Cell-seeded alginate hydrogel scaffolds promote directed linear axonal regeneration in the injured rat spinal cord.

Despite recent progress in enhancing axonal growth in the injured spinal cord, the guidance of regenerating axons across an extended lesion site remains a major challenge. To determine whether regenerating axons can be guided in rostrocaudal direction, we implanted 2mm long alginate-based anisotropic capillary hydrogels seeded with bone marrow stromal cells (BMSCs) expressing brain-derived neurotrophic factor (BDNF) or green fluorescent protein (GFP) as control into a C5 hemisection lesion of the rat spinal cord. Four weeks post-lesion, numerous BMSCs survived inside the scaffold channels, accompanied by macrophages, Schwann cells and blood vessels. Quantification of axons growing into channels demonstrated 3-4 times more axons in hydrogels seeded with BMSCs expressing BDNF (BMSC-BDNF) compared to control cells. The number of anterogradely traced axons extending through the entire length of the scaffold was also significantly higher in scaffolds with BMSC-BDNF. Increasing the channel diameters from 41µm to 64µm did not lead to significant differences in the number of regenerating axons. Lesions filled with BMSC-BDNF without hydrogels exhibited a random axon orientation, whereas axons were oriented parallel to the hydrogel channel walls. Thus, alginate-based scaffolds with an anisotropic capillary structure are able to physically guide regenerating axons. STATEMENT OF SIGNIFICANCE: After injury, regenerating axons have to extend across the lesion site in the injured spinal cord to reestablish lost neuronal connections. While cell grafting and growth factor delivery can promote growth of injured axons, without proper guidance, axons rarely extend across the lesion site. Here, we show that alginate biomaterials with linear channels that are filled with cells expressing the growth-promoting neurotrophin BDNF promote linear axon extension throughout the channels after transplantation to the injured rat spinal cord. Animals that received the same cells but no alginate guidance structure did not show linear axonal growth and axons did not cross the lesion site. Thus, alginate-based scaffolds with a capillary structure are able to physically guide regenerating axons.

AngleJ: A new tool for the automated measurement of neurite growth orientation in tissue sections.

BACKGROUND: Regeneration of axons is one means to restore function after central nervous system and peripheral nervous system injury. Besides increasing the number of regenerating axons, guidance of axons over long distances into and across a lesion site are important determinants for efficient functional restoration. Quantification of axon growth directions is therefore an important measure for the efficacy of neuroregenerative approaches. While several methods exist to manually or automatically trace neurites in images of neuronal cultures to determine their length, tools to automatically measure the effect of neurite guidance in tissue sections do not exist. NEW METHOD: Because manual measurements of the orientation of regenerating axons are labor-intensive, time-consuming and unreliable, a plugin called AngleJ for the open source software ImageJ was developed that automatically determines axonal orientation in images of immunohistochemically labeled sections of the spinal cord. RESULTS: Given user-defined filters and thresholds, the plugin accurately detects neurites in sections of the intact spinal cord white matter and a spinal cord hemisection lesion model and measures the distribution pattern of axonal angles. COMPARISON WITH EXISTING METHODS: Values of automatically measured angles strongly correlate with angles obtained by manual measurements in ImageJ (Pearson correlation 0.88-0.97 for white matter and 0.76-0.94 for axons sprouting into a lesion site). CONCLUSIONS: AngleJ can be used as a fast alternative to manual angle measurement in conjunction with ImageJ and its source code is freely available to the community.