Unidirectional Neuronal Alignment with Nanowires
Evaluation of: Hallstrom W, Prinz CN, Suyatin D, Samuelson L, Montelius L, Kanje M: Rectifying and sorting of regenerating axons by free-standing nanowire patterns: a highway for nerve fibers. Langmuir 25(8), 4343–4346 (2009) – Ross & Riehle
Cell alignment to topography is well documented Citation[1]. Previous studies of neuronal alignment have used channels etched into a substrate Citation[2], patterned onto a substrate with photoresist Citation[3] or embossed into a polymer Citation[4]. All of these studies have used relatively large feature sizes, in the micrometer region. Some work has been conducted using nanoscale ridges Citation[5] but, like the other studies, this did not give directionality to the alignment.
Now, Hallstrom et al. have for the first time achieved unidirectional neuronal guidance using patterned arrays of nanowires Citation[6]. Gold nanowires (4 µm high and 75 nm in diameter) were grown on gallium phosphide substrates coated with laminin to encourage neuronal growth. The wires were patterned in an echelon formation, each echelon measuring 10 µm with a wire-to-wire spacing of 400 nm, and the lines of wires were angled at + and -30° from the anticipated direction of axonal growth. The echelons formed 5 µm wide channels ‘pointing’ to the left or right of the substrate. To test their hypothesis, Hallstrom et al. used cells isolated from the superior cervical ganglia of wild-type mice and green fluorescent protein-expressing transgenic mice, seeding them onto opposite sides of the device Citation[6]. After allowing time for growth, the β-tubulin inside axons were stained red. The green fluorescent protein-expressing axons showed up yellow (green and red), while the wild-type axons showed only red staining, thus allowing identification of the origin of individual axons. This showed that the axons would only grow in the direction pointed to by the echelons of nanowires, in other words axons from the right would grow to the left and only be in the channels where the echelons pointed left. This effect was observed at both low and high cell densities. At higher cell densities the bundles of axons extended above the level of the wires but still remained unidirectional and aligned with the guidance channels.
There is great potential for using this nanowire guidance technique in neuronal networks for sorting sprouting neurons and avoiding clogging of guidance channels with multiple axons from multiple directions. However, the technique as presented here only has limited potential in the biomedical field, as the substrate, gallium phosphide, is hard, flat and not biodegradable; the nanowires are quite fragile and so the essential device surface could be easily damaged if transplanted into the human body. Furthermore, the effects of the nanowires on other cell types are unknown.
Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
References
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- Hallstrom W , PrinzCN, SuyatinD, SamuelsonL, MonteliusL, KanjeM: Rectifying and sorting of regenerating axons by free-standing nanowire patterns: a highway for nerve fibers.Langmuir25(8), 4343–4346 (2009).
Diamond Nanocrystal Size Affects Osteoblastic Responses
Evaluation of: Yang L, Sheldon BW, Webster TJ: The impact of diamond nanocrystallinity on osteoblast functions. Biomaterials 30, 3458– 3465 (2009) – McNamara, Riehle, Burchmore & Dalby
Chemical vapor-deposited films of nanoscale diamond crystals (nanocrystalline diamond) have potential for application as hardwearing surfaces on orthopedic devices. To fully exploit these coatings, it is vital to characterize the cellular interactions with these substrata to optimize conditions for osseointegration. Yang et al proposed that some interstudy variations in cell responses to nanocrystalline diamond could have resulted from differences in substrate surface chemistry and topography Citation[1]. To address this, the authors examined osteoblast responses to two hydrophobic diamond films with analogous hydrogen-terminated surface chemistry, but different roughness and crystal sizes in the submicron (100–600 nm) and nano (30–100 nm aggregates of smaller nanofeatures) ranges. Cell adhesion was most effective on nanoscale diamond and a control silicon surface, with numerous, expansive filopodia observed using scanning-electron microscopy. On submicron diamond, where cells were less well spread, filopodial clumping was noted around larger diamond grains, which the authors attributed to differential protein adsorption. Filopodia can respond to topographic features as small as 10 nm high Citation[2], and it seems likely that physical guidance could also have contributed to the filopodial polarization. After 21 days of culture, osteoblasts on both diamond surfaces had higher levels of collagen and alkaline phosphatase (markers of osteoblast activity) than controls, with the nanocrystalline surface inducing deposition of most extracellular calcium. It is interesting that the nanocrystalline substrate best supported these osteoblastic functions, since the hydrophilic controls should be more favorable for protein and cell adhesion. Mechanical perturbation of the cell membrane by the nanofeatures could have stimulated calcium secretion, since membrane distension may influence assembly of secretory protein complexes or vesicle release, and is postulated to affect ion channels Citation[3]. It would be useful to examine hydrophilic (oxygen terminated) nanocrystalline diamond surfaces against hydrophilic controls, since the surface chemistry of the nanocrystalline surfaces can influence cell behavior, and the study was complicated by use of different chemistries of control and test materials.
A combination of cues balancing cell adhesion, proliferation and osseointegration with implant durability would be desirable to maximize device efficacy. Use of cell-responsive, anti-abrasive nanofilms with tunable chemical and topographical surface features should assist in the production of biomedical devices tailored to elicit the apposite cell behavior and maximize therapeutic benefit.
References
- Yang L , SheldonBW, WebsterTJ: The impact of diamond nanocrystallinity on osteoblast functions.Biomaterials30, 3458–3465 (2009).
- Dalby MJ , RiehleMO, JohnstoneH, AffrossmanS, CurtisASG: Investigating the limits of filopodial sensing: a brief report using SEM to image the interaction between 10 nm high nano-topography and fibroblast filopodia.Cell Biol. Int.28(3), 229–236 (2004).
- Vogel V , SheetzM: Local force and geometry sensing regulate cell functions.Nat. Rev. Mol. Cell Biol.7(4), 265–275 (2006).
Neuronal Differentiation of Human Embryonic Stem Cells Using Nanotopography
Evaluation of: Chao TI, Xiang S, Chen CS et al.: Carbon nanotubes promote neuron differentiation from human embryonic stem cells. Biochem. Biophys. Res. Commun. 384, 426–430 (2009) – Dalby, McMurray & Gadegaard
The differentiation of stem cells, whether adult or embryonic, into various tissues holds exciting promise within the field of regenerative medicine. One key problem, however, is the ability to promote the directed differentiation of stem cells down a specific lineage without the need for potentially harmful supplements in the growth media. One potential route for addressing this issue is the use of nanotopography, whereby the nanoscale features of a substrate alone may act as a platform to promote the differentiation of stem cells down a particular lineage. In the paper under review by Chao et al., the authors look at examining the effect of one such nanotopography. In the study they examined the differentiation of human embryonic stem (hES) cells into neurons in response to culture upon polymer-grafted carbon nanotubes (CNTs). The authors aimed to show that by grafting CNTs with poly(acrylic acid) (PAA), neuronal differentiation of hES cells can be enhanced in comparison to both PAA alone and poly-L-ornithine (PLO), a more commonly used polymer substrate for neuronal differentiation of hES cells. In doing this, the authors looked at several factors including neuronal differentiation using β-tubulin III, a marker for neuronal differentiation and quantitative fluorescence detection, cell viability and cell adhesion.
In response to the PAA-grafted CNTs (PAA-g-CNTs), hES cells were shown to express the neuronal marker, β-tubulin III, and the authors went on to quantify expression levels by measuring fluorescence intensity of β-tubulin III against DAPI-stained hES cells. Although valid, further controls, such as neurons that are known to positively express β-tubulin III, may also provide a suitable basis for the analysis of fluorescence intensity. The results showed that when compared with either PAA or PLO alone, the hES cells cultured on PAA-g-CNTs had a greater fluorescence intensity of β-tubulin III.
In addition to this work, it may be valuable to perform techniques such as quantitative PCR, which would allow the authors to further analyze β-tubulin III expression, and also identify expression of more mature neuronal markers such as nestin.
Cell viability and attachment assays were carried out to examine the effect of this nanotopography on the cells. With regard to cell viability, the PAA-g-CNTs demonstrated results comparable to those of PLO. However, these were in contrast to that of PAA alone and, as suggested by the authors, this is not surprising as PAA alone is known to have a negative effect on neuronal differentiation. Furthermore, the cell attachment assays showed a significant difference between hES cell attachment to PAA-g-CNT substrates and PAA alone that may be attributed to protein absorption onto the surface, which was found only on the PAA-g-CNT substrate.
This study by Chao et al. provides potential evidence that not only can the differentiation of hES cells be promoted down a neuronal lineage, but that by grafting polymers onto a nanotopography, such as CNTs, their properties can be altered, in this case from being unfavorable for neuronal differentiation into a material that can provide an environment suitable for both cell growth and differentiation. The ability to graft PAA onto CNTs paves the way for developing many more materials for culturing stem cells using different polymers, of which one obvious example would be PLO-grafted CNTs.
Pillar-Like Titania Nanostructures on Titanium and Their Interaction with Skeletal Stem Cells
Evaluation of: Sjostrom T, Dalby MJ, Hart A, Tare R, Oreffo ROC, Su B: Fabrication of pillar-like titania nanostructures on titanium and their interactions with human skeletal stem cells. Acta Biomater. 5, 1433– 1441 (2009) – Dalby & Ahmed
Previous literature has shown that human skeletal stem cells can respond to nanoscale cues and that differentiation toward an osteoblastic lineage can be achieved through the use of nanopatterned surface topographies Citation[1]. Utilizing this knowledge to move from in vitro technology to patterning these surface topographies onto medical implant materials has proven difficult. Current implants fabricated from titanium have proven unsuccessful owing to the formation of a fibrous capsule on the implant surface. Traditionally, methods such as electron beam lithography have been used to pattern surfaces but owing to the hardness of titanium this proves unsuccessful and traditional methods of producing nanotopographies on titanium are redundant because of the lack of control over the pattern produced.
This paper looks at titanium dioxide nanopillars with heights of 15, 55 and 100 nm on titanium surfaces. These pillars are fabricated using an anodization technique. This is where a porous anodic aluminum dioxide mask is used as a template on the titanium to pattern the surface. The surfaces achieved with this technique are of a near hexagonal array of titania pillars and not a square array. This is concurrent with previous literature that showed that biomimetic surfaces encourage differentiation of human mesenchymal stem cells into an osteoblastic lineage Citation[1].
Cells were seeded on the various nanopillar heights and a polished titanium surface was used for a control. It was observed that the cells exhibited increased spreading on the 15 nm high pillars with large focal adhesions and decreased cytoskeletal organization and smaller focal adhesions on the 55 nm high pillars and an even more marked decrease in all areas on the 100 nm high nanopillars. Larger focal adhesions are associated with increased direct mechanotransductive signaling. Quantification of the cell area showed a significant increase on the 15 and 55 nm surfaces in comparison to the control, with no significance found with the 100 nm high pillar surface. Staining for the osteogenic markers of osteocalcin and osteopontin after a 21-day culture found an inversely proportional relationship between osteoid matrix nodule formation and nanopillar height, with the planar control showing a negligible presence of either marker.
This paper has demonstrated that well-defined nanostructures could be fabricated onto titanium using an anodization technique to aid in the control of human mesenchymal stem cells towards an osteoblastic lineage. This is moving on from initial studies in plastics and enabling this technology to be used on load-bearing implant surfaces.
This paper is part of a steadily growing body of work on fabricating nanoscale features into metals (e.g., Citation[2]). This is clearly important if we want to translate this technology from the laboratory to the clinic.
References
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- Oh S , BrammerKS, LiYSet al.: Stem cell fate dictated solely by altered nanotube dimension.Proc. Natl Acad. Sci. USA106, 2130–2135 (2009).
Enzyme-Assisted Self-Assembly Under Thermodynamic Control
Evaluation of: Williams RJ, Smith AM, Collins R, Hodson N, Das AK, Ulijn RV: Enzyme-assisted self-assembly under thermodynamic control. Nat. Nanotechnol. 4, 19–24 (2009) – Tsimbouri & Dalby
In biology, self-assembly is a commonly used method for the formation of functional molecular architectures under tight regulation by spatially confined molecular mechanisms. However, despite advances, it is still a major challenge to achieve similar complexity in the laboratory. Scientists are constantly searching for ways to copy these biological approaches such as the use of enzymes to control self-assembly formation Citation[1].
In this study, the Ulijn laboratory report the use of reversible enzyme-catalyzed reactions to drive self-assembly. In this approach, the self-assembly of aromatic short peptide derivatives (Fmoc peptides) provide a thermodynamic driving force that enables a protease enzyme (thermolysin) to produce building blocks in a reversible and spatially restricted manner. They demonstrate that their system combines three features:
Self-correction: a fully reversible self-assembly under thermodynamic control. Using high performance liquid chromatography to measure the time-dependent production of self-assembly components and a competition assay to investigate whether these systems evolve under thermodynamic control, they showed that enzyme-assisted self-assembly systems are totally reversible and equilibrium driven.
Component selection: the ability to amplify the most stable molecular self-assembly structures in dynamic combinatorial libraries (DCL). DCL is an approach that has been broadly used to detect molecular interactions and folding events through thermodynamically driven component selection. With the exception of the Lehn laboratory Citation[2], who worked on gelation-driven component selection in the generation of constitutional dynamic hydrogels, the use of DCL for the discovery of stable supramolecular materials is an uncharted area. Interestingly, the Ulijn group, using transmission-electron microscopy and atomic force microscopy, and in combination with Fourier transform infrared spectroscopy, showed that their system allows for the discovery of stable self-assembled nanostructures through component selection and possible use of DCL to discover other materials with similar properties.
Spatiotemporal confinement of nucleation and structure growth: using transmission-electron microscopy and atomic force microscopy to visualize the nucleation of the self-assembly process in their Fmoc systems in the presence of thermolysin and in combination with birefringence, the authors showed that enzyme-assisted self-assembly favors formation of localized structure nucleation and growth close to enzyme molecules, a property that is involved with the self-assembly kinetics of the system.
Hence, enzyme-assisted self-assembly may provide control in the fabrication of nanomaterials that could eventually lead to functional nanostructures with improved complexities and fewer defects. Consequently, these systems could be very useful with applications in 3D cell cultures Citation[3] to direct cell proliferation and differentiation, which are of critical importance in regenerative medicine.
References
- Toledano S , WilliamsRJ, JayawarnaV, UlijnRV: Enzyme triggered self-assembly of peptide hydrogels via reversed hydrolysis.J. Am. Chem. Soc.128, 1070–1071 (2006).
- Sreenivasachary N , LehnJM: Gelation-driven component selection in the generation of constitutional dynamic hydrogels based on guanine-quartet formation.Proc. Natl Acad. Sci. USA102, 5938–5943 (2005).
- Silva GA , CzeislerC, NieceKLet al.: Selective differentiation of neural progenitor cells by high-epitope density nanofibers.Science303, 1352–1355 (2004).