Microtissue engineered constructs with living axons for targeted nervous system reconstruction

Abstract

As a common feature of many neurological diseases and injury, the loss of axon pathways can have devastating effects on function. Here, we demonstrate a new strategy to restore damaged axon pathways using transplantable miniature constructs consisting of living neurons and axonal tracts internalized within hydrogel tubes. These hydrogel microconduits were developed through an iterative process to support neuronal survival and directed axon growth. The design included hollow agarose tubes providing a relatively stiff outer casing to direct constrained unidirectional outgrowth of axons through a central soft collagen matrix, with overall dimensions of 250 μm inner diameter ×500 μm outer diameter and extending up to several centimeters. The outer casing was also designed to provide structural support of neuronal/axonal cultures during transplantation of the construct. Using neuron culture conditions optimized for the microconduits, dissociated dorsal root ganglia neurons were seeded in the collagen at one end of the conduits. Over the following week, high-resolution confocal microscopy demonstrated that the neurons survived and the somata remained in a tight cluster at the original seeding site. In addition, robust outgrowth of axons from the neurons was found, with axon fascicles constrained in a longitudinal projection along the internal collagen canal and extending over 5 mm in length. Notably, this general geometry recapitulates the anatomy of axon tracts. As such, these constructs may be useful to repair damaged axon projections by providing a transplantable bridge of living axons. Moreover, the small size of the construct permits follow-on studies of minimally invasive transplantation into potentially sensitive regions of the nervous system.

FIG. 1.

Concept: three-dimensional (3D) microconduits with uniaxial axonal tracts. Our objective was to optimize 3D microconduits to promote neuronal survival at one end while facilitating unidirectional axonal extension through the conduit interior (A). Microscale tubular guidance channels were generated using an agarose exterior and a bioactive matrix interior (B). These agarose-collagen microconduits simultaneously provide comparatively rigid structural support (via agarose) and bioactive ligands (via collagen) to encourage neuronal survival, somata localization, and longitudinal neuritic extension in a 3D microenvironment. The size and geometry of these microconduits permit minimally invasive injection into neural tissue for targeted replacement of axonal tracts. Color images available online at www.liebertpub.com/tea

FIG. 2.

Verification of agarose-collagen architecture in 3D microconduits. Confocal reconstruction of a representative fluorescently labeled microconduit with collagenous core. To image the location of collagen within the agarose tubes, collagen was mixed with Lucifer Yellow (LY; green) prior to being drawn into agarose conduits that were premixed with Alexa-546 (red). Confocal microscopy revealed collagen in the center of the conduits based on hyper-intense LY signal (A). Although the Alexa-546 diffused into the collagen core during the imaging period, the borders of the agarose tubes were sufficiently denoted by the Alexa-546 signal (B) with overlay (C). The microconduit outer diameter (Alexa-546+) measured 990 μm and the collagenous core (LY+) measured 250 μm. Scale bar: 100 μm. Color images available online at www.liebertpub.com/tea

FIG. 3.

Neuronal growth and penetration within 3D microconduits. Confocal reconstructions of neurons adjacent to and within microconduits stained to denote live cells (green) and the nuclei of dead cells (red) at 6 days in vitro (DIV). Initial studies utilized larger diameter microconduits: 990 μm OD (denoted by dashed lines) with a 250 μm ID (denoted by cellular distribution). Live neurons were observed lining the interior of the microconduits (A). Robust neuronal survival was observed within the microconduits. These neurons appeared to migrate from a large cluster of cells located immediately external to the longitudinal end of the microconduit (B). Scale bars: 100 μm. Color images available online at www.liebertpub.com/tea

FIG. 4.

Neuronal polarity and axonal presence in 3D microconduits. Confocal reconstructions of neuronal constructs stained via immunocytochemistry to denote neuronal somata/dendrites (MAP-2; green) and axons (tau; red) at 6 DIV. A dense cluster of neuronal somata was located at one end with axonal projections extending longitudinally across the microconduit (A–C) (scale bar: 250 μm). Neuronal somata were restricted to a dense ganglion at the microconduit extreme, whereas the interior was composed exclusively of long axons projecting several millimeters (D, E) (scale bar: 50 μm). Color images available online at www.liebertpub.com/tea

FIG. 5.

Axonal distribution, branching, and structure in 3D microconduits. Higher magnification confocal reconstructions from demonstrative regions in figure 4. A large tightly coalesced bundle of axons (fascicle) projecting from a ganglion, suggesting axons departed ganglia in the center (collagenous) portion of the conduits (A). As growth occurred longitudinally, increased axonal branching (B) with the formation of axonal spines (C) in the leading segment was observed. Scale bar (A–C) 20 μm. Axonal spines (D, E) denoted by white arrows (scale bar: 10 μm). Confocal reconstructions from specific levels of the z-stacks were useful to determine the spatial location of extended axons. Schematic of imaging direction, sub-fields from the full-thickness z-stack, with hypothetical representative axons shown as green circles in cross section (F). This analysis methodology revealed that, following axonal branching, extension occurred at the agarose-collagen border, potentially exploiting the duel benefits of agarose stiffness and collagen presence (G–I). Scale bar (G–I) 20 μm. Color images available online at www.liebertpub.com/tea

FIG. 6.

Neuronal versus glial penetration into the microconduits. Confocal reconstructions of neuronal constructs stained via immunocytochemistry to denote neuronal somata/axons (β-tubulin III; green), cell nuclei (Hoechst; blue), and glial somata/processes (GFAP; red) at 7 DIV. Axons extended unidirectionally into the microconduit interior (A), while both neuronal and glial somata remained in a dense cluster at the end of the construct (B). Glial processes extended ∼1 mm into the microconduit interior, and were primarily colocalized with axons over this span (C, D). Scale bar (A–D) 100 μm. Higher magnification confocal reconstructions from demonstrative regions in (D) show that although glial processes were present in the initial region of the microconduit, purely axonal projections extended deep into the microconduit interior (E–G). Scale bars (E, G) 50 μm. Scale bar (F) 40 μm. Higher magnification confocal reconstructions from regions in (E) and (G) show that while there was no glial presence at the leading extremities of the axonal projections (H), glial and axonal processes initially grew in tandem, often intertwining (I). Scale bar (H, I) 40 μm. Color images available online at www.liebertpub.com/tea