The ECM provides a 3D substrate enriched in ligands that are sensed and remodeled by cells and imposes a spatial restraint to cellular migration and neurite extension.31
During development, DRG neurons innervate sensory targets by extending long axons with multiple branches that are properly distributed within target tissues.23,32
Mechanisms regulating neurite outgrowth and branching in sensory neurons are widely studied, but little is known about the mechanisms that regulate polarization events. Moreover, researchers have successfully induced sensory process outgrowth and indiscriminate branching in a diverse array of conditions, including different substrates in 2D and 3D culture.3,4,25,30,33,34
Remarkably, despite a notable report that bipolar morphology is an artifact of 2D culture,15
the unipolar structure of DRG neurons is usually overlooked.
Our work demonstrates that ontogeny of axonal structure relies on the dimensionality of the biomaterial scaffold. One of the fundamental differences between 2D and 3D culture is the distribution of cell–cell and cell–matrix interactions, which alter signaling mechanisms regulating neuronal biological response and activity.35
Therefore, we hypothesized that neurons sense the dimensionality of their environment and respond by altering their morphology in adaptation to the different arrangement of extracellular signals. Our results demonstrate that environment dimensionality plays a major role in sensory neuron development and regulation of signaling events controlling polarization, branching, growth cone motility, and neurite outgrowth. Neurons sensed the dimensionality of their environment and developed into distinct morphologies in 2D and 3D. The 3D setting also allowed elaboration of the in vivo
growth cone morphology, axonal branching pattern, and polarization features within normal ontogeny. These results suggest that a 2D substrate is insufficient to permit completion of the polarization program, whereas, in contrast, the 3D environment supports maturation past the embryonic milestone.
For neurons, as with all cells, morphogenesis occurs in a 3D environment wherein the cell dynamically responds to local environmental cues, such as cell–cell and cell–matrix interactions as well as diffusible molecules.1
These stimuli engage the neuron in a sequence of signaling events that define the characteristic neuronal morphologies and arborization patterns. In a 3D environment, a cell is completely surrounded by stimuli and thus the cell can receive similar external signals from multiple directions. Moreover, as evidenced by the results shown in – neurons are able to make unrestricted use of use all three dimensions (or degrees of freedom), resulting in longer neurites and more branches than on 2D substrates where integrin receptors must reconfigure to the planar arrangement of ligands.
We have also analyzed the involvement of soluble cues in these morphogenic processes by culturing the neurons in the absence of NGF, a molecule that plays distinct roles in the development of the peripheral and central nervous system. In the developing embryo, NGF is required for the survival of DRG neurons, as the majority of DRG neurons die of apoptosis by E14.5 in mice lacking NGF.8
Moreover, in the absence of NGF/TrkA signaling, embryonic sensory neurons fail to innervate peripheral targets.36
Herein we report that upon withdrawal of NGF, neurite outgrowth was dramatically reduced in 2D culture; this is not surprising as NGF is known to enhance sensory neurite elongation on permissive substrates.29,30,34
We also found that this response to NGF withdrawal was enhanced when the same substrate was presented as an encompassing 3D matrix ().
Many have reported that DRG neurons adapt a bipolar morphology in 2D culture regardless of the addition of growth factors29,30,37–40
; we also found this result in 3D culture conditions without NGF (). Therefore, neurons cultured in 3D without NGF lost the ability to differentiate into unipolar neurons. Since the morphology of DRG neurons in 2D culture is similar with or without NGF, we argue that presentation of soluble cues like NGF but perhaps more importantly the surrounding 3D presentation of adhesive ligands allows for realization of the innate cellular morphogenic program.
The branching patterns of sensory neurons in response to NGF withdrawal were distinct between 2D and 3D culture (). The effect of removing NGF from 2D culture was dramatic as the majority of neurons had no branches. This result supports the observed link between NGF treatment and the increased complexity of neurite patterns of adult sensory neurons grown in 2D culture.29,30
In 3D, however, DRG neurons cultured without NGF retained the capacity to form complex arbors in the 3D matrix, suggesting that regulation of branching programs in 3D are independent of NGF.
To begin to understand these findings, we propose a hypothetical model () wherein cells sense and balance cues from their environment. For DRG neurons, the end result of this process is a cell morphology that allows proper relay of sensory information from the periphery to the central nervous system. We propose that in a 3D environment, various matrix and soluble cues are presented toward all surfaces of the cell; this optimized milieu allows neurons to elaborate their genuine phenotype and follow programmed instructions that are intrinsic to the neuron, but disrupted when cells were dissected from the embryo. In other words, the embryonic neurons are competent in a 3D environment to generate mature DRG neurons with complex arbors. On a 2D substrate, matrix and soluble cues are largely segregated by the geometry of a planar system and impair receptor cross-talk and signal integration. As a result, the rearrangement of the cytoskeleton is aberrant and the transformation to a unipolar neuron is delayed for up to a month.15
The suspended transition can be partially overcome in 2D if Schwann cells are included in the culture; however, the time to transformation remains prolonged as compared to in vivo
from 1 to 2 days to up to 2 weeks.15
By contrast, the data presented herein support the hypothesis that the axonal growth pattern of the sensory neurons is intrinsic, and in a sufficient environment, the DRG neuron can recapitulate the in vivo
FIG. 5. Model of cell response to culture dimensionality. On a 2D substrate, matrix and soluble cues are segregated and provide an artificial environment that alters the integration of extracellular cues and results in a delayed polarization program. When exposed (more ...)
It is also important to recognize that tissue stiffness plays a key role in morphogenesis and that substrate mechanical properties actively affect neurite outgrowth in both 2D and 3D culture. For example, differentiation of mesenchymal stem cells into specific cell lineages is highly regulated by substrate stiffness41
and an increase in matrix stiffness influences cytoskeletal tension in mammary epithelial cells and drives the cells toward a malignant phenotype.24
Matrix stiffness influences neurite outgrowth mechanisms, resulting in different and potentially neuron-type-specific responses in 2D and 3D culture: in 2D culture, neurite outgrowth is greater on stiffer substrates,42
whereas the opposite trend occurs for 3D substrates in which softer gels result in a greater extent of neurite outgrowth.25–27
Our results indicate that DRG neurons cultured on soft 2D collagen gel substrates adapt simplified morphological features similar to those cultured on stiff 2D collagen-coated glass (). However, when the same soft substrate is presented to the cells as a surrounding 3D environment, the cells are able to extend long neurites with significant branching. Thus, after excluding the effects of stiffness in our results, we confirmed that the significant differences in total neurite length, number of branches, and primary neurites found between the 2D and 3D culture conditions are correlated with the changes in dimensionality. Engineered scaffolds (e.g., Refs.43–45
) will enable investigation of the effect of scaffold properties in 2D and 3D culture in more detail.
These findings suggest that in a 3D environment, sensory neurons are able to use the limited resources available in culture to realize their innate morphogenic program and thus mimic the phenotypic characteristics observed in vivo. While this study focused on sensory neurons, emerging data hint that the response may be shared by neurons of peripheral and central origin. These findings open new doors for neuronal regeneration and nerve repair strategies that make use of intrinsic cellular mechanisms, in collaboration with engineered environments, to reproduce the natural context and avoid situations that constrain cells into an artificial configuration, leading to temporary or unpredictable biological outcomes. Thus, our ongoing work focuses on defining the signaling mechanisms regulating polarization and axonal arbors in 2D versus 3D culture environments.