Paralyzed mice walked after one injection

Researchers at Northwestern University injected a material of constantly moving molecules into severed spinal cords; within four weeks the mice were walking.

A liquid that assembles itself into fibres

In 2001, Jeffrey Hartgerink, Elia Beniash and Samuel Stupp at Northwestern University reported in Science a molecule they called a peptide amphiphile: a short peptide attached to a fatty tail, dissolved in water, that assembled into cylindrical nanofibres when the pH changed. The fibres could be reversibly cross-linked, and once cross-linked they directed the growth of hydroxyapatite crystals along their length in the same alignment seen between collagen and mineral in bone. The paper was about making a synthetic extracellular matrix. Its practical consequence was a material that could be injected as a liquid and would become a fibrous scaffold on contact with the salts of living tissue.

First test in an injured spinal cord

Seven years later, Stupp's group and John Kessler's neurology laboratory at Northwestern put the material into a mouse spinal cord. Vicki Tysseling-Mattiace and colleagues designed the peptide to display IKVAV, a five amino acid sequence from the protein laminin that is known to promote neurite outgrowth and to steer neural stem cells away from becoming glia. Injected after a compression injury, the fibres reduced the formation of reactive astrocytes, reduced cell death, increased the number of oligodendrocytes at the injury site, and promoted regrowth of both descending motor and ascending sensory fibres through the lesion. The animals showed significant behavioural improvement. The 2008 paper in the Journal of Neuroscience established that a glial scar could be inhibited and axons coaxed across an injury with a purely synthetic nanostructure. The effect was partial, and the injury was mild.

Two signals and a mutation that had nothing to do with biology

The 2021 paper that this story is built on, by Zaida Álvarez, Stupp and colleagues at Northwestern's Simpson Querrey Institute, changed two things. First, it carried two signals instead of one: the IKVAV sequence, which activates the receptor beta-1 integrin, and a fifteen amino acid mimic of fibroblast growth factor 2, which activates the receptor FGFR1 and promotes cell survival, proliferation and blood vessel growth. Second, and this is the part that gave the material its nickname, the researchers altered the part of the molecule that has no biological function at all.

Between the fatty tail and the signal sits a four amino acid segment that controls how the molecules pack. The team built a library of eight IKVAV molecules identical in every way except this segment, which was varied among the amino acids valine, alanine and glycine. Simulations predicted, and two physical measurements confirmed, that the variants differed in how much the molecules moved inside the fibre. Fluorescence depolarisation of a dye buried in the fibre core and transverse-relaxation nuclear magnetic resonance of a proton in the IKVAV signal both identified the same two variants, called PA2 and PA5, as the most mobile. Those same two variants activated beta-1 integrin and its downstream kinases most strongly in human neural progenitor cells, and pushed the highest fraction of those cells toward a neuronal identity. Motion and bioactivity rose together.

The same logic was then applied to the FGF2 molecule. Mixed at a 90 to 10 ratio with IKVAV PA2, two FGF2 variants formed stable gels of similar stiffness. One carried a valine-valine-alanine-alanine segment and moved more; the other carried alanine-alanine-glycine-glycine and moved less. Everything else about the two mixtures was the same.

What happened in the cord

The model was a severe contusion of the mouse spinal cord producing hind limb paralysis. Twenty-four hours after the injury, a saline solution of the material was injected into the cord at the lesion. Comparison groups received saline alone, IKVAV PA2 alone, the less mobile co-assembly, or the more mobile co-assembly. Corticospinal axon regrowth was measured 12 weeks later with an anterograde tracer.

In saline-injected mice there were almost no regrown axons within the lesion. In mice given the more mobile two-signal material, corticospinal axons crossed the lesion and passed its far border. Total regrowth within the lesion was 50 times that of the saline group and twice that of the less mobile co-assembly, a difference produced entirely by a change in a segment of the molecule that no receptor recognises. The glial scar at the lesion borders was less dense in every bioactive group. Growth-associated protein 43, a marker of growing axon tips, was elevated only in the most mobile material. At three months, myelin basic protein wrapped the regrown axons. Newly formed blood vessels, labelled by BrdU given in the first week after injury, were present in the lesion of the most mobile group at 12 weeks. Ventral tissue structure was preserved, and more motor neurons survived around the lesion. There was no sign of new neurons being born locally.

The material itself did not stay. Fluorescently labelled scaffolds were imaged at 1, 2, 4, 6 and 12 weeks, and all three materials degraded gradually over that period at the same rate.

The walking measure

Locomotion was scored on the Basso Mouse Scale, an open field locomotor test, and by footprint analysis over 12 weeks. From one week after injury onward, every group given the material scored significantly higher than the saline group. At three weeks the most mobile two-signal material separated from the others: a mean score of 5.9, against 4.4 for the less mobile co-assembly and 4.3 for IKVAV PA2 alone, with a standard deviation of 0.5 in each. Footprints of the best group showed significantly longer and wider strides. Northwestern's announcement on November 11, 2021 summarised the result as paralysed mice regaining the ability to walk within four weeks of a single injection.

Why the molecules dance

Stupp's explanation, in the university's statement, was that receptors on neurons and other cells are themselves in constant motion, so molecules that move more within a fibre will encounter those receptors more often. The innovation claimed was control over the collective motion of more than 100,000 molecules within a nanofibre, a property the paper proposes as a general design variable for any synthetic signalling material. The same statement quoted Stupp as intending to go directly to the U.S. Food and Drug Administration to begin the process of approval for human use.

What the story does and does not show

The 2021 paper showed, in a mouse model of severe spinal cord contusion, that a single injection of a two-signal supramolecular material 24 hours after injury produced corticospinal axon regrowth 50 times that of saline, reduced the glial scar, restored myelin and blood vessels, preserved motor neurons, and raised open field locomotor scores above those of every comparison group by three weeks. It also showed that these outcomes depended on the internal motion of the molecules, a variable that had not previously been treated as a therapeutic parameter.

The model was a contusion, not a complete transection, and the injection was given one day after injury, not in the chronic phase where most human patients live. Group sizes are given in the supplementary material rather than the main text. The slide text's phrase about the material degrading into nutrients comes from the university's statement, not the paper, which reports only that the scaffolds degraded over 1 to 12 weeks. The statement that the team would go to the FDA was an intention expressed in November 2021; no human trial result exists. The finding is a mechanism, measured in mice, and a design principle that other materials can now be tested against.

Four weeks after one injection, they walked.