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ANISOGEL Report Summary

Project ID: 637853
Funded under: H2020-EU.1.1.

Periodic Reporting for period 2 - ANISOGEL (Injectable anisotropic microgel-in-hydrogel matrices for spinal cord repair)

Reporting period: 2016-09-01 to 2018-02-28

Summary of the context and overall objectives of the project

In many tissues of the human body, such as nerve tissue, the spatial organization of cells plays an important role. Nerve cells and their long protrusions assemble into nerve tracts and transport information throughout the body. When such a tissue is injured, an accurate spatial orientation of the cells facilitates the healing process. In Anisogel, we developed an injectable gel, which can act as a guidance system for nerve cells. Inside the body, an extracellular matrix surrounds the cells. It provides mechanical support and promotes spatial tissue organization. In order to regenerate damaged tissue, an artificial matrix can temporally replace the natural extracellular matrix. This matrix needs to mimic the natural cell environment in order to efficiently stimulate the regenerative potential of the surrounding tissue. Solid implants, however, may impair remaining healthy tissue whereas soft, injectable materials allow for a minimal invasive therapy, which is particularly beneficial for sensitive tissues, such as the spinal cord. Unfortunately, up to now, artificial soft materials did not yet reproduce the complex structures and spatial properties of natural tissues.

The newly developed minimal invasive material is termed ‚Anisogel‘. If you aim to enhance the regeneration of damaged spinal cord tissue, you need to come up with a new material concept. We use micrometer-sized building blocks and assemble them into 3D hierarchically organized structures. Anisogel consists of two gel components. Many, microscopically small, soft rod-shaped gels, incorporated with a low amount of magnetic nanoparticles, are the first component. Using a weak magnetic field, scientists can orient the gel rods, after which a very soft surrounding gel matrix is crosslinked, forming the structural guidance system. The gel rods, being stabilized by the gel matrix, maintain their orientation, even after removal of the magnetic field. Using cell culture experiments, we demonstrate that cells can easily migrate through this gel matrix, and that nerve cells and fibroblasts orient along the paths provided by this guidance system. A low amount of one percent gel rods inside the entire Anisogel volume is proven to be sufficient to induce linear nerve growth. The material is the first injectable biomaterial, which assembles into a controlled oriented structure after injection and provides a functional guidance system for cells.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

During this period in Anisogel, we have developed the methods to fabricate rod-shaped microgels with different dimensions, aspect ratios, stiffness, and biochemical modification. Depending on the amount of magnetic particles loaded inside, we achieve magnetically responsive microgels, which orient in low milli tesla magnetic fields. The microgels are sufficiently small that they can be injected in combination with a master hydrogel, which can crosslink around the aligned microgels to fix their orientation after removal of the magnetic field. A synthetic poly (ethylene based) master hydrogel was synthesized with bioactive domains to support cell attachment and growth, degradable domains that can be cleaved by the cells, and domains with the ability to bind growth factors to stimulate cell growth. In addition, a natural fibrin gel has been applied to study the guiding effect of the Anisogel. Both master hydrogels crosslink according to the same blot clotting mechanism.
Cell culture experiments revealed that cells and nerves feel the physical and mechanical anisotropy of the Anisogel, resulting in unidirectional growth. Only a minimal amount of rod-shaped guiding elements was required, all the way down to 1 vol% in the case of nerve cells. This work was recently published in the journal Nano Letters (June 2017, front cover).

Later, the effect of biofunctional molecules attached to the microgels was investigated and demonstrated that introducing cell adhesiveness of the microgels enhanced fibroblast attachment and alignment. Interestingly, modifying the microgels with cell-adhesive peptides, such as RGD, reduced the need of the cells to produce their own extracellular proteins, as a significan reduction of fibronectin was observed. This shows that the microgels nicely mimicked the natural ECM proteins available for the cells to attach to. As the produced fibronectin is also aligned, a positive feedback is presented for the cells, where the natural proteins can over time take over the function of the degradable microgels.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Due to the success of the Anisogel, an alternative method to produce rod-shaped, magneto-responsive guiding objects was established. Here, cell adhesive short fibers are applied in a crosslinked precursor solution. The micron-scale fibers are prepared by an effective high-throughput electrospinning/microcutting technique with tailorable dimensions. Encapsulation of low iron oxide nanoparticles during the spinning process also leads to magneto-responsive behavior in the presence of an external magnetic field in the milli Tesla range.The simplicity and versatility of this approach also enables the formation of unidirectional, oriented structures in situ with controlled features that stimulate fibroblasts and functional nerve cells to grow in a linear manner. The fiber-based Anisogel supports spontaneous electrical activity of the neurons, proving neuronal functionality and importantly, electrical signals that propagate along the anisotropy axis of the material. This is a crucial function for applications in linearly oriented neuronal tissues, such as spinal cord. This elegant, high-throughout fiber fabrication method and low invasiveness of this technology can enhance the clinical outcome for patients without risking further damage.
Based on this work, one additional paper was published open access in Small (September 2017, front cover plus an additional video abstract (featured on the Wiley website and https://youtu.be/136xwXsLWGg)).

In the second half of Anisogel, we will study the effect of microgel dimensions and aspect ratios on the rate of magnetic orientation and directed cell growth inside the Anisogel. We will also further modify the microgels with different peptides to affect specific cell types. Further the function of the Anisogel will be investigated to grow aligned blood vessels (angiogenesis) and glial cells, present in the nervous system to insulate the nerves with myeling and enhance electrical nerve signaling.
The animal protocol will be submitted in 2017 to hopefully start testing the Anisogels in a rat contusion spinal cord injuyr model in 2018. These in vivo studies are crucial to proof the potential of the Anisogels for clinical therapies.

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