Hillary Stoll, Il Keun Kwon, Jung Yul Lim,

1 Department of Biological Systems Engineering, University of Nebraska-Lincoln, Lincoln, NE, USA

2 The Graduate School of Dentistry, Kyung Hee University, Seoul, Korea

3 Department of Mechanical and Materials Engineering, University of Nebraska-Lincoln, Lincoln, NE, USA

Material and mechanical factors: new strategy in cellular neurogenesis

Hillary Stoll1, Il Keun Kwon2, Jung Yul Lim2,3

1 Department of Biological Systems Engineering, University of Nebraska-Lincoln, Lincoln, NE, USA

2 The Graduate School of Dentistry, Kyung Hee University, Seoul, Korea

3 Department of Mechanical and Materials Engineering, University of Nebraska-Lincoln, Lincoln, NE, USA

Since damaged neural circuits are not generally self-recovered, developing methods to stimulate neurogenesis is critically required. Most studies have examined the effects of soluble pharmacological factors on the cellular neurogenesis. On the other hand, it is now recognized that the other extracellular factors, including material and mechanical cues, also have a strong potential to induce cellular neurogenesis. This article will review recent data on the material (chemical patterning, micro/nano-topography, carbon nanotube, graphene) and mechanical (static cue from substrate stiffness, dynamic cue from stretch and fl ow shear) stimulations of cellular neurogenesis. These approaches may provide new neural regenerative medicine protocols. Scaffolding material templates capable of triggering cellular neurogenesis can be explored in the presence of neurogenesis-stimulatory mechanical environments, and also with conventional soluble factors, to enhance axonal growth and neural network formation in neural tissue engineering.

neural regenerative medicine; cellular neurogenesis; material cue; mechanical factor; soluble signal

Funding:This study was supported by NE EPSCoR Trans-disciplinary Neuroscience Research Seed Grant, NSF CAREER Award 1351570, AHA Scientist Development Grant 12SDG12030109, Osteology Foundation Grant 12-006, Nebraska Research Initiative.

Stoll H, Kwon IK, Lim JY. Material and mechanical factors： new strategy in cellular neurogenesis. Neural Regen Res. 2014;9(20)：1810-1813.

Neurogenesis is a very important topic due to its relevance to nerve injuries and neurological disorders. Since the loss of functional circuits rarely displays spontaneous recovery, fi nding successful methods to stimulate neurogenesis is very useful and important. Due to the fact that the body is run by an abundance of biochemical cascades, it is no secret there are many soluble factors that could in fl uence neurogenesis. Studies have therefore tested the effects of various soluble factors on the neurogenesis of neural precursor cells. On the other hand, it is becoming increasingly apparent that other extracellular cues, such as material and mechanical factors, can also in fl uence cellular neurogenesis (Bani-Yaghoub et al., 2005; Higgins et al., 2013).is is very exciting for neuronal regenerative medicine as it allows to explore multiple stimulatory cues for neurogenesis. It also allows for a combination of multiple stimuli, soluble and non-soluble, administered together to be tested.at soluble factors, substrate cues, and mechanical stimuli all have the capability to in fl uence neurogenesis may enlighten how we deal with neural damages and disorders.

Various soluble factors, including nerve growth factor (NGF), retinoic acid (RA), brain-derived neurotrophic factor, neuropathiazol,etc., have been identified to positively influence neurogenesis. NGF is responsible for increasing the survival and di ff erentiation of sensory and sympathetic neurons and there is evidence that endogenous NGF helps protect neurons and promotes their repair (Sofroniew et al., 2001). RA stimulates cellular neurogenesis through pathwaysviaactivating retinoic acid receptors (RARs) and also peroxisome proliferator-activated receptor β/δ (Yu et al., 2012).ese soluble factors have been shown to stimulate the neurogenesis in manyin vitroandin vivostudies, and therapeutic ways to utilize the soluble reagents for neuro regeneration have been exploited. We will now highlight key fi ndings on the material and mechanical control of cellular neurogenesis.

It is established that cell growth substrate a ff ects cell adhesion, proliferation, and di ff erentiation.e substrate control of cells has been applied to neuronal cells using chemical and topographic micropatterns. Chemical micropatterning producesviaphoto- and solithography techniques chemical/ biomolecular cell-adhesive micropatterns and cell-repellent background. Cell attachment and growth can be geometrically confined within cell-adhesive micropatterns, so the cell size, shape, and interconnectivity can be systematically manipulated (see details in our review: Poudel et al., 2012). Topographic micropatterning produces physical microscale structures (ridges/grooves, islands/protrusions,etc.). Lithographically produced master or its inverse replica can beused as topographic patterns.ese topographies have also been shown to direct cell morphology and behavior (see the review: Lim, 2009). Micropatterned surfaces, both chemical and topographic, provide useful templatesviawhich cell physiology under varying extracellular milieus could be systematically investigatedin vitro.

When applied to neuronal cells, chemical micropatterns could affect neurite outgrowth and neuronal cell orientation/migration (Yang et al., 2005; Doyle et al., 2009).ese studies took advantage of the chemical micropatterning that cells could be positioned within predetermined extracellular matrix (ECM) protein patternsviadirect recruiting of integrins through patterned peptide sequence (such as Arg-Gly-Asp, RGD). In our recent study (Poudel et al., 2013), when patterned within narrow (5 and 10 μm wide) collagen-I lanes, SH-SY5Y neuroblastoma cells showed preferred nucleus orientation along the patterned lanes and exerted signi fi cantly longer neurite extension (one of the apparent markers of cellular neurogenesis) relative to unpatterned control. Interestingly, we also observed that the neurite extension for patterned cells without soluble RA stimulation was even greater than that of unpatterned but RA-treated sample.is may suggest that extracellular cue from chemical patterning could provide even stronger signal for cellular neurogenesis than the conventional soluble cue. Another study also demonstrated that the neurite growth and axonal speci fi cation in hippocampal neurons could be controlled by the scale of chemical micropatterning (Tomba et al., 2014).

Utilizing the capability of chemical micropatterning, it was also shown that neural network formation could be manipulated.ese studies adopted combinations of cell-adhesive protein patterns and cell-non-adhesive back-filling. For example, 104μm2area poly-L-lysine squares were utilized for neural stem cell patterning with polyethylene oxide cell-repellent background (Ruiz et al., 2008). If the patterns were connected with 15 μm wide lanes, they could successfully guide axonal outgrowth along the connection lanes to ultimately form well-communicated neural network. In another study, PCC7-MzN neuroblastoma cells formed neural network with a nodal compliance of 86% when patterned on 20 μm diameter laminin dots interconnected by 4 μm wide lanes and with non-tissue culture grade polystyrene background (Lauer et al., 2001). Also, di ff erent shapes of cell patterning (circle, square, triangle) may a ff ect neurite initiation, soma shape, axonal outgrowth, and network formation (Jang and Nam, 2012). Improved cell-cell communication achievedviachemical patterning-directed network formation may further enhance neuronal cell differentiation and even the commitment of stem cells into neuronal cells (Solanki et al., 2010; Béduer et al., 2012).

Topographic substrate modi fi cation has also been utilized to direct neuronal cell behavior. Most of the e ff ort has been based on the well-established phenomenon, so-called contact guidance. Cells cultured on anisotropic topographies such as ridges and grooves tend to exhibit cell alignment/elongation along the anisotropic direction of the topography (although it is not through direct RGD recruiting of integrin as was for chemical patterning). Following the anisotropic cell shaping, related and downstream cell behaviors including nucleus orientation, focal adhesion and cytoskeletal formation, and even cell migration, proliferation, and terminal di ff erentiation are anisotropically directed (Lim, 2009). It is only natural to imagine that such an anisotropic control of cell shape and behavior by ridge and groove could be well applied to formulate neuronal cell functions, considering that axonal structure of neuronal system is intrinsically anisotropic. While it is still under investigation what geometrical shape and size (ridge and groove width, ridge height, pitch all at both micro and nanoscale) are most effective in directing the contact guidance for various cell types (Guilak et al., 2009), the use of anisotropic topographies constitute one of the important non-soluble approaches to guide neuronal cell behavior.

An earlier study showed that Xenopus neurites grew along the grooves as deep as 14 nm and as wide as 1 μm and neurites determined where to emerge from somas depending on the groove substrates (Rajnicek et al., 1997). Other studies using varying dimensions of ridges and grooves also demonstrated that neural cell orientation and neurite/axonal growth were aligned following the anisotropic direction but to a di ff erent degree depending on the size of the ridges and grooves (S?rensen et al., 2007; Fozdar et al., 2010). According to a recent study (Su et al., 2013), rat adrenal pheochromocytoma (PC-12) cells cultured on ridge and groove patterns (800 and 100 nm width respectively, 1,200 nm depth) displayed not only greater bipolar elongation and neurite extension but also higher expression of growth-associated protein-43 compared with cells on planar surfaces. However, groove topography decreased the neuronal cell movement speed, illustrating how topography may have a positive e ff ect on certain aspects of neurogenesis and a negative e ff ect on others. Another study showed that alignment of cellular elements from inner ear neural system increases with increasing ridge height and with decreasing periodicity (Tuet al., 2013). In addition to neurite alignment, anisotropic surface topographies were also demonstrated to induce bone marrow-derived mesenchymal stem cell (MSC) differentiation toward a neuronal fate (D’Angelo et al., 2010).

Data on chemical and topographic patterning imply that researchers are now able to gain more control over the desired cellular neurogenesis. This is important because it allows neural cell shape, neurite/axonal outgrowth, and terminal cellular neurogenesis to be manipulated at a more intricate level.is is expected to ultimately lead to advance the strategy to deal with neuronal injuries and disorders. For example, synthetic neurochips with fully functional neural network developed from chemical and topographic micropatterning may be used to replace damaged neural system (Bani-Yaghoub et al., 2005).

Additionally, recent developments in nanotechnology have introduced new classes of nanomaterials that can be used to trigger cellular neurogenesis. Carbon nanotube (CNT)-based nano-scaffolds were effective in cell and nuclear shapingof human MSCs and triggering gene expressions related to neurogenesis including voltage-gated ion channel formation (Park et al., 2013). Another type of nanomaterial that is of significant interest for cellular neurogenesis includes graphene, 2-dimensional monolayer of hexagonal carbon atoms, in addition to its huge applications in electronic devices. Several recent studies strongly suggest the potential of graphene to stimulate neurite sprouting (Li et al., 2011), to enhance electrical signaling in neural networks (Tang et al., 2013), and to induce MSC fate toward neuronal lineage (Wang et al., 2012).

Mechanical factors, both static and dynamic, may also play an important role in regulating neurogenesis. It is now gathering much attention that neural and axonal development and cellular neurogenesis may be driven by mechanical cues (Franze, 2013). As regards static mechanical factor (such as substrate sti ff ness), a pioneering study on MSC fate reported that on sogel MSCs tend to di ff erentiate toward neuronal cells unlike myogenesis and osteogenesis favored on stiffer gels (McBeath et al., 2004). Another study also found that soft environments promote early neurogenesis of human pluripotent stem cells while decreasing their self-renewal (Keung et al., 2012). Similarly, it was shown that differentiated neurons prefer sosubstrates as their growth milieu (Georges et al., 2006). It was proposed that growing neurons could detect substrate sti ff ness and avoid sti ff portions of the substrate as with an observation of neuronal retraction and re-extension at static stress over 274 ± 41 pN/μm2(Franze et al., 2009).ese data may give an indication as to which static mechanical condition is ideal for neurogenesis, and therefore suggest practical knowledge on what matrix sti ffness will be required for neuronal tissue regeneration.

Dynamic mechanical stimulation, tension or shear, also has a potential to a ff ect the neurogenesis. Based on traditional understanding on the role of tension in axonal elongation or retraction (Heidemann et al., 1995), studies attempted to apply direct tension to axonal growth cone for regenerative medicine purpose. Continuous mechanical tension (3.5 μm per 5 minutes) could induce stretch-induced growth (1 cm growth after 10 days of stretch) of central nervous system axons (Smith et al., 2001). Later studies reported even more effective axonal growth cone stretch and achieved up to a growth rate of 10 mm/day (Loverde et al., 2011; P fi ster et al., 2004). In our recent study, instead of direct pulling of anchored axons, we examined the stretching of neuronal cells seeded on a stretchable membrane. We observed that SHSY5Y neuroblastoma cells exposed to 10% stretch (equiaxial, 0.25 Hz, 120 minutes/day for 7 days) even without soluble stimulatory factor (RA) displayed significantly longer and more neurites than unstretched control (Higgins et al., 2013), suggesting the competitive importance of mechanical signal relative to soluble stimulation. One particular study tested the e ff ects of fl uid fl ow-induced shear stresses (0.1 to 1.5 Pa) on neurite outgrowth, in which PC-12 cells showed the longest neurite length at 0.25 Pa shear (Kim et al., 2006).ese unconventional data on dynamic mechanical (stretch, fl uid shear) stimulations of neurogenesis may suggest a new strategy to treat neuronal injuries and disorders.

The process of neurogenesis is incredibly complex and there is still much to learn. However, it is becoming increasingly evident that not only are the conventional soluble factors influencing neurogenesis important, but also the substrate cues and mechanical stimuli.e implications this knowledge has are crucial for neuronal regenerative medicine. Although much of the evidences so far are fromin vitrostudies, it can be possibly applied to deduce optimalin vivoenvironments, material and mechanical, that should be provided to accomplish successful neural regeneration. For example, a sca ff old with cells can be maintainedin vitrothen implantedin vivofor treating spinal cord injury, which should be able to provide a structural platform capable of inducing axonal growth and neural network formation. Obtained knowledge from chemical/topographic patterning and nanomaterials combined with mechanical stimulations may be applied to this situation to exert the best neural regeneration outcome.e more information that is acquired regarding not only the soluble factors but the material and mechanical factors as well, the more successful neurogenesis will be achievable in treating neural damages and diseases.

Bani-Yaghoub M, Tremblay R, Voicu R, Mealing G, Monette R, Py C, Faid K, Sikorska M (2005) Neurogenesis and neuronal communication on micropatterned neurochips. Biotechnol Bioeng 92：336-345.

Béduer A, Vieu C, Arnauduc F, Sol JC, Loubinoux I, Vaysse L (2012) Engineering of adult human neural stem cells differentiation through surface micropatterning. Biomaterials 33：504-514.

D’Angelo F, Armentano I, Mattioli S, Crispoltoni L, Tiribuzi R, Cerulli GG, Palmerini CA, Kenny JM, Martino S, Orlacchio A (2010) Micropatterned hydrogenated amorphous carbon guides mesenchymal stem cells towards neuronal differentiation. Eur Cell Mater 20：231-244.

Doyle AD, Wang FW, Matsumoto K, Yamada KM (2009) One-dimensional topography underlies three-dimensional fi brillar cell migration. J Cell Biol184：481-490.

Fozdar DY, Lee JY, Schmidt CE, Chen S (2010) Selective axonal growth of embryonic hippocampal neurons according to topographic features of various sizes and shapes. Int J Nanomed 6：45-57.

Franze K, Gerdelmann J, Weick M, Betz T, Pawlizak S, Lakadamyali M, Bayer J, Rillich K, G?gler M, Lu YB, Reichenbach A, Janmey P, K?s J (2009) Neurite branch retraction is caused by a threshold-dependent mechanical impact. Biophys J 97：1883-1890.

Franze K (2013)The mechanical control of nervous system development. Development 140：3069-3077.

Georges PC1, Miller WJ, Meaney DF, Sawyer ES, Janmey PA (2006) Matrices with compliance comparable to that of brain tissue select neuronal over glial growth in mixed cortical cultures. Biophys J 90：3012-3018.

Guilak F, Cohen DM, Estes BT, Gimble JM, Liedtke W, Chen CS (2009) Control of stem cell fate by physical interactions with the extracellular matrix. Cell Stem Cell 5：17-26.

Heidemann SR, Lamoureux P, Buxbaum RE (1995) Cytomechanics of axonal development. Cell Biochem Biophys 27：135-155.

Higgins S, Lee JS, Ha L, Lim JY (2013) Inducing neurite outgrowth by mechanical cell stretch. Biores Open Access 2：212-216.

Jang MJ, Nam Y (2012) Geometric effect of cell adhesive polygonal micropatterns on neuritogenesis and axon guidance. J Neural Eng 9：046019.

Keung AJ, Asuri P, Kumar S, Schaffer DV (2012) Soft microenvironments promote the early neurogenic differentiation but not self-renewal of human pluripotent stem cells. IntegrBiol (Camb) 4：1049-1058.

Kim IA, Park SA, Kim YJ, Kim SH, Shin HJ, Lee YJ, Kang SG, Shin JW (2006) Effects of mechanical stimuli and micro fi ber-based substrate on neurite outgrowth and guidance. J Biosci Bioeng 101：120-126.

Lauer L, Klein C, Offenh?usser A (2001) Spot compliant neuronal networks by structure optimized micro-contact printing. Biomaterials 22：1925-1932.

Li N, Zhang X, Song Q, Su R, Zhang Q, Kong T, Liu L, Jin G, Tang M, Cheng G (2011) The promotion of neurite sprouting and outgrowth of mouse hippocampal cells in culture by graphene substrates. Biomaterials 32：9374-9382.

Lim JY (2009) Topographic control of cell response to synthetic materials. Tissue Eng Regen Med 6：365-370.

Loverde JR, Tolentino RE, P fi ster BJ (2011) Axon stretch growth： the mechanotransduction of neuronal growth. J Vis Exp 54：2753.

McBeath R, Pirone DM, Nelson CM, Bhadriraju K, Chen CS (2004) Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell 6：483-495.

Park SY, Kang BS, Hong S (2013) Improved neural differentiation of human mesenchymal stem cells interfaced with carbon nanotube scaffolds. Nanomedicine (Lond) 8：715-723.

Pfister BJ, Iwata A, Meaney DF, Smith DH (2004) Extreme stretch growth of integrated axons. J Neurosci 24：7978-7983.

Poudel I, Menter DE, Lim JY (2012) Directing cell function and fate via micropatterning： Role of cell patterning size, shape, and interconnectivity. Biomed Eng Lett 2：38-45.

Poudel I, Lee JS, Tan L, Lim JY (2013) Micropatterning-retinoic acid co-control of neuronal cell morphology and neurite outgrowth. Acta Biomater 9：4592-4598.

Rajnicek A, Britland S, McCaig C (1997) Contact guidance of CNS neurites on grooved quartz： in fl uence of groove dimensions, neuronal age and cell type. J Cell Sci 110：2905-2913.

Ruiz A, Buzanska L, Gilliland D, Rauscher H, Sirghi L, Sobanski T, Zychowicz M, Ceriotti L, Bretagnol F, Coecke S, Colpo P, Rossi F (2008) Micro-stamped surfaces for the patterned growth of neural stem cells. Biomaterials 29：4766-4774.

Smith DH, Wolf JA, Meaney DF (2001) A new strategy to produce sustained growth of central nervous system axons： continuous mechanical tension. Tissue Eng 7：131-139.

Sofroniew MV, Howe CL, Mobley WC (2001) Nerve growth factor signaling, neuroprotection, and neural repair. Annu Rev Neurosci 24：1217-1281.

Solanki A, Shah S, Memoli KA, Park SY, Hong S, Lee KB (2010) Controlling differentiation of neural stem cells using extracellular matrix protein patterns. Small 6：2509-2513.

S?rensen A, Alekseeva T, Katechia K, Robertson M, Riehle MO, Barnett SC (2007) Long-term neurite orientation on astrocyte monolayers aligned by microtopography. Biomaterials 28：5498-5508.

Su WT, Liao YF, Wu TW, Wang BJ, Shih YY (2013) Microgrooved patterns enhanced PC12 cell growth, orientation, neurite elongation, and neuritogenesis. J Biomed Mater Res A 101：185-194.

Tang M, Song Q, Li N, Jiang Z, Huang R, Cheng G (2013) Enhancement of electrical signaling in neural networks on graphene fi lms. Biomaterials 34：6402-6411.

Tomba C, Bra?ni C, Wu B, Gov NS, Villard C (2014) Tuning the adhesive geometry of neurons： length and polarity control. Soft Matter 10：2381-2387.

Tuft BW, Li S, Xu L, Clarke JC, White SP, Guymon BA, Perez KX, Hansen MR, Guymon CA (2013) Photopolymerizedmicrofeatures for directed spiral ganglion neurite and Schwann cell growth. Biomaterials 34：42-54.

Wang Y, Lee WC, Manga KK, Ang PK, Lu J, Liu YP, Lim CT, Loh KP (2012) Fluorinated graphene for promoting neuro-induction of stem cells. Adv Mater 24：4285-4290.

Yang IH, Co CC, Ho CC (2005) Alteration of human neuroblastoma cell morphology and neurite extension with micropatterns. Biomaterials 26：6599-6609.

Yu S, Levi L, Siegel R, Noy N (2012) Retinoic acid induces neurogenesis by activating both retinoic acid receptors (RARs) and peroxisome proliferator-activated receptor β/δ (PPARβ/δ). J BiolChem 287：42195-42205.

Jung Yul Lim, Department of Mechanical and Materials Engineering, University of Nebraska-Lincoln, W317.3 Nebraska Hall, Lincoln, NE 68588-0526, USA, jlim4@unl.edu.

10.4103/1673-5374.143426

http://www.nrronline.org/

Accepted: 2014-09-22