Novel use of a chitosan-based hydrogel for the treatment of a nervous tissue lesion

Abstract

The present invention relates to hydrogel microparticles comprising chitosan for use in the treatment of a nervous tissue lesion in a patient who does not exhibit an improvement in at least one score of at least one neurological assessment scale.

Description

Description Title: NEW USE OF A CHITOSAN-BASED HYDROGEL FOR THE TREATMENT OF NERVE TISSUE INJURY

[0001] The present invention relates to a novel use of a hydrogel comprising chitosan for the treatment of nerve tissue injury in a patient who does not show improvement in at least one score on at least one neurological assessment scale. Context of the invention

[0002] Nerve tissue damage includes brain injuries, spinal cord injuries, and peripheral nerve damage. In all cases, these injuries result in a total or partial loss of motor and / or sensory functions, as well as, in some cases, losses of cognitive or executive functions, such as language, memory, or attention.

[0003] More specifically, spinal cord injuries lead to paralysis, loss of sensation, and chronic pain. In the vast majority of cases in humans (as in rodent preclinical models), these injuries result in the disruption of nerve connections between the brain and the rest of the body, followed by progressive neurodegeneration from the site of the injury and the formation of a cavity surrounded by glial and fibrous scar tissue. This complex constitutes both a physical and chemical barrier to axon regeneration. It should also be noted that disruption of the blood-spinal barrier and impaired vascularization further hinder the repair of damaged tissue.

[0004] The chemical barrier is due to the significant production by non-neuronal cells of toxic and inhibitory molecules (cytokines, proteoglycans (such as extracellular chondroitin sulfate proteoglycans (CSPG)), and chemorepulsive molecules). Other inhibitory molecules originate from myelin (myelin-derived protein Nogo-A, MAG (Myelin-Associated Glycoprotein), and OMgp (Oligodendrocyte myelin glycoprotein)). These latter molecules are exposed in the extracellular matrix following axon demyelination or degeneration.

[0005] In addition to inhibitory effects, the bioavailability of trophic factors promoting axonal survival and regeneration is quite limited in the adult central nervous system (CNS). These major complications—toxicity of the injury environment and reinhibition of axonal regrowth from the spinal cord injury—are associated with inappropriate inflammation that leads to tissue and vascular damage and inevitably results in hypoxia, hemorrhage, and edema. These effects accelerate necrosis of the damaged neural tissue. Disruption of the blood-spinal barrier creates an abnormal situation and promotes the infiltration of immune cells from the blood, such as monocytes / macrophages and lymphocytes, which, along with activated microglia (resident immune cells in the CNS), spread inflammation beyond the initial injury site, contributing to secondary lesion expansion and the worsening of neurological disorders. This inflammation This persists because it is not inhibited by the sequential activation of the anti-inflammatory process that contributes to tissue regeneration outside the nervous system (for example, in the case of wound healing after a skin or muscle injury). This abnormal immune response therefore constitutes one of the major obstacles to the recovery from central nervous system lesions.

[0006] These characteristics, along with the formation of a cavity, also occur in cases of cerebral trauma and following ischemia. Even if some axons attempt to regrow through the lesion in the initial stages following injury, they eventually retract as harmful factors increase at the injury site.

[0007] In comparison, the peripheral nervous system (PNS) is rather susceptible to axonal repair and regeneration after traumatic injury, thanks to the rapid inflammatory response and the action of Schwann cells (myelinating cells of the PNS, in favor of rapid removal of cellular debris and myelin (although in the case of severe injuries, complete regeneration is sometimes limited due to the difficulty in guiding nerve fibers to the appropriate peripheral targets).

[0008] Current treatments for spinal cord injuries include, in the acute stage, surgical decompression within 24 hours of the injury. Administration of methylprednisolone may sometimes be considered. Neuromodulation and rehabilitation techniques can also be used to improve symptoms, particularly after stabilization in the chronic stage.

[0009] Furthermore, the mammalian central nervous system (CNS) possesses neuronal plasticity. Thus, after a partial spinal cord injury, the undamaged axons can undergo remodeling and participate in functional recovery, primarily linked to the growth of collateral processes (or "sprouting"). Indeed, this plasticity allows for the establishment of new neural circuits, some of which can lead to recovery. In the case of a complete lesion, such plasticity is mainly manifested in the segments adjacent to the lesion. These observations regarding the existence of a plastic capacity in the CNS have spurred intense research into the development of new therapeutic strategies for repairing the damaged nervous system (Nothias F, Stimulating Axonal Regrowth, Biofutur No. 322, June 2011, pages 36-40).

[0010] Several experimental models have thus been developed to stimulate / guide axonal regrowth by counteracting the inhibitory action of proteins contained in the glial scar, such as: - Injection of an IN-1 antibody to antagonize the inhibitory effect of certain myelin components (Nogo, MAG, and OMgp) that share the same receptor on the neuron, Nogo-A. Chen MS, Huber AB, van der Haar ME, Frank M, Schnell L, Spillmann AA, Christ F, Schwab ME. Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibodies. IN-1. Nature. 2000 Jan 27;403(6768):434-9. doi:10.1038 / 35000219. PMID: 10667796); - Cell therapy, for example stem cell transplantation (mesenchymal, neural progenitor, etc.), in the presence of ABC-type chondroitinase to degrade CSPG proteoglycans, which are inhibitors of axonal growth (Ikegami T, Nakamura M, Yamane J, Katoh H, Okada S, Iwanami A, Watanabe K, Ishii K, Kato F, Fujita H, Takahashi T, Okano HJ, Toyama Y, Okano H. Chondroitinase ABC combined with neural stem / progenitor cell transplantation enhances graft cell migration and outgrowth of growth-associated protein-43-positive fibers after rat spinal cord injury. Eur J Neurosci. 2005 Dec;22(12):3036-46. doi: 10.1111 / j.1460-9568.2005.04492.x. PMID: 16367770), or Schwann cells or other glial cells (olfactory bulb sheathing cells, OEC).

[0011] Furthermore, tissue engineering has been introduced into nervous system repair through the use of biomaterials that act as polymer-based scaffolds. These biomaterials are used in spinal cord injury therapy to support damaged tissue, allowing it to regenerate rather than progressing to necrosis and ultimately leading to cavity formation. This necrotic process involves numerous cell types, including vascular, inflammatory, and neuronal cells. The creation of a hostile, poorly vascularized environment hinders cell survival and nerve fiber regeneration. Strategies for repairing injured spinal cords using biomaterial reinforcement have thus been developed in preclinical studies based on numerous experimental models.

[0012] In this regard, advances in the design of biomaterials for tissue engineering offer a promising basis for neural tissue repair. Biomaterials differ in their physical and chemical properties, their shape or structure, and whether they are of natural or synthetic origin (for further details, see the article by Soares et al. (Soares S, Y von Boxberg, and F Nothias (2020) Repair strategies for traumatic spinal cord injury; advance in bioengineering-based preclinical approaches. Review in Revue Neurologique 176 (4) 252-260). The central idea is to create a scaffold that provides both mechanical support to the injured tissue and physical guidance for axon regrowth.It is also about mitigating the toxicity of the hostile environment by acting either directly by capturing toxic residues to prevent the death of endogenous cells, or indirectly by association with cell therapy or a drug approach.

[0013] The use of a biomaterial composed of chitosan hydrogel microparticles for the treatment of traumatic injuries to nerve tissue, particularly the spinal cord, has also been considered (EP2874672). This patent demonstrates that a suspension of chitosan hydrogel microparticles can stimulate and guide axonal regrowth in traumatic spinal cord injuries during the acute phase, i.e., immediately after the injury occurs, without inducing inflammation.

[0014] Therapeutic solutions for treating spinal cord injuries do exist. However, one of the current challenges in treating nerve tissue lesions is the very short window of opportunity for action. Patent EP2874672 describes the insertion of an implant made of chitosan hydrogel microparticles immediately after the lesion forms. It has also been described that surgical decompression in the acute phase within 24 hours of the onset of spinal cord injury is recommended, and that beyond this time the benefit of this surgical intervention seems to be lost (Ramakonar H, Fehlings MG. 'Time is Spine': new evidence supports decompression within 24 h for acute spinal cord injury. Spinal Cord. 2021 Aug;59(8):933-934. doi: 10.1038 / S41393-021-00654-0. Epub 2021 Jul 3. PMID: 34218264; PMCID: PMC8338556).

[0015] These treatments thus make it possible to act immediately after the occurrence of the lesion, when it is still "unstable" (due to cellular and molecular rearrangements) and when it is considered that it can still be repaired.

[0016] To date, no therapeutic treatment has been shown to be clinically effective in the acute phase of nerve tissue lesions, particularly those affecting the spinal cord. No therapeutic treatment has been shown to be clinically effective in the absence of spontaneous functional recovery. Clinically, when there is no functional recovery and no further progression, the lesion is considered "stabilized," and the patient is described as having reached a standard state, whether or not conditioned by symptomatic treatment. When at least one of the patient's scores in a standard state, as described on a neurological assessment scale, does not change, no longer changes, or even worsens, it is considered that the neurodegeneration at the lesion site is too extensive and / or the presence of glial and fibrous scarring prevents any reconnection across the lesion sites (regeneration, axonal regrowth, etc.) in the absence of any currently proven therapeutic treatment.The present invention aims to treat neurological tissue lesions in patients who do not show improvement in at least one score on at least one neurological assessment scale. The score is generally assessed repeatedly by healthcare professionals. The same score on at least one neurological assessment scale, obtained at two successive assessments, is indeed correlated with a lack of improvement in the patient's sensory and / or motor functions. The score is considered stable and corresponds to a lesion that will not spontaneously repair itself. A worsening of at least one score on at least one neurological assessment scale between two successive assessments is also correlated with a lack of improvement in the patient's lost sensory and / or functional abilities. The score is considered worsened.

[0017] The present invention is based on the inventors' unexpected results, which showed that the use of hydrogel microparticles in experimental models of spinal cord and cortical lesions, which showed no improvement in neurological assessment tests, not only reduced the lesion but also improved the affected functions (e.g., locomotion, sensation, or pain). These results thus demonstrate that, surprisingly, the use of hydrogel microparticles results in a therapeutic benefit even when the nerve tissue damage appeared to be irreparably established and irreversible. Description of the invention

[0018] The present invention therefore relates to hydrogel microparticles comprising chitosan, the chitosan having a degree of acetylation less than or equal to 20% and its concentration in the hydrogel being between 0.25 and 5% by weight relative to the total weight of the hydrogel, for its use in the treatment of nerve tissue injury in a patient who does not show improvement of at least one score on at least one neurological assessment scale.

[0019] Chitosan is a well-known bioactive and biodegradable polymer. It is a polysaccharide derived from the deacetylation of chitin. Chitin is the organic matrix of the cuticle of arthropods and is therefore a fundamental constituent of these organisms' bodies. Chitin is one of the most abundant natural, non-toxic, and biodegradable polysaccharides, found notably in the exoskeleton of crustaceans, mollusks, and insects, in the endoskeleton of cephalopods, and also in fungi. The molecular mass of chitin can reach hundreds or even millions of kilodaltons (kDa). Chitin is a high molecular weight biopolymer and, along with cellulose, is one of the most widespread polysaccharides in nature. Chitin consists of a linear chain whose structure contains glucosamine (GLcN) and N-acetylglucosamine (GLcNAc) repeat units linked by a β,(1-4) bond.Chitin is extracted from crustacean shells and / or cephalopod endoskeletons primarily through chemical processes. Chitosan is then transformed by deacetylation of the chitin in hot, concentrated sodium hydroxide. Due to its biocompatibility with human tissues and its healing properties, chitosan has demonstrated its effectiveness in various physical forms, such as gels, powders, films, and tubes, in a range of products including dressings, artificial skin, corneal dressings, and surgical sutures that dissolve naturally after healing. It is also used in bone and cartilage repair (Montembault et al. Biochimie, 88, 2006, pages 551-564), as well as in dental surgery, implants, and gum healing.

[0020] The chitosan used in the context of the present invention has a degree of acetylation of 20% or less, i.e., between 0 and 20%. Indeed, if a higher degree of acetylation is used, the chitosan does not allow axonal regeneration and, moreover, induces a significant inflammatory reaction. In one embodiment, the chitosan used in the context of the present invention has a degree of acetylation between 0 and 15%, advantageously less than 10%. The degree of acetylation also influences the bioresorbability of the chitosan. In fact, the higher the degree of acetylation, the higher the biodegradation and bioresorption kinetics. The degree of acetylation of the chitosan can be measured by any technique, preferably proton NMR, following the Hirai methodology. (Asako Mirai, Hisashi Odani, Akio Nakajima, Polymer Bulletin (1991) Volume: 26, Issue: 1, Springer, Pages: 87-94).

[0021] In one embodiment, the molar mass of chitosan is greater than 180,000 g / mol, advantageously greater than 200,000 g / mol, and advantageously greater than 300,000 g / mol. In another embodiment, the molar mass of chitosan is less than 1,000,000 g / mol, and even more advantageously less than 600,000 g / mol, in particular around 450,000 g / mol. Indeed, high molar masses allow for a hydrogel with a low chitosan concentration, while limiting the inflammatory response and promoting the bioresorbability of the hydrogel.

[0022] According to the invention, "nerve tissue injury" means any injury to the peripheral or central nervous system. The peripheral nervous system means all parts of the nervous system that are outside the brain and spinal cord. The central nervous system, on the other hand, consists of the brain (including the retina and optic nerve) and the spinal cord.

[0023] A "peripheral nervous system injury" refers to damage to parts of the nervous system that are outside the central nervous system. This can include any damage to the peripheral nerves, whether they belong to the autonomic or somatic nervous system, that transmit information to the central nervous system. Examples include injuries to the radial nerve of the upper limb (the most common), injuries to the brachial plexus, and injuries to the nerves of the upper or lower limbs (such as the sciatic nerve).

[0024] A "central nervous system injury" refers to damage to the brain or spinal cord. In the case of the spinal cord, this damage can be caused by trauma (mechanical injury resulting, for example, from a fall or a road accident) or by non-traumatic causes such as tumors, degenerative and vascular diseases, infections, toxins, or congenital malformations. A spinal cord injury is also referred to as a "spinal cord injury." This could be a lesion at the level of O1-O4, C4-T1, T1-T12, L1-S1, S2-S4 / 5, for example at the level of O1-O3, C3-C4, O5, C6-C7, C7-T1, T1-T12, T11-L2, L3, L4-S1, S2-S4 / S5. Preferably, it is a cervical (C1-C7) or thoracic (T1-T12) lesion.At the level of the brain, damage can result from head trauma, ischemia, stroke, transient ischemic attack (TIA), neurodegenerative disease (Alzheimer's disease, Frontotemporal dementia, Parkinson's disease, Huntington's disease, Lewy body disease, Multiple sclerosis, Cerebellar (spino) ataxia, Friedreich's ataxia, Posterior cortical atrophy, Multiple system atrophy, Progressive supranuclear palsy, corticobasal degeneration (this list is not exhaustive)).

[0025] According to the invention, "treatment of nerve tissue damage" can also be understood as the repair of a peripheral nerve or the repair of the spinal cord or at the cerebral level, of the reduction of the lesion, axonal regeneration, treatment of paralysis related to spinal cord or brain injury, treatment of pain related to nerve tissue injury, and / or treatment of disorders related to neurodegenerative disease. More specifically, the treatment according to the invention aims to increase the number of axons, for example by axonal regeneration, and / or promote axonal regrowth, for example by promoting axonal plasticity and the establishment of new nerve connections. The treatment according to the present invention aims in particular to reduce the size of the cavity that has formed following the injury in the patient, to reduce the glial reaction, to modulate neuroinflammation, to restore vascularization, and / or improve the patient's motor and sensory abilities. The size of the cavity can be observed by medical imaging and by measurements of inflammation (See, for example, Nicolas Arlicot et al.)., Initial evaluation in healthy humans of [. 18 F]DPA-714, a potential PET biomarker for neuroinflammation, Nuclear Medicine and Biology, Volume 39, Issue 4, 2012, Pages 570-578).

[0026] According to the invention, a "neurological assessment scale" means any standard used to determine whether the patient's brain, spinal cord, peripheral nerves, and / or muscle and skin receptors are functioning normally. More specifically, the neurological assessment scales used in the invention are scales of the patient's motor and / or sensory functioning, namely a sensory assessment scale, a motor assessment scale, or a sensorimotor assessment scale.

[0027] Specifically, this refers to a scale based on international standards for neurological classification of spinal cord injuries (such as the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI)), the ASIA (American Spinal Injury Association) scale, the Lower Extremity Functional Scale (LEFS), the Upper Extremity Functional Scale (UEFS), the Tetraplegic Motor Capacity Scale, the Berg Balance Scale, the Functional Independence Measure, the Spinal Cord Injury Independence Measure (SCIM version III), the modified Barthel Index, or peripheral nerve injury assessments. These standards and scales are used to measure the degree of remaining intact motor and / or sensory function in the patient.

[0028] According to one embodiment of the invention, the neurological functional assessment scale used is selected from among the ASIA scale (whose AIS (ASIA Impairment Scale) grade is determined according to the ISNCSCI clinical examination), the Lower Limb Functional Scale (LEFS), the Upper Limb Functional Scale, the Tetraplegic Motor Capacity Scale, the Berg Balance Scale, the Spinal Cord Independence Measure (SCIM version III), and the modified Barthel Index. According to a preferred embodiment, the neurological functional assessment scale used is the ASIA scale.

[0029] According to one embodiment, the rating scale is a sensory rating scale for measuring neurological dysfunction and / or pain (such as the rating scale ISCIPBDS (International SCI Pain Basic Dataset), the NPSI (Neuropathic Pain Symptom Inventory), and the QST (Quantitative Sensory Testing) scale, which assesses mechanical allodynia by mechanical stimulation of the dorsal column (DCML, Dorsal Column-Medial Lemniscus) and / or by thermal stimulation of the spinothalamic tract (STT). In one embodiment, the assessment scale is a scale for measuring spastic reflexes (or spastic reflex activity of muscles) selected from flexion, extension, or clonus, for example, the SCAT (Spinal Cord Assessment Tool).

[0030] According to the invention, "at least one score on at least one neurological assessment scale" means that one or more scores can be determined by referring to one or more scales. For example, some scales determine only the patient's motor function, and in this case, only a "motor" score is determined by the scale. In other cases, the scales aim to determine the patient's sensation, and a "sensory" score is determined. Other scales incorporate both a motor score and a sensory score, which allow, for example, the definition of lesion grades. Alternatively, several scales can also be used, for example, to combine a scale for determining a motor score with a scale for determining a sensory score.When a scale allows for the determination of both a motor score and a sensory score, the absence of improvement can be understood as the absence of improvement in the motor score only, the sensory score only, or both motor and sensory scores. The motor score aims to assess muscle function, for example, to determine if there is total paralysis, if there is a visible or palpable contraction, if there is active movement through its full range of motion, etc. The sensory score aims to assess the patient's sensitivity, for example, by evaluating sensitivity to pinprick and light touch at key points in the 28 dermatomes of the human body. The sensory score can also assess the patient's pain (for example, pain at the level of the lesion or below it, neuropathic pain, musculoskeletal pain, etc.).

[0031] According to one embodiment, the invention relates to hydrogel microparticles for the aforementioned use in a patient who does not show improvement in a motor score and / or a sensory score on at least one neurological assessment scale.

[0032] According to one embodiment, the invention relates to hydrogel microparticles for the aforementioned use, wherein the absence of improvement in at least one score on at least one neurological assessment scale is determined after two successive post-lesion assessments.

[0033] According to the invention, a "lack of improvement of at least one score" means a score that is identical during two successive assessments over time using the same scale. In this case, the lesion is said to be "stable." A "lack of improvement of at least one score" also means a less favorable score during a subsequent assessment compared to the first assessment using the same scale. In this case, the lesion is said to have worsened. In one embodiment, the invention thus relates to hydrogel microparticles comprising chitosan for the aforementioned use, in which the absence of improvement is a stability of at least one score on at least one neurological assessment scale or a worsening of at least one score on at least one neurological assessment scale.

[0034] According to a particular embodiment of the invention, the absence of improvement in at least one score on at least one neurological assessment scale is evaluated using the ASIA scale. The ASIA grade, also called the ASIA impairment scale, was developed by the American Spinal Injury Association (ASIA). This classification is now widely used to assess spinal cord injuries. The ASIA scale consists of grades represented by letters from A to E and comprises the scores from three assessments: (1) Motor Assessment: The scale assesses the muscle strength of key muscle groups in different parts of the body, using a scale of 0 to 5. Scores range from "0" (no muscle contraction) to "5" (normal muscle strength). More specifically, the values ​​are as follows: 0: total paralysis; 1: visible or palpable contraction; 2: active movement without gravity; 3: active movement without gravity; 4: active movement against gravity; 5: active movement against resistance. (2) Sensorimotor Assessment: This scale evaluates the sensitivity of different body regions to tactile stimuli, using a scale from 0 to 2. A score of "0" indicates a total loss of sensation, while a score of "2" indicates normal sensitivity. More specifically, the values ​​are as follows: 0: absent; 1: diminished; 2: normal. (3) Lesion assessment: The ASIA scale identifies the level of the lesion, i.e. the vertebra where the spinal cord is damaged.

[0035] The scores obtained allow for the assignment of a grade (A, B, G, D, or E) to describe the patient's degree of functionality. Grade A signifies a total loss of sensory and motor function, while grade E indicates normal function. More specifically, the grades are as follows: A Complete: no motor function or sensation in the S4-S5 region; B Incomplete: sensation but not motor function is preserved below the level of the lesion, particularly in the S4-S5 region; G Incomplete: motor function is preserved below the level of the lesion, and more than half of the muscles tested below this level have a score < 3; D Incomplete: motor function is preserved below the level of the lesion, and at least half of the muscles tested below this level have a score > 3; E Normal: both sensation and motor function are normal.

[0036] Scales, such as the ASIA scale, allow for the establishment of several scores which then determine grades. The transition from one grade to another is generally recognized as a significant functional change. According to one embodiment, the invention relates to hydrogel microparticles for the aforementioned use in a patient who does not show an improvement of at least one grade score on at least one neurological assessment scale.

[0037] According to one embodiment, the lesion is graded AB, G, or D according to the ASIA scale. For example, a lack of improvement in at least one score on the neurological assessment scale is understood here as the same grade being found during two successive assessments, or as a more severe grade being obtained at the second assessment, indicating a worsening of the patient's condition.Thus, according to one embodiment, within the framework of the ASIA scale, a lack of improvement of at least one score means that grade D was assessed following the injury and that the same grade is found during the second assessment or that it has evolved to grade G without treatment, that grade G was assessed following the injury and that the same grade is found during the second assessment or that it has evolved to grade B without treatment, that grade B was assessed following the injury and that the same grade is found during the second assessment or that it has evolved to grade A without treatment, or that grade A was assessed following the injury and that the same grade is found during the second assessment.

[0038] According to a particular embodiment of the invention, the absence of improvement in at least one score on at least one neurological assessment scale is evaluated using the ASIA scale, combined with the IMSOP scale (functional scoring scale, International Medical Society of Paraplegia) (Lascu CF, Buhas CL, Mekeres GM, Bulzan M, Bot RB, Càità GA, Voità IB, Pogan MD. Advantages and Limitations in the Evaluation of the Neurological and Functional Deficit in Patients with Spinal Cord Injuries. Clin Pract. 2022 Dec 27;13(1):14-21.)

[0039] According to a preferred embodiment, the absence of improvement in at least one score on at least one neurological assessment scale is determined after two successive assessments. In one embodiment, these two post-injury assessments are performed, respectively, the first within 24 hours of the injury (the injury being considered the TO) and the second at TO + at least 2 days, such as TO + 2 days, TO + 3 days, TO + 4 days, TO + 5 days, TO + 7 days, TO + 2 weeks, TO + 3 weeks, TO + 4 weeks, ...

[0040] Depending on the specific manifestation, the lesion is either complete or incomplete. A spinal cord injury or a peripheral nerve injury can indeed lead to a partial or complete loss of sensory and / or motor functions downstream from the lesion. In the case of paraplegia, arm function is preserved (this can be described as a lesion in the lower part of the spinal cord), whereas in the case of tetraplegia, this function is also affected (this can also be described as a lesion in the upper part of the spinal cord). Similarly, in the case of a peripheral nerve injury, for example, an injury to a nerve in the hand (median, radial, or ulnar nerve), the lesion can be complete, with total loss of motor and sensory functions, or incomplete, with loss of mobility in some fingers or phalanges, or loss of sensation in all or part of the fingers.

[0041] According to one embodiment, nerve tissue damage is either traumatic or non-traumatic. Indeed, the damage may be due to trauma or may not result from a traumatic event. These non-traumatic lesions are not due to a physical impact but to a cause that will lead to progressive trauma to the spinal cord. This is sometimes referred to as spinal cord injury without radiographic abnormalities (or SCIWORA (Spinal Cord Injury Without Radiography Abnormalities)). For example, this includes lesions due to the progressive growth of a cell mass (compression by a tumor, for example), progressive degeneration (myelopathy, for example), or a herniated disc. Neurodegenerative lesions result in the loss of neurons in specific locations; axons from other regions are then left without target neurons and can therefore no longer perform their function. Implanting the microparticles according to the present invention at the lesion site can create a favorable environment for regenerating nerve tissue and stimulating / guiding axonal regrowth from damaged neurons to other, undamaged neurons.Particle implantation can also stimulate / guide the regrowth of collaterals from neighboring axons (“Sprouting”, PTJA Williams, et al., Combined biomaterial scaffold and neuromodulation strategy to promote tissue repair and corticospinal connectivity after spinal cord injury in a rodent model, Experimental Neurology, Volume 382, ​​2024) and promote neuronal connections.

[0042] According to the invention, "hydrogel" means a viscoelastic mass (having a complex shear modulus G* = G' + jG” such that G' > 10G” over a frequency range greater than one decade, where G' is the conservation modulus (elastic modulus), G” is the loss modulus (viscous modulus), and j is an imaginary number (sometimes denoted i)) comprising at least 80% water by mass, preferably at least 90% water by mass, and preferably at least 95% water by mass. There are two categories of hydrogels: chemical hydrogels and physical hydrogels. A hydrogel is considered physical when the interactions responsible for interchain cross-linking are physical, and are in particular hydrogen bonds and / or hydrophobic interactions, as opposed to a chemical hydrogel in which the interchain interactions are covalent.

[0043] According to the invention, the hydrogel is chitosan-based. In the context of the present invention, the concentration of chitosan in the hydrogel is between 0.25 and 5% by weight relative to the total weight of the hydrogel. In one embodiment, the concentration of chitosan in the hydrogel is less than 4% by weight relative to the total weight of the hydrogel. In another embodiment, the concentration of chitosan in the hydrogel is greater than 0.5% by weight relative to the total weight of the hydrogel, in particular between 1 and 3% by weight relative to the total weight of the hydrogel, more particularly approximately 2.5% by weight relative to the total weight of the hydrogel. Indeed, when the chitosan solution has a concentration of less than 0.25% by weight, it is not possible to gel the solution to obtain a macroscopic hydrogel.Furthermore, beyond 5% by weight of chitosan in the solution, its dissolution becomes difficult and the viscosity of the solutions obtained makes the preparation of materials difficult.

[0044] In a preferred embodiment, the hydrogel is a physical chitosan hydrogel. Advantageously, such a hydrogel does not contain a chemical crosslinking agent that could be toxic. The formation of such hydrogels is, for example, described in the following publications: A Montembault; G Viton and A. Dornard in Rheometryc study of the gelation of chitosan in a hydroalcoholic medium. Biomateria / s 26(14), 1633-1643, 2004; Montembault A, Viton C, Dornard A, Rheometnc study of the gelation of chitosan in aqueous water without cross-linking agent, Biomacromolecules, 6(2): 653-662, 2005; Chedly J, Soares S, Montembault A, von Boxberg Y, Veron-Ravaille M, Mouffle C, Benassy MN, Taxi J, David L, Nothias F. (2017) Physical chitosan microhydrogels as scaffolds for spinal cord injury restoration and axon regeneration. Biomaterials. 138:91-107.

[0045] According to a particular embodiment, the physical hydrogel according to the present invention is obtained by aqueous or hydroalcoholic means, advantageously by aqueous means such as, for example, the process comprising the following successive steps: - preparation of a chitosan solution with a concentration between 0.25% and 5% by weight relative to the total weight of the solution, by mixing purified and lyophilized chitosan and an aqueous solution of acetic acid; - gelling by contact of the solution obtained with ammonia vapors or in an ammonia solution; and - Washing the hydrogel obtained to remove ammonia and salts formed during gelation. The advantage of the aqueous route is that the hydrogels obtained are more rapidly biodegraded in the human body.

[0046] According to one embodiment, the microparticles usable within the scope of the present invention have a median size d50, obtained from a number distribution, ranging from 1 to 300 pm, preferably from 5 to 150 pm, and even more advantageously from 10 to 100 pm, in particular around 20 pm. The median size d50 of the microparticles is measured, for example, by observation using a Zeiss phase-contrast optical microscope (Axiovert 200M) with the Linkys32 image acquisition software. The size distribution of the hydrogel microparticles can be obtained by image processing with the ImageJ software (https: / / imagej.net / ), or as described in C. Igathinathane et al. (Computers and Electronics in Agriculture 63 (2008) 168-182). The size and number of particles can also be determined by particle size analysis.The number of particles can be distributed according to their size, for example by passing the particles through a capillary at a determined speed.

[0047] Chitosan microparticles with the desired median size d50 can, for example, be obtained by grinding the hydrogel, particularly using an IKA ULTRA-TURRAX® homogenizer (operating at a speed of 11000 rpm for 3 x 10 seconds with a 30-second pause between cycles), and recovering the microparticles of satisfactory size, for example, by centrifugation (e.g., 13000 rpm for 3 minutes with a Sigma 3K30 type device). The homogenizer can be used with a dispersion tool such as the IKA S25N-8G-ST disperser. Advantageously, the process according The present invention includes a sterilization step, in particular by autoclave, for example at 121 °C for 20 minutes, before the recovery of the microparticles.

[0048] According to one embodiment, said hydrogel microparticles are in the form of an aqueous suspension, advantageously having a viscosity greater than 1000 Pa.s measured in continuous mode at 22 °C for a shear rate of 0.001 s- 1According to a preferred embodiment, the hydrogel microparticles are in the form of an aqueous suspension having a viscosity greater than 10,000 Pa·s, or more advantageously greater than 100,000 Pa·s, in particular approximately 330 kPa·s, as measured in continuous mode at 22°C for a shear rate of 0.001 s⁻¹. The viscosity is advantageously measured by cone-to-plate rheometry with a TA Instruments Advanced Rheometer AR2000 stress-imposed rheometer. As an example, the characteristic viscosity value of the suspension can be 330 kPa·s after centrifugation at 13,000 rpm for s minutes from a hydrogel concentrated with 2.5% chitosan and a molar mass of 450,000 g / mol.

[0049] The suspension according to the invention is sufficiently liquid to be injected directly into the lesion site where the axons have been cut or damaged by the impact that damaged the nerve tissue and sufficiently thick to remain confined to the implantation site.

[0050] This suspension can be obtained from a pellet after centrifugation containing the microparticles according to the present invention, obtained by the microparticle manufacturing process indicated above, advantageously so as to obtain a viscosity greater than 1000 Pa·s (as measured in continuous mode at 22°C for a shear rate of 0.001 s⁻¹). For injection, the pellet can be resuspended in physiological saline or artificial cerebrospinal fluid.

[0051] According to one embodiment, in said hydrogel microparticles according to the invention, the hydrogel comprises a mixture of chitosan with at least one other biocompatible and biodegradable biomaterial, such as hyaluronic acid, collagen, alginate, fibrin, fucoidan, ...

[0052] According to one embodiment, said microparticles are mixed with cells; for example, Schwann cells, stem cells, neural cell progenitors, exosomes, and / or trophic or neurotrophic factors, polynucleotides of interest (siRNA, MIRs..., vectors, ...), and / or molecules of interest (neuroprotective, ...).

[0053] In one embodiment, the hydrogel microparticles or suspension are injectable or surgically implantable. Specifically, the microparticles can be implanted (i) at the site of the lesion, particularly a traumatic lesion, to fill it and serve as a scaffold, (ii) at the site where axonal regrowth is to occur, and (iii) at the site where nerve tissue is to be restored. The microparticles are therefore advantageously implanted by injection, and even more advantageously by surgery. In one embodiment, the implant used comprises a The implant used may also comprise an aqueous suspension comprising the hydrogel microparticles according to the present invention and cells of interest and / or factors of interest. These include, for example, Schwann cells, olfactory ensheathing cells (OECs), stem cells (such as mesenchymal stem cells, neural progenitor cells, or stem cell-derived cells (glial, vascular, etc.)), and / or tropic or neurotrophic factors. The cells can be added one to two hours before implantation, allowing time for them to adhere to the microparticles, given that chitosan has adhesive properties. Advantageously, an implant such as that used according to the present invention does not contain chondrocytes.An implant such as that used according to the present invention is advantageously intended to be implanted in the nervous system, advantageously intended to be implanted in the brain and / or spinal cord or a peripheral nerve. It must be biocompatible and not elicit immune rejection or an inflammatory reaction.

[0054] One of the advantages of the microparticles according to the present invention is that they provide a suitable surface for both cellular and axonal adhesion and motility on the one hand, and on the other hand, axons and cells can use the inter-particle porosity (between the microparticles of the suspension) to invade the lesion site.

[0055] The suspension or microparticles according to the invention can be used in multi-membrane systems such as, for example, the multi-membrane chitosan fibers described in patent application W02009 / 044053 for the constitution of implants, for example at the level of the traumatic lesion.

[0056] The present invention also relates to the use of hydrogel microparticles as described herein (e.g., the hydrogel comprising chitosan, the chitosan having a degree of acetylation less than or equal to 20% and its concentration in the hydrogel being between 0.25 and 5% by weight relative to the total weight of the hydrogel), for the preparation of a drug or an implant intended for the treatment of a nerve tissue lesion in a patient who does not show improvement of at least one score on at least one neurological assessment scale of said lesion.

[0057] The present invention also relates to a method of neuronal regeneration and / or nerve tissue repair and / or nerve tissue treatment, comprising the implantation of microparticles according to the present invention in a patient who does not show improvement of at least one score on at least one neurological assessment scale for said lesion, particularly at the site of the lesion in order to fill it, or at the location where axonal regrowth or repair is to take place. Advantageously, this implantation is performed by deposition, injection, or surgery. Brief description of the Figures

[0058] Other features, details and advantages of the invention will become apparent from reading the attached Figures.

[0059] [Fig. 1] represents the extension of the post-injury time window for the implantation of the chitosan-based biomaterial according to the described formula. The injury alone (untreated) induces the formation of one to several cavities (as in humans) (A). These cavities are visible in the rat model from 3 to 4 weeks post-injury. When chitosan particles are implanted at 2 (B), 4 (C), or 6 (D) weeks post-injury, the cavities are replaced by restored tissue, thanks to cell migration and significant axonal regrowth. Analysis is performed 6 to 8 weeks post-implantation. Following an injury alone (A), a cavity forms from the third or fourth week onward, the axons that have exhibited plasticity retract, and a glial and fibrous scar forms around the cavity. Furthermore, spontaneous functional recovery stabilizes.Implantation of the biomaterial fills the cavities and serves as a scaffold for renewed plasticity and dynamic regeneration of the damaged tissue. This is manifested at the level of (1) axons, which regain dynamics and the ability to regrow through the damaged tissue (see axons labeled with neuronal beta-tubulin-specific Tuj1); (2) cells (see DAPI nuclear labeling in C) that migrate again, as observed during implantation immediately after injury (Chedly et al., 2017), and occupy the lesion; (3) astrocyte remodeling (see GFAP in B), as a result of which the glial belt around the lesion is no longer visible. Astrocytic processes (GFAP+) accompany (closely associated with) the axons at the interface between the damaged tissue and the adjacent segment (boxed in B / right). This also demonstrates compatibility between the biomaterial and nerve tissue. This structural repair is identical to that observed after acute injury.

[0060] [Fig. 2] represents spontaneous recovery after thoracic injury (CT alone), using two types of impactors (IH Impactor or Benchmark™). This type of injury results in loss of motor function in both hind legs and therefore affects locomotion (so-called "open-field" test with the BBB score). This spontaneous recovery stabilizes from the 3rd to the 4th week. 1 J = 1 day; 1 S = 1 week; 2 S = 2 weeks, ...up to 8 S = 8 weeks, n represents the number of animals.

[0061] Figure 3 represents the evolution of locomotion recovery (open-field test with BBB scores) over an 8-week period (W8) following a contusion injury (day of injury = W0, 175kd-1s), comparing the effects of implantation performed at 4 weeks (in a chronic lesion, Chro28 Chito, 175kd-1s) with the results of a single lesion and a reopened lesion at 4 weeks without implantation (Chro28 sham, 175kd-1s). The results show, on the one hand, no deterioration of function and, on the other hand, improved locomotion after implantation. W+1 = W0 + 1 day; W1 = 1 week, W2 = 2 weeks, ...up to W9 = 9 weeks from W0. n represents the number of animals.

[0062] [Fig. 4] represents the data from the analysis of mechanical sensation, using the von Frey test. Following a contusion to the chest and without treatment, hypersensitivity was noted in both hind legs of the animals. This hypersensitivity was significant compared to that of the unaffected animal (4A). This hypersensitivity persisted until the lesion stabilized (from the 4th èmeweek). Implantation of the biomaterial (chitosan particles) at 24 hours was not followed by this hypersensitivity (4B). Similarly, implantation of the biomaterial at 2 weeks (4D) was not followed by this hypersensitivity compared to the control cohorts (shams) (4C). The baseline represents the mean sensitivity of the same cohort before the lesion. 2S = 2 weeks, ...up to 8S = 8 weeks. In Figures 4A and 4B, the data are combined for both legs. In Figures 4C and 4D, the data show the results for the left leg on the left and the results for the right leg on the right.

[0063] Figure 5 illustrates the effects of chitosan particle implantation on nervous system repair following traumatic brain injury, using a rat model of controlled cortical injury induced by impactor contusion. The injury causes progressive neural cell death and necrosis, resulting in cavity formation (Nissl lesion alone). Chitosan microparticle implantation prevents cavity formation. In the untreated lesion alone, neuronal death also spreads to the unaffected region, whereas this progressive death is not observed in treated animals (lesion-chitosan). GFAP labeling shows the formation of an astrocytic boundary around the lesion alone, while after biomaterial implantation, this boundary is not formed, and astrocytic processes accompany the axons growing within the lesion.The characteristics of this structural restoration are similar to those obtained after a spinal cord injury.

[0064] Figure 6 shows an example of the effects of chitosan particle implantation on functional recovery after traumatic brain injury, using the contusion-controlled cortical lesion model. Figure 6A shows the joint use score for both legs, and Figure 6B shows the use score for the ipsilesional (intact) leg. Here, the lesion is unilateral, located in the motor cortex of the forelimbs. The connections between the cortex and the spinal cord are crossed (the right cortex projects into the left spinal segments, or a cortical lesion in the right hemisphere results in a deficit in the left leg). The lesion here is of moderate intensity. The cylinder test forces the animals to stand only on their hind legs, placing both forelimbs on the wall. Before the lesion, the probability of placing both forelimbs is greater.It decreases after injury because the motor function of the leg contralateral to the cortical lesion is affected (the one that receives connections from the damaged cortex). Animals that have been implanted show less motor deficit (in placing both legs on the body). The significant difference is noted only in lesioned animals. Similarly, only lesioned animals show a significant increase compared to the intact (pre-injury) state in using only the ipsilateral leg (which receives connections from the damaged cortex). the undamaged hemisphere). Therefore, the implantation of the biomaterial particles restores the motor deficit induced by the cortical lesion. 1 S = 1 week, 2 S = 2 weeks, ...up to 6 S = 6 weeks. Examples

[0065] Example 1: Materials & methods: animal model of the lesion, obtaining microparticles and tests performed

[0066] ANIMALS

[0067] All surgical procedures and postoperative care were performed in accordance with the European Community and French Council Directive of 22 September (2010 / 63 / EU) and approved by the Ethics and Animal Welfare Committee. Consequently, the number of animals in our study was kept to the minimum necessary, and every effort was made to minimize discomfort. Female Wistar rats (Janvier, France) weighing between 230 and 250 g were used throughout this study. All animals were housed in groups of three under controlled temperature (22 ± 1 °C), relative humidity (55 ± 10%), and a 12-hour light-dark cycle. Food and water were available ad libitum before and after surgery.The rats were randomly divided into three groups with different conditions: a contusion group (lesion only), a contusion + treatment group (biomaterial implantation), and a control group (Sham, undergoing the same procedure without treatment). All surgical procedures were performed at the thoracic level (T8-T9).

[0068] Animal model of spinal cord injury

[0069] The injury model is a severe contusion which at the T8-T9 thoracic level induces complete paraplegia. It is performed using the Benchmark™ impaction device, according to a previously described approach (Beliard, B., Ahmanna, G., Tiran, E. et al. Ultrafast Doppler imaging and ultrasound localization microscopy reveal the complexity of vascular rearrangement in chronic spinal lesion. Sci Rep 12, 6574 (2022)). Briefly, after laminectomy, the dorsal surface of T8 to T9 is exposed by laminectomy (which involves exposing the dorsal portion of the vertebra to expose the spinal cord while preserving the dura mater). The spine was stabilized by fixing the vertebral bodies rostrally and caudally to the impact zone using clamps attached to the base of the impaction device. The exposed dorsal surface of the spinal cord is subjected to the impaction of a 2.5 mm diameter spike at a velocity of 1.96 m / s, with retraction after 100 ms.

[0070] At the end of the procedure, the spinal cord segment containing the lesion is subjected to histological and biochemical analysis.

[0071] The surgical procedures were performed under reversible and continuous isoflurane anesthesia (2%-2.5%, Isoflurane®), and sterile precautions were taken throughout the procedure. The surgical conditions for biomaterial implantation are identical to those of the First procedure. Briefly, the skin is incised along the spine (intervention beyond 2-3 days after the contusion, subacute state 1 and 2; entry into the chronic state at 3-4 weeks and chronic state at 6 weeks). The paravertebral muscles are re-exposed to access the primary injury site, i.e., the segment exposed after the initial laminectomy. A small incision in the dura mater is made to inject 2-5 µL of the biomaterial suspension (using a Microman® pipette with a piston cone). The animals were housed on additional bedding to prevent the development of friction sores. Until normal urination was restored, the bladders were manually emptied twice daily, and the health of the operated rats was monitored by regular weight assessments and visual inspection of the surgical wound.

[0072] Animal model of lesions in the cerebral cortex

[0073] The skull is shaved and disinfected; then an analgesic is applied topically, as a solution under the scalp (Lurocaine® 20 mg / ml) and as an ointment (Lidocaine / Prilocaine Biogaran® 5%) to the ears. The animal is then placed in a stereotaxic frame (KOPF®): the mouth is held in the mask delivering isoflurane using the retaining clamp, and the head is secured with ear bars. An anteroposterior incision is made in the scalp, followed by a craniectomy of approximately 3-4 mm in diameter (slightly larger than the tip of the impactor) using a dental burr (Foredom®) in the motor area controlling one of the two forelimbs. The stereotaxic coordinates of the impact were determined so as to induce a cortical lesion at the level of the motor cortex causing a unilateral motor deficit at the level of one of the two forelegs, measurable using behavioral tests.Thus the tested coordinates range from 0.6 to 2 mm anteriorly and 2.5 mm laterally with respect to the Bregma point mainly adapted from the following references: Combs, HL, Jones, TA, Kozlowski, DA, and Adkins, DL (2016). Combinatorial Motor Training Results in Functional Reorganization of Remaining Motor Cortex after Controlled Cortical Impact in Rats. Journal of Neurotrauma 33, 741 -747; Fonoff, ET, Pereira Jr., JF, Camargo, LV, Dale, CS, Pagano, RL, Ballester, G., and Teixeira, MJ (2009). Functional mapping of the motor cortex of the rat using transdural electrical stimulation. Behavioral Brain Research 202, 138-141 ; Schônfeld, L.- M., Jahanshahi, A., Lemmens, E., Schipper, S., Dooley, D., Joosten, E., Temel, Y., and Hendrix, S. (2017). Long-Term Motor Deficits after Controlled Cortical Impact in Rats Can Be Detected by Fine Motor Skill Tests but Not by Automated Gait Analysis. Journal of Neurotrauma 34, 505-516.according to the rat brain atlas (Paxinos, G.and Watson, C. (2007) The Rat Brain in Stereotaxic Coordinates. 6th Edition, Academic Press, San Diego.). The lesion is thus induced at the level of the motor cortex of the forelimb of one of the two hemispheres, near the overlap zone between the primary motor area and the sensory area of ​​the caudal area of ​​the forelimbs (Donoghue, JP, and Wise, SP (1982). The motor cortex of the rat: cytoarchitecture and microstimulation mapping. J Comp Neurol 212, 76-88; Fonoff, ET, Pereira Jr., JF, Camargo, LV, Dale, CS, Pagano, RL, Ballester, G., and Teixeira, MJ (2009). Functional mapping of the. Motor cortex of the rat using transdural electrical stimulation. Behavioural Brain Research 202, 138–141. The lesion aims to induce a motor deficit in the forelimb contralateral to the lesion (contralesional).

[0074] The biomaterial is implanted 3 days (or 1 to 4 weeks) after the injury. A 2 µL volume of the biomaterial is injected using a pipette (Gilson®) directly into the lesion after puncturing the dura mater with a needle. The skin is closed with sutures (Ethicon® Monocryl™ 4-0) between the two procedures and then after implantation with staples (Michel 7.5 x 1.75 mm). The skin is then disinfected with Vetedine.

[0075] HYDROGEL-BASED MICROPARTICLES

[0076] The biomaterial comprising hydrogel microparticles including chitosan can be obtained as described below or according to the process of patent EP2874672.

[0077] FUNCTIONAL ANALYSES: BEHAVIORAL TESTS IN THE SPINAL CORD INJURY MODEL

[0078] Behavioral tests are performed in both examples throughout the procedure (after pre-operative habituation) to analyze the recovery of motor skills and assess sensitivity (from the 3rd day after surgery and once a week until the end of the procedure).

[0079] The BBB Test (evaluation of motor recovery, locomotion) is performed: the animal is filmed in an open field for 5 minutes, once a week, to assess the recovery of hind limb function, using the hierarchical Basso-Beattie-Bresnahan scale (Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma. 1995 Feb;12(1):1-21. doi: 10.1089 / neu.1995.12.1. PMID: 7783230.). Each point represents a significant stage of locomotion, from complete paralysis (BBB score = 0) to full recovery of joint mobility, weight-bearing capacity, and forelimb / hindlimb coordination (BBB score = 21, the best score, generally corresponding to each animal's score before the injury, i.e., an "intact state"). Three qualified evaluators independently assessed the rats' locomotor performance using the BBB scale.

[0080] The Von Frey Test (assessment of mechanical nociceptive sensation): the animal is placed on a raised grid, and the experimenter applies a plastic cone to the plantar surface, with a known force increasing until the paw is withdrawn. This test demonstrates mechanoceptive hypersensitivity secondary to the injury or treatment.

[0081] The Hargreaves Test (assessment of sensitivity to thermal nociception): a 50°C beam is applied to the paw, and the time elapsed until its removal is recorded. The animal's reaction is also taken into account (withdrawal followed or not by licking of the paw).

[0082] The cylinder test: analysis of motor function and motor deficits of the forelimbs (cortical lesion model of the area responsible for forelimb motor function). The cylinder test is performed weekly after the lesion until the end of the experiments, i.e., for 6 weeks. This test measures asymmetry in the use of the animal's forelimbs during the exploration of an unknown vertical surface (Farr and Trueman, Farr, 2011. Functional assessment of subcortical ischemia. Animal Models of Movement Disorders, Vol. II. Neuromethods, vol. 62. Springer Science + Business Media, Berlin, Germany, pp. 91-114). The rat is placed in a transparent plexiglass cylinder 19 cm in diameter and 40 cm high and filmed from above for 10 minutes using a camera (Canon Legria HF R406). The diameter of the cylinder does not allow the rat to easily stand on its four paws, which often forces it to stand upright against the wall.The first 20 contacts with the forelegs against the wall are counted and used for analysis. The number of contacts made with both forelegs and the number of individual contacts with the ipsilateral versus contralateral paw to the lesion are recorded. A new contact can only be counted after both of the animal's paws have left the wall. When the animal begins by making contact with one paw, but the other paw reaches the wall before the first has left, this event is considered a single contact with both paws. When both paws remain against the wall and cross irregularly, this is also considered a single contact with both paws. This distinction allows us to focus on the individual contacts of the animal. The data collected from this test were analyzed in two different ways.Scores representing the percentage of joint use of both paws, and of the ipsi- and contra-lesional paw, were calculated in relation to the total number of contacts recorded (example: (number of contacts with the ipsilesional paw / total number of contacts) * 100). An asymmetry score was calculated as follows: (number of contacts made with the ipsilesional paw +. 1 (number of contacts made with both paws) / (total number of contacts) * 100 (Jones et al., 2012). An increasing asymmetry score indicates a decrease in the use of the contra-lesional paw, the one receiving projections from the injured site.

[0083] The cylinder test was used to determine whether the cortical lesion method employed induced a unilateral motor deficit and, if so, whether implantation of the biomaterial could modulate the onset of the deficit. This test measures the use of the forelimbs jointly and individually and determines whether the ipsilateral lesion limb (whose afferent cortical neurons are not directly affected by the impact) is used more frequently than the contralesional limb after the lesion.

[0084] Description of results

[0085] Example 2: Severe spinal cord contusion injury in rats

[0086] Reactivation of tissue repair of the spinal cord injury: implantation of the biomaterial composed of chitosan microparticles at different times after the injury (here tested at 2, 4 and 6 weeks post-injury) shows that this treatment allows reactivation of an already stabilized lesion. Indeed, even after the formation of the cavity and the establishment of a fibrous border around the lesion, the biomaterial allows reactivation of a repair response, similar to that which occurs when the implantation of the biomaterial is performed immediately after the injury: 1 / Astrocytes (labeled with GFAP, Fig.1 B) undergo further morphological remodeling to orient their extension towards the lesion; 2 / Impressively, massive regrowth of axons is noted in the lesion site accompanied by astrocytic extensions at the interface between intact (not directly damaged) and damaged tissue; the cavity(ies) that forms in a lesion (Fig. IA) is / are no longer observed after implantation. Similarly, several cells (highlighted by the presence of DAPI-labeled nuclei (Fig.IC) migrate and colonize the lesion site; 3 / the newly formed environment thanks to the biomaterial allows cell survival; no new cavity is produced after implantation; 4 / Most remarkable is that the reactivation by injection of the biomaterial particles of neural plasticity at late stages, once the lesion is stabilized at the structural and functional level, does not require surgical removal of the glial and fibrous scar before implantation of the biomaterial.

[0087] Functional analysis of the spinal cord lesion:

[0088] Evaluation of locomotion recovery (BBB scores): The evolution of locomotion recovery (BBB scores) over an 8-week period following a contusion injury (day of injury = D0; Fig. 2) is compared to that obtained after particle implantation performed 4 weeks post-injury. Preliminary data on the absence of functional deterioration and the improvement in locomotion after implantation are shown in Fig. 3. Furthermore, the group of animals that received biomaterial implantation showed greater improvement than the groups that underwent wound reopening without implantation. It should be noted that neither group (implanted and sham) exhibited any deterioration in the animal's health. For example, no urinary deficit was observed in these animals, demonstrating that the surgical procedure did not cause further damage.

[0089] Evaluation of sensory recovery, von Frey test: In 70% of patients with spinal cord injury, there is hypersensitivity and neuropathic pain. This type of sensitivity is measured in rodents using the von Frey test. Data obtained from this test show that following a thoracic contusion and without treatment, hypersensitivity is noted in both hind legs (Fig. 4A). This hypersensitivity is significant compared to that of the uninjured animal and persists until the injury stabilizes (from the 4th ème week). Implantation of the biomaterial (chitosan particles) at 24 hours is not followed by this hypersensitivity (Fig. 4B). Similarly, implantation of the The biomaterial observed at 2 weeks (Fig. 4D), and also observed 4 weeks after implantation following injury, was not followed by this hypersensitivity compared to the sham cohorts, which underwent the same surgery as the implanted animals. The latter did, in fact, exhibit hypersensitivity. These data are remarkable and clearly demonstrate that the application of the chitosan-based biomaterial has a protective effect against the development of pain.

[0090] Example 3: Treatment of cortical lesions by implantation of chitosan particles

[0091] Tissue restoration, anatomical data: The rat model of controlled cortical injury, induced by impactor contusion, was used. The injury causes progressive neural cell death and necrosis, resulting in the formation of a cavity that enlarges over time. Implantation of chitosan microparticles 3 days after the injury prevents cavity formation (Fig. 5) or halts its progression if injected after a delay. In the untreated lesion alone, neuronal death also spreads to the unaffected area. Injection of chitosan particles prevents the progression of this cell death in treated animals.Furthermore, in a solitary (untreated) cortical lesion, GFAP labeling of astrocytes reveals the formation of an astrocytic border around the lesion (cavity), whereas after implantation this border is not formed, and the astrocytic processes accompany the axons growing into the lesion. The characteristics of this structural restoration are similar to those obtained after injection of the biomaterial particles into a spinal cord lesion.

[0092] Functional Restoration: The cylinder test was performed weekly after injury until the end of the experiments, i.e., for 6 weeks. This test measures asymmetry in the use of the animal's forelimbs during exploration of an unknown vertical surface (see Methods). Before injury, animals predominantly (80-85%) used both limbs together when exploring the vertical surface (Figure 6A, before injury). After unilateral injury, the score for use of both limbs (Figure 6A) decreased over time and was significantly different (p = 0.0377) at 2 weeks post-injury; this difference was observed only in the injury-only group. The treated animals did not show significant changes. At the same post-injury time point, the score for use of the ipsilesional limb increased significantly (p = 0.0352) compared to before injury (Figure 6B at 2 weeks).This significant increase (p = 0.0390) in the ipsilesional leg use score, also noted at 6 weeks post-lesion (6s) compared to pre-lesion, was observed in the lesion-only group (Figure 6B), with a non-significant decrease in the combined use score of both legs (Figure 6A). Animals in the lesion-Chito group showed an increase in the use of this leg, but this increase was not significant at any post-lesion time point. These data clearly demonstrate that biomaterial implantation prevents the development of the deficit. Motor function is measured using the cylinder test. Note that the applied lesion impact is adapted for a moderate lesion followed by a slight motor deficit.

Claims

Demands [Claim 1] Hydrogel microparticles comprising chitosan, the chitosan having a degree of acetylation less than or equal to 20% and its concentration in the hydrogel being between 0.25 and 5% by weight relative to the total weight of the hydrogel, for its use in the treatment of nerve tissue injury in a patient who does not show improvement in at least one score on at least one neurological rating scale. [Claim 2] Hydrogel microparticles comprising chitosan for its use according to claim 1, wherein the absence of improvement in at least one score on at least one neurological assessment scale is determined after two successive assessments. [Claim 3] Hydrogel microparticles comprising chitosan for use according to any one of the preceding claims, wherein the absence of improvement is a stability of at least one score on at least one neurological rating scale or a worsening of at least one score on at least one neurological rating scale. [Claim 4] Hydrogel microparticles comprising chitosan for use according to any one of the preceding claims, wherein the nerve tissue injury is an injury to the peripheral nervous system or the central nervous system, preferably an injury to the spinal cord. [Claim 5] Hydrogel microparticles comprising chitosan for use according to any one of the preceding claims, wherein the neurological assessment scale is a sensory assessment scale, a motor assessment scale, or a sensorimotor assessment scale. [Claim 6] Hydrogel microparticles comprising chitosan for use according to any one of the preceding claims, wherein the neurological assessment scale used is selected from: - the ASIA scale, the Lower Limb Functional Scale (LEFS), the Upper Limb Functional Scale, the Tetraplegic Motor Capacity Scale, the Berg Balance Scale, the Functional Independence Measure, the Spinal Cord Injury Independence Measure (SCIM version III), the modified Barthel Index, preferably the ASIA scale, - a sensory assessment scale allowing the measurement of neurological dysfunction and / or pain, such as the ISCIPBDS assessment scale, the NPSI scale, the QST scale), in particular which assesses mechanical allodynia by mechanical stimulation of the dorsal spine and / or which assesses mechanical allodynia by thermal stimulation of the spinothalamic tract (STT), - a scale allowing measurement of spastic reflexes chosen from flexion, extension or clonus, for example the SCAT scale. [Claim 7] Hydrogel microparticles comprising chitosan for use according to any one of the preceding claims, said microparticles having a median size d50, obtained from a number distribution, of between 1 and 300 µm, preferably between 5 and 150 µm. [Claim 8] Hydrogel microparticles comprising chitosan for use according to any one of the preceding claims, characterized in that they are in the form of an aqueous suspension, the suspension preferably being injectable or surgically implantable. [Claim 9] Hydrogel microparticles comprising chitosan for use according to any one of the preceding claims, characterized in that the hydrogel comprises a mixture of chitosan with at least one other biocompatible and biodegradable biomaterial, such as collagen, hyaluronic acid, fibrin, alginate, fucoidan [Claim 10] Hydrogel microparticles comprising chitosan for use according to any one of the preceding claims, characterized in that they are mixed with Schwann cells, stem cells, neural cell progenitors, and / or tropic or neurotrophic factors, exosomes, polynucleotides of interest, and / or molecules of interest.

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