WO2019200152A1 - Compositions and methods for the treatment of brain damage - Google Patents

Abstract

Described herein are compositions and methods for treating brain damage from disease or injury using neuroregenerative implants.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF

BRAIN DAMAGE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/657,693, filed April 13, 2018, the entire content of which is incorporated herein by reference in its entirety.

BACKGROUND

Stroke is a leading cause of death and long-term disability in United States. Approximately one third of stroke survivors develop permanent disabilities and one fifth require institutional care at three months after onset. Stroke has become a global health burden as existing treatments are minimally effective at improving the long term functional deficits that result from this debilitating disease. Currently, thrombolytics are the only approved pharmacotherapy for ischemic stroke, yet only 2-8% of patients qualify as candidates for thrombolysis. In addition, thrombolysis must be performed only within a narrow therapeutic time window following stroke onset (3 to 7 hours depending on guidelines) and is associated with life-threatening systemic complications. To date, experimental therapeutics targeting the chronic impairments of ischemic stroke have been unsuccessful; for the majority of patients, physical and occupational rehabilitation therapies are the only effective means to manage long-term functional deficits.

Brain-derived neurotrophic factor (BDNF), the most abundant neurotrophic factor in brain, regulates neural stem cell survival and differentiation, axon and dendrite differentiation, synapse development and maturation, as well as refinement of developing neural connections. Despite its promising therapeutic potential, BDNF has poor blood-brain barrier (BBB) permeability. To overcome this limitation, alternative methods for BDNF delivery have been explored, such as: systemically-administered molecular carriers, small molecule mimetics, and chimeric BDNF peptides targeting tyrosine kinase B (TrkB) receptors. While these approaches are promising in principle, they are often accompanied by adverse off-target effects such as altered glucose metabolism and neuropathic pain.

The present disclosure addresses these and other shortcomings in the field of therapies for the recovery of brain injuries and diseases.

SUMMARY

Ischemic stroke continues to be a leading cause of adult disability and is characterized by the development of long-term functional impairments. To date, preclinical testing of novel therapeutics aimed at restoring chronic functional deficits have been unsuccessful, including attempts to translate these to effective clinical treatments. Furthermore, these studies have not been able to show a relationship between improvements in functional outcome and reductions in ischemic pathology, indicating a need for more targeted therapies.

In certain embodiments, a method of treating brain tissue damage in a subject includes, administering a therapeutically effective amount of a neuroregenerative implant. In other embodiments, the neuroregenerative implant comprises a biocompatible matrix and a neuroregenerative agent. In yet other embodiments, the biocompatible matrix comprises a hydrogel. In yet other embodiments, the hydrogel comprises thiol-modified hyaluronan, thiol- modified gelatin, and polyethylenegycol diacrylate (PEGDA). In various other embodiments, the hydrogel is made by a method comprising: (a) reconstituting the thiol-modified hyaluronan, thiol- modified gelatin, and polyethylenegycol diacrylate (PEGDA); and (b) mixing the thiol-modified hyaluronan, thiol-modified gelatin, and polyethylenegycol diacrylate (PEGDA) together. In other embodiments, the hydrogel is made by a method comprising: (a) contacting a first thiolated monomer with GSSG; (b) allowing the first thiolated monomer and the GSSG to react; and (c) adding a second thiolated monomer to the reaction of step (b), thereby forming a hydrogel comprising the first and second thiolated monomers, but not comprising glutathione or GSSG. In some embodiments, the first thiolated monomer is thiolated carboxymethylated hyaluronan and wherein the second thiolated monomer is thiolated gelatin.

In some embodiments, the biocompatible matrix comprises SLF. In some embodiments, SLF is made by a method comprising: (a) thawing a combination of thiol-modified hyaluronan and thiol- modified gelatin at a temperature of approximately 35 °C or greater; and (b) adding polyethylenegycol diacrylate (PEGDA) to the thawed combination of thiol-modified hyaluronan and thiol-modified gelatin.

In various embodiments, the neuroregenerative agent comprises chemo-attractants and trophic factors. In various embodiments, the neuroregenerative agent comprises a growth factor. In various embodiments, the neuroregenerative agent comprises BDNF. In other embodiments, the neuroregenerative implant is formulated with BDNF is at a concentration of between about 0.01 pg/pL and about 0.5 pg/pL, about 0.02 pg/pL and about 0.4. In some aspects, the neurodegenerative implant is formulated with BDNF at about any of the following concentrations: 0.001 pg/pL, 0.01 pg/pL, 0.02 pg/pL, 0.03 pg/pL, 0.04 pg/pL, 0.05 pg/pL, 0.051 pg/pL, 0.052 pg/pL, 0.053 pg/pL, 0.054 pg/pL, 0.055 pg/pL, 0.056 pg/pL, 0.057 pg/pL, 0.058 pg/pL, 0.059 pg/pL, 0.06 pg/pL, 0.07 pg/pL, 0.08 pg/pL, 0.09 pg/pL, 0.1 pg/pL, 0.12 pg/pL, 0.13 pg/pL, 0.14 pg/pL, 0.15 pg/pL, 0.16 pg/pL, 0.165 pg/pL, 0.166 pg/pL, 0.167 pg/pL, 0.168 pg/pL, 0.169 pg/pL, 0. 0.2 pg/pL, 0.3 pg/pL, 0.4 pg/pL, and 0.5 pg/pL. In certain embodiments, a therapeutically effective amount of the neuroregenerative implant results in a reduction of neuroinflammation. In certain other embodiments, the administration of the therapeutically effective amount of the neuroregenerative implant results in an improvement in sensorimotor function as measured from a baseline. In certain other embodiments, administration of the neuroregenerative implant results in a decrease of an infarct cavity volume as measured from a baseline or compared to non-treated subjects.

In some embodiments, the infarct cavity volume is decreased by between about 1% to about 20% compared to non-treated subjects. In some embodiments, the infarct cavity volume is decreased by about any of the following amounts: 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, and 50%.

In some embodiments, the neuroregenerative implant is administered into an infarct cavity. In some embodiments, the implant is administered adjacent to the infarct cavity.

In some embodiments, the neuroregenerative implant comprises a hydrogel and BDNF and wherein the hydrogel is fully gelated before administration. In some embodiments, the neuroregenerative implant comprises a hydrogel and BDNF and wherein the hydrogel is partially gelated before administration.

In some embodiments, the BDNF is released from the hydrogel over at least about 1 day to about 2 months.

In some embodiments, the neuroregenerative implant is administered at about 1 day to about 1 year following a stroke. In some embodiments, the neuroregenerative implant is administered at about 5 minutes to about 24 hours following a stroke. In some embodiments, the neuroregenerative implant is administered at about 30 minutes to about 12 hours following a stroke. In some embodiments, the neuroregenerative implant is administered at about any of the following number of days following a stroke: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, and 14 days following a stroke.

In some embodiments, the brain tissue damage is caused by a stroke. In some embodiments, the brain tissue damage is caused by traumatic brain injury (TBI). In some embodiments, the brain tissue damage is caused by one or more of: tumor, surgical procedure, radiation therapy, chemotherapy, acquired brain injury (ABI), neurological illness, birth trauma, poison, infection, strangulation, choking, drowning, heart attack, aneurysm, illegal drug abuse, neurological illness.

As used herein, the term“about” in reference to a numeric value means within 10 % of the numeric value. A number is“about” a numeric value if the number is within a range that is + or - 10 % of the numeric value.

Further aspects of the technology described herein will be brought out in the following portions of the specification, wherein the detailed description is for the purpose of fully disclosing preferred embodiments of the technology without placing limitations thereon.

BRIEF DESCRIPTION OF THE DRAWINGS

The technology described herein will be more fully understood by reference to the following drawings which are for illustrative purposes only:

Figure 1A shows photomicrographs of cresyl violet-stained brain sections containing hydrogel implants (arrows) at 5 x magnification. Regions of interest for immunohistochemistry analysis are indicated by circles. Grey = anterior motor cortex, black = corpus striatum, broken-line circle = cingulate cortex.

Figure 1B shows a timeline of procedures, treatment administration, and behavioral testing.

Figure 2A shows a graph illustrating the effects of BDNF and delivery vehicle on sensorimotor function following dMCAo. Sensorimotor deficits, measured using the 28-PN test, improve following treatment with hydrogel-only, BDNF-only, or hydrogel+BDNF_HiGH· Improvement measured as % recovery compared to Day 7. Vehicle (n=4), BDNF-only (n=7-9), hydrogel-only (n=7-9), hydrogel+BDNF_LOw (n=7-9), hydrogel+BDNF_HiGH (n=6-l l). Error bars indicate the mean±SEM; *p<0.05, **/?<0.0l, ***/?<0.00l.

Figure 2B shows a graph illustrating the effects of BDNF and delivery vehicle on sensorimotor function following dMCAo. Performance on the ART contralateral to injury. Contact time recovery index is significantly improved in the hydrogel+BDNF_HIGH group on Days 42 and 56. Vehicle (n=4), BDNF-only (n=7-9), hydrogel-only (n=7-9), hydrogel+BDNF_LOw (n=7-9), hydrogel+BDNF_HiGH (n=6-l l). Error bars indicate the mean±SEM; *p< 0.05, **/?<0.0l,

***p<0.00l. Figure 2C shows a graph illustrating the effects of BDNF and delivery vehicle on sensorimotor function following dMCAo. Performance on the ART contralateral to injury. Removal time recovery index is significantly improved in the hydrogel+BDNF_HIGH group on Day 42. Vehicle (n=4), BDNF-only (n=7-9), hydrogel-only (n=7-9), hydrogel+BDNF_LOw (n=7-9), hydrogel+BDNF_HiGH (n=6-l l). Error bars indicate the mean±SEM; *p<0.05, **p<0.0l, ***p<0.00l.

Figure 3A shows photomicrographs of sections stained with cresyl violet at 5 x magnification illustrating the effects of BDNF and delivery vehicle on infarct volume.

Figure 3B shows a graph illustrating the quantification of infarct volume. Infarct volume is reduced in rats treated with hydrogel+BDNF_HIGH. Vehicle (n=2l), BDNF-only (n=l2), hydrogel-only (n=l2), hydrogel+BDNF_LOw (n=l2), hydrogel+BDNF_HIGH (n=l2); **/?<0.0l.

Figure 4A shows a graph and corresponding photomicrographs of Ibal (bright white) and DAPI nuclear stain (light grey) at x 20 magnification from each treatment group and ROI illustrating the effects of BDNF and delivery vehicle on microgliosis following dMCAo. Flydrogel+BDNF_HIGH treatment reduces Ibal IR in the ipsilateral corpus striatum. Vehicle (n=5), BDNF-only (n=5), hydrogel-only (n=5), hydrogel+BDNF_LOw (n=4-5), hydrogel+BDNF_HIGH (n=5). Scale bar = 20 pm. IR = immunoreactivity. Error bars indicate the mean+SEM; *p<0.05.

Figure 4B shows a graph and corresponding photomicrographs of Ibal (bright white) and DAPI nuclear stain (light grey) at x 20 magnification from each treatment group and ROI illustrating the effects of BDNF and delivery vehicle on microgliosis following dMCAo. Hydrogel+BDNF_HiGH treatment reduces Ibal IR in the ipsilateral cingulate cortex. Vehicle (n=5), BDNF-only (n=5), hydrogel-only (n=5), hydrogel+BDNF_LOw (n=4-5), hydrogel+BDNF_HiGH (n=5). Scale bar = 20 pm. IR = immunoreactivity. Error bars indicate the mean+SEM; *p<0.05.

Figure 5 shows a graph and corresponding photomicrographs of CD68 (bright white) and DAPI nuclear stain (grey) at x 20 magnification from each treatment group in neighboring panels, illustrating the effects of BDNF and delivery vehicle on phagocytosis following dMCAo. Hydrogel+BDNF_HIGH treatment reduces levels of CD68 in the ipsilateral corpus striatum. Vehicle (n=5), BDNF-only (n=5), hydrogel-only (n=5), hydrogel+BDNF_LOw (n=5), hydrogel+BDNF_HIGH (n=5). Scale bar = 20 pm. IR=immunoreactivity. Error bars indicate the mean±SEM; *p<0.05. Figure 6 A shows a graph and corresponding photomicrographs of GFAP (Cy5, bright white) and DAPI nuclear stain (light grey) at x 20 magnification from each treatment group and ROI illustrating the effects of BDNF and delivery vehicle on astrogliosis following dMCAo. Hydrogel+BDNF_HIGH treatment reduces GFAP IR in the ipsilateral corpus striatum. Vehicle (n=5), BDNF-only (n=5), hydrogel-only (n=5), hydrogel+BDNF_LOw (n=5), hydrogel+BDNF_HIGH (n=5). Scale bar = 20 pm. IR=immunoreactivity. Error bars indicate the mean±SEM; *p<0.05, ***/?<0.00l.

Figure 6B shows a graph and corresponding photomicrographs of GFAP (Cy5, bright white) and DAPI nuclear stain (light grey) at x 20 magnification from each treatment group and ROI illustrating the effects of BDNF and delivery vehicle on astrogliosis following dMCAo. Hydrogel+BDNF_HIGH treatment reduces GFAP IR in the ipsilateral anterior motor cortex. Vehicle (n=5), BDNF-only (n=5), hydrogel-only (n=5), hydrogel+BDNF_LOw (n=5), hydrogel+BDNF_HIGH (n=5). Scale bar = 20 pm. IR=immunoreactivity. Error bars indicate the mean±SEM; *p<0.05,

***/?<0.00l.

DETAILED DESCRIPTION

Neuroregenerative implants described herein may be used to promote reparative processes, reduce neuroinflammation, and improve sensorimotor function in subjects who have suffered from brain tissue damage caused by a stroke or other brain injury or disease. In some embodiments, the neuroregenerative implants comprise a biocompatible matrix and neuroregenerative compound.

In certain embodiments, hydrogels that have all of the characteristics required for successful delivery of complex, fragile cells and neuroregenerative molecules can be used as the biocompatible matrix.

Hyaluronan based hydrogels have been developed that mimic the natural extracellular matrix environment (ECM) for applications in 3-D cell culture, stem cell propagation and differentiation, tissue engineering, regenerative medicine, and drug delivery systems. These hydrogels were designed to recapitulate the minimal composition necessary to obtain a functional extracellular or biocompatible matrix. The individual components of the hydrogels are cross-linkable in situ, and may be seeded with cells prior to injection in vivo, without compromising either the cells or the recipient tissues.

Embodiments described herein address the clinical needs of treating stroke, including the aspect of delayed treatment administration. Following dMCAo in rats, non-viable tissue is promptly cleared by resident macrophages within 8 days and the resultant cavity provides a depot for targeted treatment delivery. Prolonged treatment with neuroregenerative implants delivered to the cavity of the ischemic (e.g., stroke) core following distal middle cerebral artery occlusion (dMCAo) demonstrated an increase in reparative processes, reduced neuroinflammation, and improved sensorimotor function. Furthermore, neuroinflammation was shown to play a key role in processes linking functional improvement to neuronal connectivity following BDNF treatment.

In various embodiments, the hydrogels contemplated herein are designed to crosslink into the hydrogel form, for example, starting from a liquid form after it is injected into the body. In some embodiments, the hydrogel begins to crosslink and is becoming more viscous as it is being administered. The liquid and delayed self-assembly of these hydrogels and the surprising discovery that they permit injection without shearing forces that would destroy cells allows a very small needle to be used for delivery of cells into the body. In some embodiments, a 30 gauge syringe needle may be used.

The technology underlying certain hydrogels described herein is based on a unique thiol cross- linking strategy to prepare hyaluronan based hydrogels from thiol-modified hyaluronan and other ECM constituents. Building upon this platform, a family of unique, biocompatible resorbable hydrogels have been developed. The building blocks for these hydrogels are hyaluronan and gelatin, each of which has been thiol-modified by carbodiimide mediated hydrazide chemistry. These hydrogels are formed by cross-linking mixtures of these thiolated macromolecules with polyethylene glycol diacrylate (PEGDA) (see US Patent No. 7,928,069 and 7,981,871, incorporated herein by reference in their entirety). The rate of gelation and hydrogel stiffness can be controlled by varying the amount of cross-linker. An attribute of these hydrogels is their large water content, >98%, resulting in high permeabilities for oxygen, nutrients, and other water-soluble metabolites.

Collectively, hydrogels are a versatile tool that can be used to inform the development of therapeutics and intervention strategies for the treatment of stroke. In addition, they can be used to improve our understanding of the pathophysiology underlying acute and chronic neurodegenerative disorders. Flydrogels offer an advantageous replacement for damaged brain tissue. The hyaluronate component of the hydrogel provides the necessary 3-dimensional space filling framework while the gelatin component provides the requisite amino acid sites for cell attachment and proliferation. As an implantable scaffold, resorbable matrices such as the hydrogels described herein can provide a safe and consistently uniform matrix with which to deliver neuroregenerative agents for the treatment of brain damage caused by, for example, ischemic stroke. In some embodiments, the biocompatible matrix composition can have a storage modulus of about 1 Pa to about 5 Pa, about 1 Pa to about 5,000 Pa, about 20 Pa to about 5,000 Pa, about 50 Pa to about 5,000 Pa, about 60 Pa to about 1,200 Pa, about 75 Pa to about 1,000 Pa, about 80 Pa to about 120 Pa, about 15 Pa to about 100 Pa, about 20 Pa to about 150 Pa, or any value in a range bounded by, or between, any of these values.

In some embodiments, the hydrogel may contain cellular attachment sites to prevent anoikis of anchorage-dependent cells. They may also have functionalizable groups on its component biopolymers allowing not only the one-step covalent linking of macromolecular therapeutic cargo by the user, but also provide for matrix customization for specific cell types requiring a unique collection of cellular attachment sites. Finally, the hydrogels described infra may have validated and desired syringeability with the gauge of the needle determined by the placement location. These properties may be achieved by varying the concentration of one or more of the monomers and/or the oxidizing agent.

In certain embodiments, the biocompatible matrix is resorbable. In certain embodiments, the biocompatible matrix composition may be mixed with neuroregenerative agents to be administered to a subject in need of brain tissue regeneration. In some embodiments, the biocompatible matrix/ neuroregenerative agent composition may be administered about 5 minutes to about 180 minutes, about 10 minutes to about 150 minutes, or about 20 minutes to about 120 minutes post mixing of the components and prior to the final crosslinking or curing of the biocompatible resorbable matrix/cell composition. In some embodiments, the biocompatible resorbable matrix/cell composition has a storage modulus of between about 1 Pa and about 10 Pa at the time the biocompatible matrix/neuroregenerative agent composition is administered to the subject and a storage modulus of about 50 Pa to about 150 Pa once the biocompatible matrix/neuroregenerative agent composition crosslinks or cures, in situ.

In various embodiments, the neuroregenerative implant is administered when the composition is at about 0.1 to about 5 Pa; or at about 0.3 to about 20 Pa; or at about 0.5 to about 10 Pa; or at about 0.75 to about 7.5 Pa. The neuroregenerative implant composition can be administered when it is at about 1% to about 100% of its final stiffness; or about 0.1 to about 50% of its final stiffness; about 5% to about 85 % of its final stiffness; or about 50% to about 95% of its final stiffness.

In certain embodiments, the biocompatible matrix crosslinks before, during and/or after administration. In others, the matrix crosslinks before, during and/or after the neuroregenerative agent is mixed with the biocompatible matrix. In certain embodiments, the biocompatible matrix begins to crosslink before the neuroregenerative agent is mixed with the biocompatible matrix. In certain embodiments, the matrix continues to crosslink after administration of the neuroregenerative implant composition. In one embodiment, the resorbable matrix crosslinks before, during and/or after administration.

In certain embodiments, the neuroregenerative implant is administered by injection.

In certain embodiments, the neuroregenerative implant is administered about 5 to about 50 minutes, about 10 to about 30 minutes or about 15 to about 20 minutes post mixing of components.

In certain embodiments, the components comprise, a neuroregenerative agent, a thiol-modified hyaluronan and a thiol-modified collagen. In certain embodiments, the components further comprise a crossl inker. In certain embodiments, the crosslinker comprises one or more of bi-, tri-, multi-functionalized molecules that are reactive to thiols, and/or oxidation agents that initiate crosslinking. In certain embodiments, the crosslinker comprises polyethylene glycol diacrylate.

In certain embodiments, the thiol-modified hyaluronan has a molecular mass of at least about 55000 g/mol; at least about 100,000 g/mol; at least about 120,000 g/mol; at least about 150,000 g/mol; at least about 170,000 g/mol; at least about 175,000 g/mol; or at least about 200,000 g/mol.

In certain embodiments, the thiol-modified hyaluronan comprises more than about 150 pmol/g of polymer; more than about 200 pmol/g of polymer; more than about 1000 pmol/g of polymer; more than about 10,000 pmol/g of polymer.

In certain embodiments, the thiol-modified hyaluronan comprises from about 1% to about 75% of the thiol groups in the resorbable matrix. In certain embodiments, the thiol-modified collagen comprises from about 1% to about 75% of the thiol groups in the resorbable matrix.

Crosslinkers may comprise, for example, a bi-, tri-, multi-functionalized molecule that is reactive to thiols (e.g. maleimido groups), oxidation agents that initiate crosslinking (e.g., GSSG), glutaraldehydes, and environment influences (e.g., heat, gamma/e-beam radiation). In some embodiments, there are no cross-linkers necessary. In some embodiments, the crosslinking agent is not present in the final hydrogel composition.

RENEVIA^® is another example of an implantable biocompatible matrix that can be used in certain embodiments described in the present disclosure. RENEVIA^® is in a lyophilized format comprised of four components - individual vials of Glycosil (thiol-modified hyaluronan), Gelin (thiol- modified gelatin), crosslinker (Extralink, e.g., polyethylene glycol diacrylate), and a user-supplied vial of sterile water for reconstitution. There are limitations of this format, for example, having four separate components that must be combined requires more manipulation than is preferred. Second, the lyophilized components (Glycosil and Gelin) require a heated 37°C shaking incubator in order to reconstitute the components, a piece of equipment few physicians have and will require purchase and setup. In addition, each vial also requires about 30 to about 60 minutes to reconstitute, slowing down the pace of the procedure. Lastly, certain procedures could be simplified if only one kit per procedure was required instead of the two (containing the 5 mL vials) currently being used.

In certain embodiments, the biocompatible matrix comprises a polysaccharide based polymer, (for example, a hyaluronan based, chitosin based) with a polysaccharide concentration of about 1 mg/mL to about 20 mg/mL, about 2 mg/mL to about 10 mg/mL, about 3 mg/mL, about 4 mg/mL, or about 5 mg/mL.

In other embodiments, the biocompatible matrix includes a gelatin component (for example, collagen) with a gelatin concentration of between about 1 mg/mL to about 20 mg/mL, about 2 mg/mL to about 10 mg/mL, about 3 mg/mL, about 4 mg/mL, or about 5 mg/mL.

In certain embodiments where the biocompatible resorbable matrix comprises a hyaluronan and gelatin hydrogel composition, the hyaluronan: gelatin weight ratio can be between 1:1 and 10:1; the hyaluronan: gelatin weight ratio can be between about 1:1 to about 1:10; about 1:1.5; about 1.5:1; about 1:2; about 2:1; or from between about 0.5:5 to about 5:0.5.

In certain embodiments, a solution to the problems posed by the lyophilized formats of hydrogels are presented. In one embodiment, the glycosil, or hyaluronan component and the gelin, or collagen, or gelatin components are supplied as a liquid mixture in one vial. The liquid mixture may be frozen in certain embodiments. First, this stable liquid format (SLF) reduces the number of components from 4 to 2 since one vial now contains a glycosil/gelin mixture and sterile reconstitution solution is no longer required. Second, since refrigerators and freezers are typical equipment in a medical setting, no new equipment is needed for purchase and set-up. While some time is required to thaw the frozen liquid, this step is less cumbersome since the SLF either can be thawed at the time of use at room temperature or in a water bath; alternatively, it can be thawed at refrigerated temperatures. Third, SLF kits can provide 10 cc of material.

The collagen, in some embodiments comprises a porcine derived collagen. In other embodiments, the collagen comprises human, bovine, porcine, or other mammalian derived collagen. An example of a SLF comprises about 80 mg (in, for example, about 10 ml) of glycosil/gelin mixture, wherein there are about 40 mg of glycosil and about 40 mg of gelin. In another example of SLF, there are from about 0.025 to about 200 mg of glycosil and about 0.025 to about 200 mg of gelin in a vial.

Table 1: SLF Formulations

In other embodiments, the biocompatible matrix includes an SLF composition with non-thiol- modified polysaccharides. In other embodiments, the biocompatible matrix includes an SLF composition with non-thiol-modified collagen or gelatin, for example. In yet other embodiments, the resorbable matrix includes an SLF composition with both non-thiol-modified polysaccharides and non-thiol-modified collagen components.

Although specific examples of hydrogels that are suitable for providing resorbable matrices are described for use with embodiments of the present disclosure, it will be understood that any suitable biocompatible matrix delivery system may be used. For example, gels made using oxidized glutathione (GSSG) as a cross-linking agent may be used (see US Patent Application Publication No. US 2014-0341842, incorporated herein by reference in its entirety).

The SLF may comprise a pFl of from about 7 to about 8. In some embodiments, the pFl is between about 7.2 and about 7.6.

In some embodiments, the SLF resorbable matrix composition may be stored from between about - 80 degrees C to about 45 degrees C, or from between about -20 degrees C to about 25 degrees C, from between about -10 degrees C to about 4 degrees C, or from between about 0 degrees C to about 10 degrees C. Neuroregenerative agents may include, but are not limited to, growth factors (e.g., BDNF, HBEGF, VEGF, IGF-l, bFGF, and the like), and trophic factors, and chemo-attractants to facilitate axon guidance promote synaptogenesis.

By an "effective" amount or a "therapeutically effective amount" of a drug or pharmacologically active agent is meant a nontoxic but sufficient amount of the drug or agent to provide the desired effect. In various embodiments, the neuroregenerative implant comprises a hydrogel and an effective amount of a neuroregenerative agent. In some embodiments, the neuroregenerative agent comprises the growth factor, BDNF. In various embodiments, the neuroregenerative implant is formulated at a concentration of between about 0.01 pg/pL and about 0.5 pg/pL. In various embodiments, the neuroregenerative implant is formulated at a concentration of between about 0.1 pg/pL and about 1.0 pg/pL, about 0.1 pg/pL and about 2.0 pg/pL, about 0.5 pg/pL and about 5 pg/pL.

The concentration of growth factor(s) and/or other neuroregenerative agent can vary widely, and will typically be selected primarily based on activity of the active ingredient(s), body weight and the like in accordance with the particular mode of administration and/or formulation selected and the subject's needs (see, e.g., Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania (1980), Remington: The Science and Practice of Pharmacy, 21st Ed. 2005, Lippincott Williams & Wilkins, and the like). In certain embodiments amounts, however, will typically be selected to provide dosages ranging from about 0.001, 0.01, 0.1 1, or 10 mg/kg/day to about 50 mg/kg/day and sometimes higher. In certain embodiments typical dosages range from about 1 mg/kg/day to about 3 mg/kg/day, preferably from about 3 mg/kg/day to about 10 mg/kg/day, more preferably from about 10 mg/kg/day to about 20.0 mg/kg/day, and most preferably from about 20 mg/kg/day to about 50 mg/kg/day. In certain preferred embodiments, dosages range from about 10 mg/kg/day to about 50 mg/kg/day.

In certain embodiments, the one or more neuroregenerative agents are released from the biocompatible matrix over a period of at least one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year.

In certain embodiments, methods of treating or preventing further brain tissue damage by administering a neuroregenerative implant are presented. In some embodiments, the brain tissue damage is caused by, for example, a stroke, traumatic brain injury, genetic defect, or neural disease. In some embodiments, the brain disease or injury causes an infarct cavity. Because BDNF and other neural (brain) growth factors do not easily pass the blood brain barrier and are likely to have significant toxicity if given systemically, in various embodiments, local administration into the infarct cavity (stroke cavity) is contemplated. Methods of delivering a neuroregenerative implant into a region of the brain (e.g., an infarct cavity) are known to those of skill in the art. In certain embodiments the implant or device is surgically implanted into the desired site. In other embodiments, the composition can be delivered into the desired cite by direct injection or through an implanted cannula.

In certain embodiments the dosage of cells useful in the delivery of the secreted factors (e.g., BDNF, IGF1, VEGF, F1BEGF, bFGF, and the like) or combinations of said factors will depend upon the size and location that would be determined at the time of treatment by a person skilled in the art. From large clinical studies the mean stroke size in humans is 42 mL with a standard deviation of +/- 48 mL and median 21 mL at acute time points (24 hours or less). These volumes are mean 83 mL with a standard deviation of +/- 75 mL and median 60 mL at 3 months (MR Stroke Collaborative Group (2006) Stroke, 3 7: 2521-2525). From another study 35.22% of patients have a lesion size of 5-50mL (at 90d); 30 40.87% have a lesion size from 5-75 cc (Whitehead et al. (2009), 40: 1347-1352). In certain embodiments, a typical formulation may provide a neuroregenerative implant volume of between about one -half to the entire lesion cavity. Thus, clinically relevant volumes may range from about 2.5 mL to about 80 mL.

In some embodiments, the infarct volume is decreased by about 1% to about 20%, about 5% to about 50%, about 10% to about 75% as compared with that of untreated subjects or a baseline measurement.

In various embodiments, the methods described herein can be practiced as stroke recovery therapy in humans or non-human mammals. In certain embodiments a hydrogel/growth factor (e.g., BDNF) formulation can be given to stroke patients once they are clinically stable from their stroke. The stroke cavity can be identified by brain MRI, and used for stereotaxic neurosurgical delivery. In various embodiments, the window for hydrogel/BDNF therapy after stroke is from about 1 to about 5 days after stroke to one year after stroke. However, in certain circumstances earlier administration/delivery may be merited. Accordingly, in certain embodiments, neuroregenerative implants may be administered from about 1 hour to about 1 week, from about 1 day to about 1 month, from about 2 weeks to about 1 year after stroke.

Ischemic brain injury leads to a cascade of aberrant cellular events in the brain, including inflammatory cell migration and a surge in the release of trophic factors. Brain-derived neurotrophic factor (BDNF) is the most abundant neurotrophic factor in the brain. It constitutively regulates neural stem cell differentiation and survival, synapse development and maturation, and refines the development of neural connections. Post-stroke, BDNF is also implicated in processes such as synaptic remodeling, which can occur for several weeks following injury. This regional plasticity may play a role in promoting functional recovery following stroke.

In certain embodiments, a therapeutically effective amount of the neuroregenerative implant causes a reduction of neuroinflammation in about 10% to about 100% of subjects.

In other embodiments, a therapeutically effective amount of the neuroregenerative implant causes improvement of sensorimotor function as measured from a baseline in 1% to 100% of subjects.

Example 1

Neuroregenerative implants comprising hydrogel and BDNF improve functional recovery following distal middle cerebral artery occlusion (dMCAO) surgery

The effects of neuroregenerative implants with a low-dose or high-dose of BDNF (hydrogel+BDNF_LOw and hydrogel+BDNF_HIGH, respectively), delivered via a hydrogel or vehicle, on functional recovery in rats following dMCAo. Prior to implantation in the following examples, the hydrogel was allowed to fully gelate in order to maximize treatment delivery to the ischemic core and minimize dissipation to surrounding tissues. Due to its constitutive elasticity, the hydrogel readily conformed to fit the cavity, thereby preventing the collapse of surrounding ischemic tissue (see Figure 1A). Treatments were administered on Day 8 following permanent dMCAo, and the effects of dose and vehicle were evaluated up to nine weeks following injury. Improved sensorimotor function was observed in rats treated with hydrogel+BDNF_HiGH, particularly six to eight weeks following treatment implantation. Infarct volume was also reduced, as were levels of Ibal, CD68, and GFAP throughout the ipsilateral injured cortex and striatum.

To evaluate sensorimotor function, the 28-Point Neuroscore (28-PN) and Adhesive Tape Removal Test (ART) were used prior to and following dMCAo. Ischemia results in damage to subcortical regions known to govern sensorimotor behavior. As a result, rats develop significant functional deficits that can be detected by assessments such as the 28-PN and ART.

A total of 96 male Sprague Dawley rats ages 64-69 days were used as subjects to model cortical ischemic stroke (Charles River, Hollister, CA, catalog # 001). Rats were housed two per cage in a reverse light-controlled environment, at standard temperature (22 ± 1 C), with ad libitum access to food and water. Prior to all surgical procedures, rats were anesthetized with 3-4% isoflurane in an induction chamber then transferred to a nose cone emitting 1.5 - 2% isoflurane for anesthesia maintenance. All animal experiments were carried out according to the National Institute of Health (NIH) guidelines for the care and use of laboratory animals and approved by the IACUC of Stanford University (APLAC 30704). The studies described herein were conducted in compliance with all applicable sections of the current version of the Final Rules of the Animal Welfare Act Regulations (9 CFR) and the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, 2010.

Focal cerebral infarcts were made by permanent occlusion of the distal right middle cerebral artery (MCA). The bilateral common carotid arteries (CCA) were occluded for 60 min as described by Tamura and colleagues with minor modifications (Tamura A., et al. Focal cerebral ischemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. J Cereb Blood Flow Metab. 1981 ; l( l):53-60, incorporated herein by reference in its entirety). The temporalis muscle was bisected and reflected through an incision made midway between the eye and the ear canal. The proximal middle cerebral artery was exposed through a subtemporal craniectomy without removing the zygomatic arch and without transecting the facial nerve. The artery was then occluded by microbipolar coagulation from just proximal to the olfactory tract to the inferior cerebral vein and then transected. Rats in the sham group received an incision in the ventral neck region and midway between the eye and the ear.

After ischemic stroke induction and treatment administration surgeries, animals received supplemental wet food, 0.9% saline and 0.03 mg/kg buprenorphine was administered subcutaneously and every 12 - 24 hours thereafter as needed based on animal physical condition. Following surgery, rats were placed in cages partially atop heating pads and allowed to fully recover.

A recombinant human/mouse/rat/canine/equine brain derived neurotrophic factor (BDNF) (Lot# NG731602A and NG731603B, R&D systems, Minneapolis, MN) was included as a component in the preparation of a neuroregenerative implant. BDNF was supplied in vials containing 58.4 pL of a 0.2 pm filtered solution in 100 mM sodium citrate and 300 mM NaCl at pH 3 and a concentration of 1.730 mg/mL. BDNF was stored at -80° C until use.

Hydrogels were prepared according to the manufacturer’s protocol (HYSTEM^®-C, BioTime, Alameda, CA). One cc of Reconstitution solution was added to the vial containing Extralink and vortexed. Two cc of Reconstitution solution was added to the vials containing Glycosil and Gelin, the vials vortexed, and placed on an orbital shaker at 37 °C for > 30 min or until completely dissolved. Aliquots of 1:1 Glycosil and Gelin were made for each animal. Glycosil, Gelin, and Extralink were combined in a 2:2:1 ratio, and Extralink added prior to syringe loading. Syringes were left to gelate for 15-30 min after loading of the BDNF (1.73 mg/ml, pH 3.0) and hydrogel components to prepare implants. The 30 minute incubation time allows for gel formation and avoids free distribution of the implant, so that treatment delivery is localized and remains at the site of implantation. The pH of solutions containing BDNF (0.057 pg/pL or 0.167 pg/pL) were adjusted using 1M NaOH prior to the addition of the hydrogel solution, and the complete suspension adjusted to a pH between 6.9 and 9.0.

Eight days following dMCAo surgery and after randomization of experimental groups based on their behavioral performance on Day 7, rats received a neuroregenerative implant with hydrogel+BDNF with a BDNF concentration of 0.057 pg/pL or a BDNF concentration of 0.167 pg/pF, referred to as‘hydrogel+BDNF_LOw’ and‘hydrogel+BDNF_HiGH,’ respectively), hydrogel- only, vehicle (buffered or unbuffered saline), or BDNF-only (0.167 pg/pF) in vehicle via stereotaxic injections. Sham control animals did not receive treatment.

Rats were anesthetized as described above. After fixing the rat’s head in the stereotaxic apparatus (David Kopf Instruments, Tujunga, CA), the scalp was disinfected and a midline skin incision along the rostrocaudal axis of the skull was made. After removing the periosteum, Bregma was located and a 50 - 100 pF gas-tight syringe with a 26-gauge needle (Hamilton, Reno, NV) was filled with treatment and the stereotaxic apparatus zeroed. After adjusting needle location to the Anterior/Posterior and Medial/Fateral coordinates relative to Bregma, a burrhole in the skull was made exposing dura mater and the tip of the needle zeroed to dura level to avoid effects of skull thickness differences and lowered to the respective Dorsal/Ventral coordinates (Paxinos G, Watson, C. The Rat Brain in Stereotaxic Coordinates - The New Coronal Set 5th ed. Fondon: Academic Press; 2005. Incorporated herein by reference in its entirety). Treatment was infused into four sites in the right hemisphere (Medial/Fateral +3.00, Anterior/Posterior -3.14, Dorsal/Ventral - 1.50; Medial/Fateral +3.00, Anterior/Posterior -3.14, Dorsal/Ventral -1.00; Medial/Fateral +5.50, Anterior/Posterior -3.14, Dorsal/Ventral -3.00; Medial/Fateral +5.50, Anterior/Posterior -3.14, Dorsal/Ventral -2.50) targeting the location of infarction cavity using a Micro4 microsyringe pump controller (World Precision Instruments, Sarasota, FF) at a rate of 167 nF/s (10 pF/min). There was a 1 min wait between injections. A total of 100 pF of neuroregenerative implant was injected between the four sites (25 pF per Dorsal/Ventral coordinate).

To evaluate sensorimotor function following dMCAO as well as prior to and following treatment, the 28-point neuroscore (28-PN) and adhesive tape removal test (ART) were used. All behavioral tests were performed as blinded tests throughout the study. Rats were habituated to the testing area 1 h prior to testing. Treatment groups were pseudo-randomized based on body weight. Baseline performance was assessed 7 days prior to treatment, and again on days 14, 28, 42, and 56 following treatment. A timeline of the behavioral tests used for these experiments is shown in Figure 1B.

In the adhesive tape removal test (ART), two pieces of adhesive tape (6x6 mm) were applied to the palmar side of the front paws and the rat placed in a transparent chamber. The adhesive elicits grooming behavior and rats are naturally inclined to remove the tape. Testing took place in a single day comprised of three trials, each lasting 120 s, separated by inter-trial intervals of 6-10 min. Performance was scored from videos recorded by a camera positioned below the chamber. For each trial, latency to initial contact and time to remove the adhesive from the left (contralateral to injury) and right (ipsilateral to injury) paws was recorded. Tactile response was assessed by comparing the time between contact and removal for each paw. These parameters allow sensory deficits to be distinguished from motor impairments. Rats were tested 7, 14, 28, and 56 days following dMCAo or sham surgery. Data collected from sham control rats served as an internal control for injury and are not shown. Rats performing < mean (M) + 1 standard deviation (SD) of sham controls 7 days following dMCAO were excluded from the study.

The 28-PN test was used to assess neurological and sensorimotor function as previously described. Eleven parameters were assessed and scored as follows: circling and paw placement (0-4); motility, general condition, ability to pull body onto a horizontal bar, and ability to ascend an inclined platform (0-3); visual paw reaching, grip strength, and contralateral rotation (0-2); contralateral and righting reflexes (0-1). The maximum score is 28, with a score of 0 indicating severe impairment. Rats were assessed 7, 14, 28, and 56 days following dMCAo or sham surgery. Rats scoring > 27 points 7 days following dMCAo were excluded from the study.

Statistical tests used for analysis included two-way ANOVA, unpaired two-sample t- test, and Mann-Whitney U. To correct for multiple comparisons, Tukey’s or Dunnett’s correction was applied. Outliers were identified using Grubb’s test (extreme studentized deviate method). Data are presented as the mean ± standard error of the mean (SEM) and statistical significance defined at the level of p<0.05.

Within groups, the main effect of time (two-way ANOVA; F( 4, 145)=11, p<0.000l) on sensorimotor recovery, measured as % improvement on the 28-PN compared to Day 7, was statistically significant and the results are shown in Figure 2A. Posthoc analysis using Dunnett’s multiple comparison test (MCT) revealed rats treated with BDNF-only (n=8) had significantly higher recovery percentages on Day 42 (r=0.0109) and 56 (p=0.004l). Rats treated with hydrogel- only (n=9) and hydrogel+BDNF_HIGH (n=6) also showed significant improvements on Days 28 (p= 0.0049, =0.0338, respectively), 42 (p= 0.0125, =0.0141 ), and 56 (p=0.0009, =0.0049).

Significant functional improvements at each time point measured using the 28-PN were demonstrated in the hydrogel+BDNF_HIGH group.

Because the 28-PN test utilizes an ordinal level of measurement, it provides a less sensitive means to detect functional differences compared to the ART. In addition, separation between groups on the 28-PN increased over time and differences between treatment groups may have resolved to reach statistical significance at time points later than those examined presently. Collectively, these data indicate that intracerebral BDNF therapy can improve functional recovery.

The main effect of treatment on the sensory contact time recovery index of the ART was found to be significant (two-way ANOVA; F(3,104)=7.5, p=0.0001) and the results are shown in Figure 2B. The hydrogel+BDNF_HIGH group (n=8) showed significant improvement on Day 42 compared to BDNF-only (n=7, p=0.0024) and hydrogel+BDNF_LOw groups (n=7, p=0.0456), and on Days 42 (p=0.0007) and 56 (j>= 0.0233) compared to hydrogel-only (n=8). The hydrogel+BDNF_HIGH group (n=l l) also required less time to remove the adhesive (F(3,128)=4.1, p=0.0080) compared to the BDNF-only (n=9, p=0.0126) and hydrogel+BDNF_LOw (n=9, p =0.0356) groups on Day 42 as shown in Figure 2C. These results suggest that neuroregenerative implants with hydrogel and higher doses of BDNF improve sensorimotor function in a time-dependent manner, particularly at time points later than four weeks following injury. Performance on the ART was significantly improved in rats receiving hydrogel+BDNF_HIGH. Comparatively, rats receiving BDNF in vehicle or hydrogel+BDNF_Low failed to show significant improvements.

Example 2

Neuroregenerative implants comprising hydrogel and BDNF reduce infarct volume following dMCAo

To examine the effects of prolonged BDNF treatment, dose, and delivery vehicle on the developing infarction, infarct volume was quantified 9 weeks following dMCAo. Upon completion of behavioral testing, rats were sacrificed and their brains assessed for stroke -related pathology using immunohistochemistry (IF1C). Rats were anesthetized with isoflurane and transcardially perfused with buffered saline followed by 4% paraformaldehyde (PFA). Brains were post-fixed in 4% PFA for 48 h at 4 °C then transferred to 30% sucrose for 5 days. Brains were flash frozen in isopentane and cryo-sectioned at 40 pm (Microm F1M-550, Thermo Scientific, Waltham, MA), mounted on slides (Fisherbrand Superfrost Plus, Fisher Scientific, Pittsburgh, PA), and left to dry overnight. Sections were re -hydrated through a series of graded ethanol washes and incubated for 10-14 min in 0.5% cresyl violet acetate (150727 MP Biomedicals, Burlingame, CA; 405760100 Acros Organics, Geel, Belgium) and glacial acetic acid solution. Sections were washed with H₂0 for 5 min and placed in an acidic formalin solution (10% neutral buffered formalin (16004-126, VWR, Radnor, PA, 0.2% glacial acetic acid, A38S Fisher Scientific, Fair Lawn, NJ)) in distilled H₂0 for 2 min and washed with Fl₂0. Sections were then dehydrated through a series of graded ethanol baths, clarified with xylene, and cover-slipped (534056 Sigma-Aldrich, Saint Louis, MO) with DPX mounting medium (360294F1 VWR, Radnor, PA; 06522 Sigma-Aldrich, Saint Louis, MO).

Representative photomicrographs of cresyl violet-stained sections used for quantification are shown in Figure 3A. Compared to the hydrogel+BDNF_LOw group (n=l2), rats receiving hydrogel+BDNF_HiGH (n=l2) had reduced infarct volume (t test, Z(22)=3.l4, p=0.0048) and the results are shown in Figure 3B. These data demonstrate that higher doses of the neuroregenerative implants (BDNF delivered via hydrogels) exert neuroprotective effects on the parenchyma following dMCAo, and that treatment delivery via hydrogel is more efficacious than vehicle.

Images of cresyl violet-stained sections from all treatment groups were obtained using a photo scanner (Epson Perfection V550) and the whole section, injured (ipsilateral) hemisphere, uninjured (contralateral) hemisphere, and each ventricle traced and analyzed using NIF1 ImageJ 1.49 software. The ventricular areas were subtracted from their respective hemispheres to control for ventricular enlargement (hydrocephalus ex vacuo ). Flealthy and injured parenchyma volumes were obtained using formulas based on the Cavalieri principle as follows: V_hhp=(åA_hhp-åA_hv) x T and Vi_hp=(åAi_hp-åA_iv) x T, where V_hh|1 is the volume of healthy parenchyma, V_ihp is the volume of injured parenchyma, åA_hh|1 is the sum of healthy hemisphere area of all sections, åA_hv is the sum of healthy ventricle areas for all sections, SA_¾R is the sum of injured hemisphere area for all sections, åA_iv is the sum of injured ventricle areas for all sections, and T is the distance between sections (~l mm). The values of the injured hemisphere were subtracted from the healthy hemisphere to determine tissue loss due to stroke (infarct) using formula V_i=V_hhp-V_ihp where V; is the infarct volume. The volume of whole parenchyma analyzed for each brain was obtained using the formula V„_pa=V_hhp+Vi_hp+Vi=V_hhp x 2, where V_wpa is the volume of whole parenchyma analyzed (analyzed sections represent the area from -3 to +3 relative to Bregma). These values define the total volume of brain parenchyma (functional tissue), because the ventricles were excluded. The volume of tissue lost due to infarct is expressed as % of the whole brain parenchyma analyzed using formula Vi_%„_pa=(V_ihpxl00)/V_wpa, where V_i%wpa is the infarct volume % of the whole (total) parenchyma analyzed. Example 3

Neuroregenerative implants comprising hydrogel and BDNF reduce Ibal in the striatum and cingulate cortex, CD68 in the striatum, and GFAP in the anterior motor cortex and striatum following dMCAo

Levels of microgliosis following dMCAo in the ipsilateral (injured) and contralateral cortices were assessed to determine the neuroregenerative implant’s ability to reduce levels of pro-inflammatory cytokines and promote the release of anti-inflammatory factors.

Sections were rinsed with buffered saline for 20 min and then transferred to 50°C heated solution containing 2.94 g tri-sodium citrate dihydrate (Lot#BCBC8643V, Sigma-Aldrich, St.Louis, MO), 0.125 ml Tween 20, and 250 ml distilled H₂0 in a water bath between 98-l00°C for 20 min. Sections were rinsed with water for 10 min, washed three times with buffered saline, and pre incubated for 90 min in a blocking solution of 0.3% Triton X-100 (X100 Sigma-Aldrich, Saint Louis, MO) and 6% normal donkey serum (017-000-121, Jackson Immunoresearch, West Grove, PA) in buffered saline. The following primary antibodies were used: rabbit anti-ionized calcium binding adapter molecule-l (Ibal, 1:1000, 019-19741, WAKO Chemicals USA, Richmond, VA), mouse anti-rat anti-cluster of differentiation 68 (CD68, 1:300, MCA341GA, Bio-Rad, Hercules, CA), or chicken anti-glial fibrillary acidic protein (GFAP, 1:1080, Ab4674, Abeam, Cambridge, UK). All primary antibody solutions were prepared in blocking buffer+buffered saline. Sections were incubated overnight in primary antibody, washed 3x, and incubated for 90 min in the appropriate secondary antibody solution: CY3-conjugated donkey anti-rabbit (711-165-152, Jackson Immunoresearch, West Grove, PA), AlexaFluor 488 donkey anti-mouse (715-545-151, Jackson Immunoresearch, West Grove, PA) IgG secondary, or CY5 donkey anti-chicken (703-175- 155, Jackson Immunoresearch, West Grove, PA). Secondary antibodies were diluted 1:250 and 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (D9542, Sigma-Aldrich, St. Louis, MO) diluted 1:5000 in buffered saline. Sections were washed twice with buffered saline and briefly with 0.1 M phosphate buffer. Sections were left to dry overnight before being cover-slipped with polyvinyl alcohol mounting medium with DABCO anti-fade (10981, Sigma-Aldrich, St. Louis, MO).

Five brains per treatment group representing the mean infarct volume were selected to avoid the bias of natural variability of infarct volume in vivo, and to heighten the sensitivity of tests to detect differences between groups. Areas directly or indirectly affected by the experimental ischemic insult within the ipsilateral and contralateral cortices were imaged and included: the dorsal and ventral cortical border of the infarct cavity (data not shown), anterior motor cortex, cingulate cortex, corpus striatum, and internal capsule (data not shown). Analysis was performed in a total of 27 images per hemisphere across seven sections to include regions of interest (ROI) within -3 to +3 of Bregma (see Figure 1A). Ibal, CD68, and GFAP immunoreactivity (IR) was assessed based on the average of each anatomical region and expressed as % area of threshold. Images were acquired using a Zeiss Axioscope M2 microscope with Stereo Investigator 10.0 software (MicroBrightField Bioscience, VT) and quantified using NIH ImageJ 1.49.

Within the corpus striatum, rats treated with hydrogel+BDNF_HiGH (n=5) had reduced Iba-1 IR compared to those treated with BDNF-only (n=5), although the difference was not statistically significant (p=0.0556, see Figure 4A). No differences in levels of Ibal were found between groups in the contralateral cortex or striatum. Ibal IR was reduced in the ipsilateral cingulate cortex of rats treated with hydrogel+BDNF_HiGH (n=5) compared to those treated with hydrogel-only (n=5, Z(8)=3.01, =0.0167) and the results are shown in Figure 4B. Rats treated with hydrogel+BDNF_HiGH also had significantly less Ibal in the cingulate cortex of the contralateral hemisphere compared to the BDNF-only group (data not shown (n=5); Z(8)=2.96, p=0.0182). Qualitatively, reactive microgliosis was evidenced by marked soma enlargement and retracted processes. The greatest reactivity was observed in the perilesional cortex and regions proximal to the injury site. These data indicate that hydrogel+BDNF_HIGH reduces microgliosis following dMCAo by mitigating cellular reactivity. These results also demonstrate that BDNF reduces neuroinflammation in a manner that is both dose- and vehicle -dependent.

To assess the phagocytic fraction of Ibal-positive cells, levels of CD68 were assessed using IHC. Because the corpus striatum was one of few regions to display positive CD68 labeling, and showed high levels of microgliosis, subsequent analyses focused on this region. CD68 IR was significantly reduced in the corpus striatum of rats receiving hydrogel+BDNF_HiGH (M=0.19, SD=0.08) compared to hydrogel-only (M=0.55, SD=0.31) and the results are shown in Figure 5 (Mann-Whitney U=2, nl=n2= 5, p=0.0317, two-tailed). No significant differences were observed between groups in the contralateral hemisphere. These results demonstrate that hydrogel+BDNF_HiGH reduces dMCAo- induced phagocytic activation of CD68+ macrophages in the corpus striatum.

Astrocyte activation is directly associated with GFAP upregulation (Barreto G, White RE, Ouyang Y, Xu L, Giffard RG. Astrocytes: targets for neuroprotection in stroke. Cent Nerv Syst Agents Med Chem. 2011 ;l l(2): 164-73.) and while early astrocytic activity may provide neuroprotection, prolonged astrogliosis can lead to glial scarring within the ischemic penumbra. BDNF can affect astrocyte reactivity either directly via its actions as a neurotrophin, or indirectly by reducing local inflammation following dMCAo. Because the corpus striatum contains fibrous white matter astrocytes which are less sensitive to ischemia, and the anterior motor cortex contains protoplasmic astrocytes that are particularly vulnerable to ischemic injury, these regions were the focus of the analyses. The hydrogel+BDNF_HIGH group (n=5) showed reduced GFAP IR in the corpus striatum compared to the BDNF-only (n=5, Z(8)=5.l7, p=0.0009) and hydrogel+BDNF_LOw (n=5, i(8)=2.98, p=0.0111) groups and the results are shown in Figure 6A. Rats treated with hydrogel+BDNF_HIGH also had significantly less GFAP in the corpus striatum of the contralateral hemisphere compared to those given BDNF-only (data not shown; Z(8)=2.62, p= 0.0306). Within the anterior motor cortex, GFAP IR was reduced in the hydrogel+BDNF_HiGH group (n=5, i(8)=3.08, p= 0.0152) compared to hydrogel-only (n=5) and the results are shown in Figure 6B. No significant differences in GFAP IR were found in the anterior motor cortex of the contralateral hemisphere. These results indicate that hydrogel+BDNF_HiGH reduces levels of GFAP in regions characteristically prone and resistant to the development of reactive astrocytosis in response to ischemic insult.

Levels of Ibal, CD68, and GFAP were most reduced in rats receiving hydrogel+BDNF_HiGH, particularly compared to hydrogel only and BDNF-only groups, indicating that both vehicle and dose affect these neuroinflammatory markers. Impressively, these reductions were not exclusive to sites proximal to the injury but spanned multiple regions as caudal as the M2/cingulate border. Microgliosis in the cingulate cortex was most attenuated by the neuroregenerative implant comprising hydrogel+BDNF_HIGH; however, the effect of treatment on levels of Ibal in the striatum did not reach statistical significance. Because the striatum lies just below the injury site and was one of few regions to show positive CD68 labeling, our sensitivity to detect differences may have been insufficient to overcome the high level of gliosis in this region. Still, hydrogel+BDNF_HIGH treatment reduced astrogliosis and infarct volume, possibly by mitigating glial-scar formation. In addition, the doses used presently are less than those reported to provide neuroprotection by bolus dosing. Therefore, a higher dose of BDNF, administered at an earlier time point, may have further improved functional recovery and reduced neuroinflammation.

Following ischemic injury, the release of inhibitory chemokines from neighboring glial scars can limit the regeneration of neuronal processes. Because focal cortical ischemia has been found to increase endogenous BDNF mRNA 5-fold, the administration of exogenous BDNF may have a cumulative effect to aid regenerative processes. These results described herein demonstrate that BDNF in a hydrogel administered as neuroregenerative implants may potentiate the recovery of functional deficits through additional mechanisms, mainly those that mitigate the development of neuroinflammation and gliosis.

As a therapeutic modality, hydrogels provide a surrogate matrix for the delivery of customized therapeutics to targeted regions of the brain. Presently, the deposition of hydrogel+BDNF_HIGH in the ischemic core following dMCAo improved sensorimotor function and reduced levels of neuroinflammation. Advantageously, BDNF delivery using fully gelated hydrogels provides sustained treatment release at concentrations higher than those achieved using ungelated liquid hydrogel. In addition, hydrogels may serve as a viable substrate for the growth and development of neural progenitors in vivo.

Although the description herein contains many details, these should not be construed as limiting the scope of the disclosure but as merely providing illustrations of some of the presently preferred embodiments. Therefore, it will be appreciated that the scope of the disclosure fully encompasses other embodiments which may become obvious to those skilled in the art.

In the claims, reference to an element in the singular is not intended to mean "one and only one" unless explicitly so stated, but rather "one or more." All structural, chemical, and functional equivalents to the elements of the disclosed embodiments that are known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the present claims. Furthermore, no element, component, or method step in the present disclosure is intended to be dedicated to the public regardless of whether the element, component, or method step is explicitly recited in the claims. No claim element herein is to be construed as a "means plus function" element unless the element is expressly recited using the phrase "means for". No claim element herein is to be construed as a "step plus function" element unless the element is expressly recited using the phrase "step for".

Claims

1. A method of treating brain tissue damage in a subject, the method comprising administering a therapeutically effective amount of a neuroregenerative implant.

2. The method of claim 1, wherein the neuroregenerative implant comprises a biocompatible matrix and a neuroregenerative agent.

3. The method of claim 1, wherein the biocompatible matrix comprises a hydrogel.

4. The method of claim 3, wherein the hydrogel comprises thiol-modified hyaluronan, thiol-modified gelatin, and polyethylenegycol diacrylate (PEGDA).

5. The method of claim 4, wherein the hydrogel is made by a method comprising:

(a) reconstituting the thiol-modified hyaluronan, thiol-modified gelatin, and polyethylenegycol diacrylate (PEGDA); and

(b) mixing the thiol-modified hyaluronan, thiol-modified gelatin, and polyethylenegycol diacrylate (PEGDA) together.

6. The method of claim 3, wherein the hydrogel is made by a method comprising:

(a) contacting a first thiolated monomer with GSSG;

(b) allowing the first thiolated monomer and the GSSG to react; and

(c) adding a second thiolated monomer to the reaction of step (b), thereby forming a hydrogel comprising the first and second thiolated monomers, but not comprising glutathione or GSSG.

7. The method of claim 6, wherein the first thiolated monomer is thiolated carboxymethylated hyaluronan and wherein the second thiolated monomer is thiolated gelatin.

8. The method of claim 2, wherein the biocompatible matrix comprises SLF.

9. The method of claim 8, wherein SLF is made by a method comprising:

(a) thawing a combination of thiol-modified hyaluronan and thiol-modified gelatin at a temperature of about 35°C or greater; and

(b) adding polyethylenegycol diacrylate (PEGDA) to the thawed combination of thiol- modified hyaluronan and thiol-modified gelatin.

10. The method of claim 1, wherein the neuroregenerative agent comprises chemo attractants and trophic factors.

11. The method of claim 1 , wherein the neuroregenerative agent comprises a growth factor.

12. The method of claim 11, wherein the neuroregenerative agent comprises BDNF.

13. The method of claim 12, wherein BDNF is at a concentration of between about 0.01 pg/pL and about 0.5 pg/pL.

14. The method of claim 1, wherein the administering of the therapeutically effective amount of the neuroregenerative implant results in a reduction of neuroinflammation.

15. The method of claim 1, wherein the administration of the therapeutically effective amount of the neuroregenerative implant results in an improvement in sensorimotor function as measured from a baseline.

16. The method of claim 1, wherein administration of the neuroregenerative implant results in a decrease of an infarct cavity volume as measured from a baseline or compared to non- treated subjects.

17. The method of claim 16, wherein the infarct cavity volume is decreased by between about 1% to about 20% compared to non-treated subjects.

18. The method of claim 1, wherein the neuroregenerative implant is administered into an infarct cavity.

19. The method of claim 18, wherein the neuroregenerative implant comprises a hydrogel and BDNF and wherein the hydrogel is fully gelated before administration.

20. The method of claim 18, wherein the BDNF is released from the hydrogel over at least about 1 day to about 2 months.

21. The method of claim 18, wherein the neuroregenerative implant is administered at about 1 day to about 1 year following a stroke.

22. The method of claim 1 , wherein the brain tissue damage is caused by a stroke.

23. The method of claim 1, wherein the brain tissue damage is caused by traumatic brain injury (TBI).

24. The method of claim 1 , wherein the brain tissue damage is caused by one or more of: tumor, surgical procedure, radiation therapy, chemotherapy, acquired brain injury (ABI), neurological illness, birth trauma, poison, infection, strangulation, choking, drowning, heart attack, aneurysm, illegal drug abuse, neurological illness.