/ day) result in significant neurite extension of stem cells in a quick time frame (3 days). This ES modality is potentially advantageous for promoting axon re-growth at an injury site using delivered adult stem cells; however, significant work still remains to understand both the delivery approach of cells as well as ES application in vivo.

Expression, isolation, and purification of soluble and insoluble biotinylated proteins for nerve tissue regeneration.

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing  a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Specific immobilization of biotinylated fusion proteins NGF and Sema3A utilizing a photo-cross-linkable diazirine compound for controlling neurite extension.

In this study we report the successful synthesis of N-(2-mercaptoethyl)-3-(3-methyl-3H-diazirine-3-yl) propanamide (N-MCEP-diazirine), with sulfhydryl and amine photoreactive ends to allow recombinant protein tethering to chitosan films. This regimen allows mimicry of the physiological endeavor of axon pathfinding in the nervous system where neurons rely on cues for guidance during development and regeneration. Our strategy incorporates strong covalent and noncovalent interactions, utilizing N-MCEP-diazirine, maleimide-streptavidin complex, and two custom biotinylated-fusion proteins, nerve growth factor (bNGF), and semaphorin3A (bSema3A). Synthetic yield of N-MCEP-diazirine was 87.3 ± 1.9%. Characteristic absorbance decrease at 348 nm after N-MCEP-diazirine exposure to UV validated the photochemical properties of the diazirine moiety, and the attachment of cross-linker to chitosan films was verified with Fourier transform infrared spectroscopy (FTIR). Fluorescence techniques showed no significant difference in the detection of immobilized proteins compared to absorbing the proteins to films (p < 0.05); however, in vitro outgrowth of dorsal root ganglia (DRG) was more responsive to immobilized bNGF and bSema3A compared to adsorbed bNGF and bSema3A over a 5 day period. Immobilized bNGF significantly increased DRG length over time (p < 0.0001), but adsorbed bNGF did not increase in axon extension from day 1 to day 5 (p = 0.4476). Immobilized bSema3A showed a significant decrease in neurite length (524.42 ± 57.31 µm) at day 5 compared to adsorbed bSema3A (969.13 ± 57.31 µm). These results demonstrate the superiority of our immobilization approach to protein adsorption because biotinylated-fusion proteins maintain their active confirmation and their tethering can be spatially controlled via a UV activated N-MCEP-diazirine cross-linker.

Neural regenerative strategies incorporating biomolecular axon guidance signals.

There are currently no acceptable cures for central nervous system injuries, and damage induced large gaps in the peripheral nervous system have been challenging to bridge to restore neural functionality. Innervation by neurons is made possible by the growth cone. This dynamic structure is unique to neurons, and can directly sense physical and chemical activity in its environment, utilizing these cues to propel axons to precisely reach their targets. Guidance can occur through chemoattractive factors such as neurotrophins and netrins, chemorepulsive agents like semaphorins and slits, or contact-mediated molecules such as ephrins and those located in the extracellular matrix. The understanding of biomolecular activity during nervous system development and injury has generated new techniques and tactics for improving and restoring function to the nervous system after injury. This review will focus on the major neuronal guidance molecules and their utility in current tissue engineering and neural regenerative strategies.