Engineered in vitro/in silico models to examine neurite target preference.

Research on spinal cord injury (SCI) repair focuses on developing mechanisms to allow neurites to grow past an injury site. In this article, we observe that numerous divergent paths (i.e., spinal roots) are present along the spinal column, and hence guidance strategies must be devised to ensure that regrowing neurites reach viable targets. Therefore, we have engineered an in vitro micropatterned model in which cultured E7 dorsal root ganglia (DRG) explants may enter alternate pathways (?roots?) along a branching micropattern. Alongside this in vitro model, we have developed an in silico simulation that we validate by comparison with independent experiments. We find in both in silico and in vitro models that the probability of a neurite entering a given root decreases exponentially with respect to the number of roots away from the DRG; consequently, the likelihood of neurites reaching a distant root can be vanishingly small. This result represents a starting point for future strategies to optimize the likelihood that neurites will reach appropriate targets in the regenerating nervous system, and provides a new computational tool to evaluate the feasibility and expected success of neurite guidance in complex geometries.

Varying assay geometry to emulate connective tissue planes in an in vitro model of acupuncture needling.

During traditional acupuncture, fine needles are inserted subcutaneously and rotated, which causes loose fascial tissue to wind around the needle. This coupling is stronger at acupuncture points, which tend to fall above intermuscular fascial planes, than control points, which lay above skeletal muscle. These different anatomical constraints may affect the mechanical coupling. Fascia at acupuncture points is bounded on two sides by skeletal muscle, but at control points is essentially unbounded. These differences were approximated in simple in vitro models. To emulate the narrower boundary within the intermuscular plane, type I collagen was cast in circular gels of different radii. To model the channel-like nature of these planes, collagen was cast in elliptical gels with major and minor axes matching the large and small circular gels, respectively, and in planar gels constrained on two sides. Acupuncture needles were inserted into the gels and rotated via a computer-controlled motor while capturing the evolution of fiber alignment under cross-polarization. Small circular gels aligned faster, but failed earlier than large circular gels. Rotation in elliptical and planar gels generated more alignment-per-revolution than circular gels. Planar gels were particularly resistant to failure. Fiber alignment in circular gels was isotropic, but was stronger in the direction of the minor axis in elliptical and planar gels. In fibroblast-populated gels, cells followed the alignment of the collagen fibers, and also became denser in regions of stronger alignment. These results suggest that the anatomy at acupuncture points provides unique boundaries that accentuate the mechanical response to needle manipulation.

An in vitro assay of collagen fiber alignment by acupuncture needle rotation.

BACKGROUND: During traditional acupuncture therapy, soft tissues attach to and wind around the acupuncture needle. To study this phenomenon in a controlled and quantitative setting, we performed acupuncture needling in vitro. METHODS: Acupuncture was simulated in vitro in three-dimensional, type I collagen gels prepared at 1.5 mg/ml, 2.0 mg/ml, and 2.5 mg/ml collagen, and either crosslinked with formalin or left untreated. Acupuncture needles were inserted into the gels and rotated via a computer-controlled motor at 0.3 rev/sec for up to 10 revolutions while capturing the evolution of birefringence under cross-polarization. RESULTS: Simulated acupuncture produced circumferential alignment of collagen fibers close to the needle that evolved into radial alignment as the distance from the needle increased, which generally matched observations from published tissue explant studies. All gels failed prior to 10 revolutions, and the location of failure was near the transition between circumferential and radial alignment. Crosslinked collagen failed at a significantly lower number of revolutions than untreated collagen, whereas collagen concentration had no effect on gel failure. The strength of the alignment field increased with increasing collagen concentration and decreased with crosslinking. Separate studies were performed in which the gel thickness and depth of needle insertion were varied. As gel thickness increased, gels failed at fewer needle revolutions. For the same depth of insertion, alignment was greater in thinner gels. Alignment increased as the depth of insertion increased. CONCLUSION: These results indicate that the mechanostructural properties of soft connective tissues may affect their response to acupuncture therapy. The in vitro model provides a platform to study mechanotransduction during acupuncture in a highly controlled and quantitative setting.

Neurite outgrowth is directed by schwann cell alignment in the absence of other guidance cues.

Schwann cells enhance axonal regeneration following nerve injury in vivo and provide a favorable substrate for neurite outgrowth in vitro. However, much remains unknown about the nature of interactions that occur between Schwann cells and growing neurites. In this paper, we describe direct evidence of the ability of Schwann cell alignment alone to direct neurite outgrowth. Previously, we reported that laminin micro-patterns can be used to align Schwann cells and thus create oriented Schwann cell monolayers. In the current study, dissociated rat spinal neurons were seeded onto oriented Schwann cell monolayers, whose alignment provided the only directional cue for growing neurites, and neurite alignment with the underlying Schwann cells was analyzed. The orientation of neurite outgrowth mimicked that of the Schwann cells. Associations observed between neurites and Schwann cells suggest that Schwann cells may guide neurite outgrowth through both topographical and molecular mechanisms. This work demonstrates that Schwann cell alignment can direct neurite outgrowth in the absence of other directional cues, and provides a new method for examining neuronal-Schwann cell interactions in vitro.

Oriented Schwann cell monolayers for directed neurite outgrowth.

Schwann cells are an important component of the peripheral nervous system and participate in peripheral nerve regeneration. They create a supportive environment for neurite outgrowth by releasing trophic factors and up-regulating permissive molecules on their surface. In addition, Schwann cells are able to self-organize into linear arrays in vitro and in vivo, suggesting a possible role in neurite guidance. Previously, we showed that Schwann cell placement and orientation in subconfluent cultures can be controlled using microlithographically patterned laminin substrates (Thompson, D. M., and H. M. Buettner. Tissue Eng. 7(3):247-266, 2001). In the current study, these substrates were used to create oriented Schwann cell monolayers. Both Schwann cell orientation and coverage were quantified in response to seeding density, culture medium, and micropattern dimensions. In serum-free medium, increasing the seeding density yielded a linear increase in coverage of the substrate area but decreased cell alignment. In an alternate approach, Schwann cells were first seeded in serum-free medium at moderate seeding density, allowed to align, then expanded in serum-containing growth medium. This produced complete coverage without large seeding densities while preserving alignment to the micropattern. Alignment and coverage were unaffected by micropattern dimensions. This work provides a useful methodology for investigating Schwann cell guidance effects on growing neurites.

Growth cone pathfinding: a competition between deterministic and stochastic events.

BACKGROUND: Growth cone migratory patterns show evidence of both deterministic and stochastic search modes. RESULTS: We quantitatively examine how these two different migration modes affect the growth cone's pathfinding response, by simulating growth cone contact with a repulsive cue and measuring the resultant turn angle. We develop a dimensionless number, we call the determinism ratio Psi, to define the ratio of deterministic to stochastic influences driving the growth cone's migration in response to an external guidance cue. We find that the growth cone can exhibit three distinct types of turning behaviors depending on the magnitude of Psi. CONCLUSIONS: We conclude, within the context of these in silico studies, that only when deterministic and stochastic migration factors are in balance (i.e. Psi ~ 1) can the growth cone respond constructively to guidance cues.