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Neural pathfinding on uni- and multidirectional photopolymerized micropatterns.

Tuft BW, Xu L, White SP, Seline AE, Erwood AM, Hansen MR, Guymon CA - ACS Appl Mater Interfaces (2014)

Bottom Line: SGN neurites orient randomly on unpatterned photopolymer controls, align and consistently track unidirectional patterns, and are substantially influenced by, but do not consistently track, multidirectional turning cues.Neurite lengths are 20% shorter on multidirectional substrates compared to unidirectional patterns while neurite branching and microfeature crossing events are significantly higher.Developing methods to understand neural pathfinding and to guide de novo neurite growth to specific stimulatory elements will enable design of innovative biomaterials that improve functional outcomes of devices that interface with the nervous system.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biochemical Engineering, University of Iowa , Iowa City, Iowa 52242, United States, United States.

ABSTRACT
Overcoming signal resolution barriers of neural prostheses, such as the commercially available cochlear impant (CI) or the developing retinal implant, will likely require spatial control of regenerative neural elements. To rationally design materials that direct nerve growth, it is first necessary to determine pathfinding behavior of de novo neurite growth from prosthesis-relevant cells such as spiral ganglion neurons (SGNs) in the inner ear. Accordingly, in this work, repeating 90° turns were fabricated as multidirectional micropatterns to determine SGN neurite turning capability and pathfinding. Unidirectional micropatterns and unpatterned substrates are used as comparisons. Spiral ganglion Schwann cell alignment (SGSC) is also examined on each surface type. Micropatterns are fabricated using the spatial reaction control inherent to photopolymerization with photomasks that have either parallel line spacing gratings for unidirectional patterns or repeating 90° angle steps for multidirectional patterns. Feature depth is controlled by modulating UV exposure time by shuttering the light source at given time increments. Substrate topography is characterized by white light interferometry and scanning electron microscopy (SEM). Both pattern types exhibit features that are 25 μm in width and 7.4 ± 0.7 μm in depth. SGN neurites orient randomly on unpatterned photopolymer controls, align and consistently track unidirectional patterns, and are substantially influenced by, but do not consistently track, multidirectional turning cues. Neurite lengths are 20% shorter on multidirectional substrates compared to unidirectional patterns while neurite branching and microfeature crossing events are significantly higher. For both pattern types, the majority of the neurite length is located in depressed surface features. Developing methods to understand neural pathfinding and to guide de novo neurite growth to specific stimulatory elements will enable design of innovative biomaterials that improve functional outcomes of devices that interface with the nervous system.

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SGN neurite alignment on variations in topographiccues. (A–C) Immunofluorescent images of neurite growth fromdissociated SGNs on unpatterned (A), parallel (B), and 90° angle(C) substrates. (D–F) Distribution of SGN neurite segment anglesrelative to the horizontal plane on unpatterned (D), parallel (E),and 90° angle (F) substrates. Regenerative neurite growth orientsrandomly on unpatterned substrates as evidenced by a nearly equaldistribution of neurite segment angles relative to the horizontalplane. Neurites strongly align to unidirectional topographic cueswith 70% of the neurite segment angles at or below 20° from thepattern direction. Neurites on repeating 90° angle patterns donot closely track multidirectional cues as demonstrated by the lowincidence of 45° angle neurite segments. They do align somewhatto the horizontal plane, although with a broader distribution of anglesthan on parallel patterns. Dissociated cultures were stained withanti-NF200 antibodies. Micropatterned substrates have a channel amplitudeof 7 μm.
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fig5: SGN neurite alignment on variations in topographiccues. (A–C) Immunofluorescent images of neurite growth fromdissociated SGNs on unpatterned (A), parallel (B), and 90° angle(C) substrates. (D–F) Distribution of SGN neurite segment anglesrelative to the horizontal plane on unpatterned (D), parallel (E),and 90° angle (F) substrates. Regenerative neurite growth orientsrandomly on unpatterned substrates as evidenced by a nearly equaldistribution of neurite segment angles relative to the horizontalplane. Neurites strongly align to unidirectional topographic cueswith 70% of the neurite segment angles at or below 20° from thepattern direction. Neurites on repeating 90° angle patterns donot closely track multidirectional cues as demonstrated by the lowincidence of 45° angle neurite segments. They do align somewhatto the horizontal plane, although with a broader distribution of anglesthan on parallel patterns. Dissociated cultures were stained withanti-NF200 antibodies. Micropatterned substrates have a channel amplitudeof 7 μm.

Mentions: Developing precisespatial control of de novo neurite growth from neurons that are relevantto neural prosthetics, such as inner ear SGNs, will lead to enhancedprosthesis performance and improved functional outcomes for patients.Directing neurite growth in this manner will require multiple typesof biologically actionable cues including biophysical cues that caneither induce or inhibit neurite turning events. Accordingly, to compareneurite pathfinding ability on varied biophysical cues, SGNs werecultured on unpatterned, unidirectional, and multidirectional photopolymerizedsubstrates (Figure 5). Qualitative immunofluorescenceimaging illustrates that SGN neurite outgrowth extends randomly onunpatterned substrates with unpredictable turning events. Conversely,neurites on unidirectional patterns are observed to strongly orientto and grow parallel to the microfeature direction (horizontal) whileexhibiting few if any turning events per neurite (Figure 5B). Interestingly, while SGN neurites on multidirectionalpatterns do not extend randomly, they also do not closely track therepeating sequence of 90° turning cues along a micropattern pathdespite encountering physical microfeatures that are comparable inwidth and depth to those of the unidirectional patterns (Figure 5C). Rather, extending neurites are often observedto align horizontally and elongate down the length of a feature path.It is interesting that the neurites extend in this fashion, even thoughthey must cross multiple feature transitions near the angle turningpoints. Furthermore, neurites on multidirectional patterns also turnmuch more frequently than on unidirectional substrates with the accompanyingbehavior of crossing over a sequence of microfeatures prior to realigningto the horizontal plane. These microfeature crossing events are rarein the case of neurite growth on unidirectional patterns, especiallyover multiple transitions.


Neural pathfinding on uni- and multidirectional photopolymerized micropatterns.

Tuft BW, Xu L, White SP, Seline AE, Erwood AM, Hansen MR, Guymon CA - ACS Appl Mater Interfaces (2014)

SGN neurite alignment on variations in topographiccues. (A–C) Immunofluorescent images of neurite growth fromdissociated SGNs on unpatterned (A), parallel (B), and 90° angle(C) substrates. (D–F) Distribution of SGN neurite segment anglesrelative to the horizontal plane on unpatterned (D), parallel (E),and 90° angle (F) substrates. Regenerative neurite growth orientsrandomly on unpatterned substrates as evidenced by a nearly equaldistribution of neurite segment angles relative to the horizontalplane. Neurites strongly align to unidirectional topographic cueswith 70% of the neurite segment angles at or below 20° from thepattern direction. Neurites on repeating 90° angle patterns donot closely track multidirectional cues as demonstrated by the lowincidence of 45° angle neurite segments. They do align somewhatto the horizontal plane, although with a broader distribution of anglesthan on parallel patterns. Dissociated cultures were stained withanti-NF200 antibodies. Micropatterned substrates have a channel amplitudeof 7 μm.
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fig5: SGN neurite alignment on variations in topographiccues. (A–C) Immunofluorescent images of neurite growth fromdissociated SGNs on unpatterned (A), parallel (B), and 90° angle(C) substrates. (D–F) Distribution of SGN neurite segment anglesrelative to the horizontal plane on unpatterned (D), parallel (E),and 90° angle (F) substrates. Regenerative neurite growth orientsrandomly on unpatterned substrates as evidenced by a nearly equaldistribution of neurite segment angles relative to the horizontalplane. Neurites strongly align to unidirectional topographic cueswith 70% of the neurite segment angles at or below 20° from thepattern direction. Neurites on repeating 90° angle patterns donot closely track multidirectional cues as demonstrated by the lowincidence of 45° angle neurite segments. They do align somewhatto the horizontal plane, although with a broader distribution of anglesthan on parallel patterns. Dissociated cultures were stained withanti-NF200 antibodies. Micropatterned substrates have a channel amplitudeof 7 μm.
Mentions: Developing precisespatial control of de novo neurite growth from neurons that are relevantto neural prosthetics, such as inner ear SGNs, will lead to enhancedprosthesis performance and improved functional outcomes for patients.Directing neurite growth in this manner will require multiple typesof biologically actionable cues including biophysical cues that caneither induce or inhibit neurite turning events. Accordingly, to compareneurite pathfinding ability on varied biophysical cues, SGNs werecultured on unpatterned, unidirectional, and multidirectional photopolymerizedsubstrates (Figure 5). Qualitative immunofluorescenceimaging illustrates that SGN neurite outgrowth extends randomly onunpatterned substrates with unpredictable turning events. Conversely,neurites on unidirectional patterns are observed to strongly orientto and grow parallel to the microfeature direction (horizontal) whileexhibiting few if any turning events per neurite (Figure 5B). Interestingly, while SGN neurites on multidirectionalpatterns do not extend randomly, they also do not closely track therepeating sequence of 90° turning cues along a micropattern pathdespite encountering physical microfeatures that are comparable inwidth and depth to those of the unidirectional patterns (Figure 5C). Rather, extending neurites are often observedto align horizontally and elongate down the length of a feature path.It is interesting that the neurites extend in this fashion, even thoughthey must cross multiple feature transitions near the angle turningpoints. Furthermore, neurites on multidirectional patterns also turnmuch more frequently than on unidirectional substrates with the accompanyingbehavior of crossing over a sequence of microfeatures prior to realigningto the horizontal plane. These microfeature crossing events are rarein the case of neurite growth on unidirectional patterns, especiallyover multiple transitions.

Bottom Line: SGN neurites orient randomly on unpatterned photopolymer controls, align and consistently track unidirectional patterns, and are substantially influenced by, but do not consistently track, multidirectional turning cues.Neurite lengths are 20% shorter on multidirectional substrates compared to unidirectional patterns while neurite branching and microfeature crossing events are significantly higher.Developing methods to understand neural pathfinding and to guide de novo neurite growth to specific stimulatory elements will enable design of innovative biomaterials that improve functional outcomes of devices that interface with the nervous system.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biochemical Engineering, University of Iowa , Iowa City, Iowa 52242, United States, United States.

ABSTRACT
Overcoming signal resolution barriers of neural prostheses, such as the commercially available cochlear impant (CI) or the developing retinal implant, will likely require spatial control of regenerative neural elements. To rationally design materials that direct nerve growth, it is first necessary to determine pathfinding behavior of de novo neurite growth from prosthesis-relevant cells such as spiral ganglion neurons (SGNs) in the inner ear. Accordingly, in this work, repeating 90° turns were fabricated as multidirectional micropatterns to determine SGN neurite turning capability and pathfinding. Unidirectional micropatterns and unpatterned substrates are used as comparisons. Spiral ganglion Schwann cell alignment (SGSC) is also examined on each surface type. Micropatterns are fabricated using the spatial reaction control inherent to photopolymerization with photomasks that have either parallel line spacing gratings for unidirectional patterns or repeating 90° angle steps for multidirectional patterns. Feature depth is controlled by modulating UV exposure time by shuttering the light source at given time increments. Substrate topography is characterized by white light interferometry and scanning electron microscopy (SEM). Both pattern types exhibit features that are 25 μm in width and 7.4 ± 0.7 μm in depth. SGN neurites orient randomly on unpatterned photopolymer controls, align and consistently track unidirectional patterns, and are substantially influenced by, but do not consistently track, multidirectional turning cues. Neurite lengths are 20% shorter on multidirectional substrates compared to unidirectional patterns while neurite branching and microfeature crossing events are significantly higher. For both pattern types, the majority of the neurite length is located in depressed surface features. Developing methods to understand neural pathfinding and to guide de novo neurite growth to specific stimulatory elements will enable design of innovative biomaterials that improve functional outcomes of devices that interface with the nervous system.

Show MeSH
Related in: MedlinePlus