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Title:  Nerve regeneration employing keratin biomaterials
United States Patent: 
7,892,573
Issued: 
February 22, 2011

Inventors:
 Van Dyke; Mark E. (Winston-Salem, NC)
Assignee:
  Wake Forest University Health Sciences (Winston-Salem, NC)
Appl. No.:
 11/673,212
Filed:
 February 9, 2007


 

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Abstract

A keratin hydrogel matrix serves as an effective acellular scaffold for axonal regeneration and facilitates functional nerve recovery.

Description of the Invention

SUMMARY OF THE INVENTION

A first aspect of the present invention is a device for promoting the growth of a nerve in a mammal, comprising:

a support structure having an elongate opening therein, such as a tubular encasing structure, configured for placement adjacent or around a damaged region of a nerve; and

a physiologically acceptable matrix composition for placement in the elongate opening, through which or in which the nerve may grow. The matrix composition typically comprises a suitable amount (e.g., from 5 to 95 percent by weight) of keratin such as one or more keratin derivatives, e.g., alpha keratose, gamma keratose, kerateine, fractions thereof, and/or mixtures thereof, typically hydrated in a liquid (e.g., from 5 to 95 percent by weight) such as water (optionally containing physiologically acceptable salts), and the matrix may optionally contain other active ingredients such as one or more growth factors.

A further aspect of the present invention is the use of a matrix composition comprising keratin or a keratin material as described herein for the preparation of a device for carrying out a method as described herein.

Another aspect of the present invention is a kit comprising a support structure and a container, wherein, said support structure is packaged in said container in sterile form. The kit may also comprise a keratin matrix composition, in hydrated or dehydrated form (e.g., for subsequent hydration once opened for use), or the keratin matrix composition may be packaged separately.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The disclosures of all United States patents cited herein are hereby incorporated by reference herein in their entirety.

Keratins extracted from hair are a novel group of biomaterials that may provide an alternative to other nerve conduit fillers. Porous keratin scaffolds in the form of a matrix promote cell binding, and contain multiple growth factors. In many instances the biocompatibility of keratin was found to exceed that of other naturally-derived biomaterials (Lee S. J. et al., Polym Prep 2005; 46(1):112). Also, certain keratin preparations have the ability to self-assemble into complex morphologies amenable to cell infiltration (Lee S. J. et al., Polym Prep 2005; 46(1):112). Keratose, an oxidized derivative of keratin, can self-assemble into nanofilaments when placed in solution. These nanofilaments then can further self-assemble into a fibrous micro-architecture on gelation. Important aspects of this self-assembly mechanism are: 1) it occurs spontaneously under benign conditions, and 2) it results in a homogeneous, porous morphology which facilitates the infiltration of regenerative cells. Cell binding to porous keratin scaffolds is facilitated by fibronectin-like binding domains (Tachibana A. et al., J Biotech 2002; 93: 165-7). Additionally, human hair has been identified as a depot of growth factors involved in normal follicle cycling, including nerve growth factor (Stenn K. S. et al., J Dermatology Sci 1994; 7S:S109-24).

One of the main purposes of using a biomaterial in tissue regeneration is to provide a surrogate extracellular matrix (ECM) for cells to attach and grow. Specific interactions to ECM binding sites through cell receptors are important in maintaining proper cell function (Ingber D., Curr Opin Cell Biol 1991; 3(5):841-8; Tooney P. A. et al., Immunol Cell Biol 1993; 71(2):131-9; Jockusch B. M. et al., Annu Rev Cell Dev Biol 1995; 11:379-416; Ruoslahti E., Annu Rev Cell Dev Biol 1996; 12:697-715). Cells attach to the ECM through more than 20 known integrin receptors, more than half of which bind to the Arginine-Glycine-Aspartic Acid (RGD) peptide motif (Ruoslahti E., Annu Rev Cell Dev Biol 1996; 12:697-715). An inspection of the more than 70 known human hair keratin protein sequences reveals that 78% contain at least one binding domain specific to the integrins expressed on many cell types, and 23% contain two or more such domains (Entrez Protein Database, National Center for Biotechnology Information (NCBI).

Through the use of biomaterials that are both neuroconductive and neuroinductive, regeneration across large nerve defects may be possible. Hair follicle morphogenesis is a highly regenerative process that is mediated by a host of regulatory and matrix molecules. Genes for several neurotrophic factors have been shown to be expressed in both human and animal hair follicle, including growth factors prominent in nerve regeneration such as insulin-like growth factor, nerve growth factor and fibroblast growth factor (Ishii D. N. et al., Pharmacol Ther 1994; 62(1-2):125-44; Fu S. Y. and Gordon T., Mol Neurobiol 1997; 14(1-2):67-116; Frostick S. P. et al., Microsurgery 1998; 18(7):397-405; Grothe C. and Nikkhah G. Anat Embryol (Berl) 2001; 204(3):171-7). The presence of some of the actual proteins has been confirmed by immunohistochemical techniques (Little J. C. et al., J Invest Dermatol 1994; 103(5):715-20; Mitsui S. et al., Br J Dermatol 1997; 137(5):693-8).

Subjects to be treated by the present invention include both human and animal subjects, particularly mammalian subjects such as dogs, cats, horses, cattle, mice, monkeys, baboons, etc., for both human and veterinary medicine purposes and drug and device development purposes.

Nerves to be treated by the methods of the invention include afferent, efferent and mixed peripheral nerves such as somatic nerves, sensory-somatic nerves (including the cranial and spinal nerves), and autonomic nerves, which include sympathetic nerves, and parasympathetic nerves. Examples of nerves to be treated include, but are not limited to, cranial nerves, spinal nerves, nerves of the brachial plexus, nerves of the lumbar plexus, musculocutaneous nerve, femoral nerve, obturator nerve, sciatic nerve, the intercostal nerves, subcostal nerve, ulnar nerve, radial nerve, median nerve, pudendal nerve, saphenous nerve, common peroneal nerve, deep peroneal nerve, superficial peroneal nerve, and tibial nerve.

Damaged regions of nerves to be treated by the invention include those that have been subjected to a traumatic injury, such as crushed regions and severed (including fully and partially severed) regions in a limb, as well as nerves damaged in the course of a surgical procedure, e.g., as necessary to achieve another surgical goal. Damaged regions also include nerve regions that have degenerated due to a degenerative nerve disorder or the like, creating a "bottleneck" for axonal activity that can be identified by techniques such as electromyography and treated by use of the methods and devices of the present invention.

Enhancing cell migration has important implications in many regenerative processes. Functional repair of tissues is often size limited, due primarily by the inability of regenerative cells to migrate over long distances. In the case of nerve regeneration, infiltrating Schwann cells are driven by chemotactic mechanisms to migrate into the damaged nerve's provisional matrix and initiate the repair process. Materials that have the ability to mediate this process can be tested using the modified Boyden method (Boyden S. J Exp Med 1962; 115: 543-66).

Keratin Preparations.

After extrusion through the skin, the hair fiber is formed into a highly stable and robust structural tissue that is relatively impervious to environmental insult. The hair fiber contains structural macromolecules, crosslinkers, plasticizers, and UV stabilizers, which serve to protect the regulatory molecules contained within it. The useful contents of the hair fiber can be retrieved using chemical methods that break down the constituent matrix proteins called keratins.

The matrix compositions may comprise a keratin, including alpha keratose, gamma keratose, kerateine, kerateine fractions, mixtures thereof, etc., typically hydrated with a physiologically acceptable aqueous medium such as sterile water, sterile saline solution, etc. In some embodiments the keratin of the matrix compositions comprises a mixture of alpha and gamma keratose. In some embodiments the alpha and/or gamma keratose is acidic alpha and/or gamma keratose. In some embodiments the alpha and/or gamma keratose is basic alpha and/or gamma keratose. General procedures for the preparation of useful keratins are set forth below.

A preferred method for the production of keratoses is by oxidation with hydrogen peroxide, peracetic acid, or performic acid. A most preferred oxidant is peracetic acid. Preferred concentrations range from 1 to 10 weight/volume percent (w/v %), the most preferred being approximately 2 w/v %. Those skilled in the art will recognize that slight modifications to the concentration can be made to effect varying degrees of oxidation, with concomitant alterations in reaction time, temperature, and liquid to solid ratio. It has also been discussed by Crewther et al. that performic acid offers the advantage of minimal peptide bond cleavage compared to peracetic acid. However, peractic acid offers the advantages of cost and availability. A preferred oxidation temperature is between 0 and 100 degrees Celsius (.degree. C.). A most preferred oxidation temperature is 37.degree. C. A preferred oxidation time is between 0.5 and 24 hours. A most preferred oxidation time is 12 hours. A preferred liquid to solid ratio is from 5 to 100:1. A most preferred ratio is 20:1. After oxidation, the hair is rinsed free of residual oxidant using a copious amount of distilled water.

The keratoses are extracted from the oxidized hair using an aqueous solution of a denaturing agent. Protein denaturants are well known in the art, but preferred solutions include urea, transition metal hydroxides (e.g. sodium and potassium hydroxide), ammonium hydroxide, and tris(hydroxymethyl)aminomethane (tris base). A preferred solution is Trizma.RTM. base (a brand of tris base) in the concentration range from 0.01 to 1M. A most preferred concentration is 0.1M. Those skilled in the art will recognize that slight modifications to the concentration can be made to effect varying degrees of extraction, with concomitant alterations in reaction time, temperature, and liquid to solid ratio. A preferred extraction temperature is between 0 and 100 degrees Celsius. A most preferred extraction temperature is 37.degree. C. A preferred extraction time is between 0.5 and 24 hours. A most preferred extraction time is 3 hours. A preferred liquid to solid ratio is from 5 to 100:1. A most preferred ratio is 40:1. Additional yield can be achieved with subsequent extractions with dilute solutions of tris base or deionized (DI) water. After extraction, the residual solids are removed from solution by centrifugation and/or filtration.

The crude extract can be isolated by first neutralizing the solution to a pH between 7.0 and 7.4. A most preferred pH is 7.4. Residual denaturing agent is removed by dialysis against DI water. Concentration of the dialysis retentate is followed by lyophilization or spray drying, resulting in a dry powder mixture of both gamma- and alpha-keratose. Alternately, alpha-keratose is isolated from the extract solution by dropwise addition of acid until the pH of the solution reaches approximately 4.2. Preferred acids include sulfuric, hydrochloric, and acetic. A most preferred acid is concentrated hydrochloric acid. Precipitation of the alpha fraction begins at around pH 6.0 and continues until approximately 4.2. Fractional precipitation can be utilized to isolate different ranges of protein with different isoelectric properties. Solid alpha-keratose can be recovered by centrifugation or filtration.

The alpha-keratose can be further purified by re-dissolving the solids in a denaturing solution. The same denaturing solutions as those utilized for extraction can be used, however a preferred denaturing solution is tris base. Ethylene diamine tetraacetic acid (EDTA) can be added to complex and remove trace metals found in the hair. A preferred denaturing solution is 20 mM tris base with 20 mM EDTA or DI water with 20 mM EDTA. If the presence of trace metals is not detrimental to the intended application, the EDTA can be omitted. The alpha-keratose is re-precipitated from this solution by dropwise addition of hydrochloric acid to a final pH of approximately 4.2. Isolation of the solid is by centrifugation or filtration. This process can be repeated several times to further purify the alpha-keratose.

After removal of the alpha-keratose, the concentration of gamma-keratose from a typical extraction solution is approximately 1-2%. The gamma-keratose fraction can be isolated by addition to a water-miscible non-solvent. To effect precipitation, the gamma-keratose solution can be concentrated by evaporation of excess water. This solution can be concentrated to approximately 10-20% by removal of 90% of the water. This can be done using vacuum distillation or by falling film evaporation. After concentration, the gamma-keratose solution is added dropwise to an excess of cold non-solvent. Suitable non-solvents include ethanol, methanol, acetone, and the like. A most preferred non-solvent is ethanol. A most preferred method is to concentrate the gamma-keratose solution to approximately 10 w/v % protein and add it dropwise to an 8-fold excess of cold ethanol. The precipitated gamma-keratose can be isolated by centrifugation or filtration and dried. Suitable methods for drying include freeze drying (lyophilization), air drying, vacuum drying, or spray drying. A most preferred method is freeze drying.

A preferred method for the production of kerateines is by reduction of the hair with thioglycolic acid or beta-mercaptoethanol. A most preferred reductant is thioglycolic acid (TGA). Preferred concentrations range from 1 to 10M, the most preferred being approximately 1.0M. Those skilled in the art will recognize that slight modifications to the concentration can be made to effect varying degrees of reduction, with concomitant alterations in pH, reaction time, temperature, and liquid to solid ratio. A preferred pH is between 9 and 11. A most preferred pH is 10.2. The pH of the reduction solution is altered by addition of base. Preferred bases include transition metal hydroxides, sodium hydroxide, and ammonium hydroxide. A most preferred base is sodium hydroxide. The pH adjustment is effected by dropwise addition of a saturated solution of sodium hydroxide in water to the reductant solution. A preferred reduction temperature is between 0 and 100.degree. C. A most preferred reduction temperature is 37.degree. C. A preferred reduction time is between 0.5 and 24 hours. A most preferred reduction time is 12 hours. A preferred liquid to solid ratio is from 5 to 100:1. A most preferred ratio is 20:1. Unlike the previously described oxidation reaction, reduction is carried out at basic pH. That being the case, keratins are highly soluble in the reduction media and are expected to be extracted. The reduction solution is therefore combined with the subsequent extraction solutions and processed accordingly.

Reduced keratins are not as hydrophilic as their oxidized counterparts. As such, reduced hair fibers will not swell and split open as will oxidized hair, resulting in relatively lower yields. Another factor affecting the kinetics of the reduction/extraction process is the relative solubility of kerateines. The relative solubility rankings in water is gamma-keratose>alpha-keratose>gamma-kerateine>alpha-kerateine from most to least soluble. Consequently, extraction yields from reduced hair fibers are not as high. This being the case, subsequent extractions are conducted with additional reductant plus denaturing agent solutions. Preferred solutions for subsequent extractions include TGA plus urea, TGA plus tris base, or TGA plus sodium hydroxide. After extraction, crude fractions of alpha- and gamma-kerateine can be isolated using the procedures described for keratoses. However, precipitates of gamma- and alpha-kerateine re-form their cystine crosslinks upon exposure to oxygen. Precipitates must therefore be re-dissolved quickly to avoid insolubility during the purification stages, or precipitated in the absence of oxygen.

Residual reductant and denaturing agents can be removed from solution by dialysis. Typical dialysis conditions are 1 to 2% solution of kerateines dialyzed against DI water for 24 to 72 hours. Those skilled in the art will recognize that other methods exist for the removal of low molecular weight contaminants in addition to dialysis (e.g. microfiltration, chromatography, and the like). The use of tris base is only required for initial solubilization of the kerateines. Once dissolved, the kerateines are stable in solution without the denaturing agent. Therefore, the denaturing agent can be removed without the resultant precipitation of kerateines, so long as the pH remains at or above neutrality. The final concentration of kerateines in these purified solutions can be adjusted by the addition/removal of water.

Regardless of the form of the keratin (i.e. keratoses or kerateines), several different approaches to further purification can be employed to keratin solutions. Care must be taken, however, to choose techniques that lend themselves to keratin's unique solubility characteristics. One of the most simple separation technologies is isoelectric precipitation. In this method, proteins of differing isoelectric point can be isolated by adjusting the pH of the solution and removing the precipitated material. In the case of keratins, both gamma- and alpha-forms are soluble at pH>6.0. As the pH falls below 6, however, alpha-keratins begin to precipitate. Keratin fractions can be isolated by stopping the precipitation at a given pH and separating the precipitate by centrifugation and/or filtration. At a pH of approximately 4.2, essentially all of the alpha-keratin will have been precipitated. These separate fractions can be re-dissolved in water at neutral pH, dialyzed, concentrated, and reduced to powders by lyophilization or spray drying. However, kerateine fractions must be stored in the absence of oxygen or in dilute solution to avoid crosslinking.

Another general method for separating keratins is by chromatography. Several types of chromatography can be employed to fractionate keratin solutions including size exclusion or gel filtration chromatography, affinity chromatography, isoelectric focusing, gel electrophoresis, ion exchange chromatography, and immunoaffinity chromatography. These techniques are well known in the art and are capable of separating compounds, including proteins, by the characteristics of molecular weight, chemical functionality, isoelectric point, charge, or interactions with specific antibodies, and can be used alone or in any combination to effect high degrees of separation and resulting purity.

A preferred purification method is ion exchange (IEx) chromatography. IEx chromatography is particularly suited to protein separation owning to the amphiphilic nature of proteins in general and keratins in particular. Depending on the starting pH of the solution, and the desired fraction slated for retention, either cationic or anionic IEx (CIEx or AIEx, respectively) techniques can be used. For example, at a pH of 6 and above, both gamma- and alpha-keratins are soluble and above their isoelectric points. As such, they are anionic and can be bound to an anionic exchange resin. However, it has been discovered that a sub-fraction of keratins does not bind to a weakly anionic exchange resin and instead passes through a column packed with such resin. A preferred solution for AIEx chromatography is pure keratin, isolated as described previously, in purified water at a concentration between 0 and 5 weight/volume %. A preferred concentration is between 0 and 4 w/v %. A most preferred concentration is approximately 2 w/v %. It is preferred to keep the ionic strength of said solution initially quite low to facilitate binding to the AIEx column. This is achieved by using a minimal amount of acid to titrate a purified water solution of the keratin to between pH 6 and 7. A most preferred pH is 6. This solution can be loaded onto an AIEx column such as DEAE-Sepharose.RTM. resin or Q-Sepharose.RTM. resin columns. A preferred column resin is DEAE-Sepharose.RTM. resin. The solution that passes through the column can be collected and further processed as described previously to isolate a fraction of acidic keratin powder.

In some embodiments the activity of the keratin matrix is enhanced by using an AIEx column to produce the keratin to thereby promote cell adhesion. Without wishing to be bound to any particular theory, it is envisioned that the fraction that passes through an anionic column, i.e. acidic keratin, promotes cell adhesion. In addition, nerve growth factor (NGF), thought to be present in hair extracts, has an isoelectric point of 9.5-10. That being the case, NGF would also flow through the column under the stated conditions. The resulting acidic fraction provides an optimized matrix for nerve regeneration because it is capable of stimulating cell attachment in general, and nerve growth in particular.

Another fraction binds readily, and can be washed off the column using salting techniques known in the art. A preferred elution medium is sodium chloride solution. A preferred concentration of sodium chloride is between 0.1 and 2M. A most preferred concentration is 2M. The pH of the solution is preferred to be between 6 and 12. A most preferred pH is 12. In order to maintain stable pH during the elution process, a buffer salt can be added. A preferred buffer salt is Trizma.RTM. base. Those skilled in the art will recognize that slight modifications to the salt concentration and pH can be made to affect the elution of keratin fractions with differing properties. It is also possible to use different salt concentrations and pH's in sequence, or employ the use of salt and/or pH gradients to produce different fractions. Regardless of the approach taken, however, the column eluent can be collected and further processed as described previously to isolate fractions of basic keratin powders.

A complimentary procedure is also feasible using CIEx techniques. Namely, the keratin solution can be added to a cation exchange resin such as SP Sepharose.RTM. resin (strongly cationic) or CM Sepharose.RTM. resin (weakly cationic), and the basic fraction collected with the pass through. The retained acid keratin fraction can be isolated by salting as previously described.

The formation of a matrix comprising keratin materials such as described above can be carried out in accordance with techniques long established in the field or variations thereof that will be apparent to those skilled in the art. In some embodiments, the keratin preparation is dried and rehydrated prior to use. See, e.g., U.S. Pat. No. 2,413,983 to Lustig et al., U.S. Pat. No. 2,236,921 to Schollkipf et al., and U.S. Pat. No. 3,464,825 to Anker. In preferred embodiments, the matrix, or hydrogel, is formed by re-hydration of the lyophilized material with a suitable solvent, such as water or phosphate buffered saline (PBS). The gel can be sterilized, e.g., by .gamma.-irradiation (800 krad) using a Co60 source. Other suitable methods of forming keratin matrices include, but are not limited to, those found in U.S. Pat. Nos. 6,270,793 (Van Dyke et al.), 6,274,155 (Van Dyke et al.), 6,316,598 (Van Dyke et al.), 6,461,628 (Blanchard et al.), 6,544,548 (Siller-Jackson et al.), and 7,01,987 (Van Dyke).

The matrix may optionally contain one or more active ingredients such as one or more growth factors (e.g., in an amount ranging from 0.0000001 to 1 or 5 percent by weight of the matrix composition) to facilitate nerve growth. Examples of suitable active ingredients include, but are not limited to, nerve growth factor, vascular endothelial growth factor, fibronectin, fibrin, laminin, acidic and basic fibroblast growth factors, testosterone, ganglioside GM-1, catalase, insulin-like growth factor-I (IGF-I), platelet-derived growth factor (PDGF), neuronal growth factor galectin-1, and combinations thereof. See, e.g., U.S. Pat. No. 6,506,727 to Hansson et al. and U.S. Pat. No. 6,890,531 to Horie et al.

As used herein, "growth factors" include molecules that promote the regeneration, growth and survival of nervous tissue. Growth factors that are used in some embodiments of the present invention may be those naturally found in keratin extracts, or may be in the form of an additive, added to the keratin extracts or formed keratin matrices. Examples of growth factors include, but are not limited to, nerve growth factor (NGF) and other neurotrophins, platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF-8), growth differentiation factor-9 (GDF9), basic fibroblast growth factor (bFGF or FGF2), epidermal growth factor (EGF), hepatocyte growth factor (HGF), granulocyte-colony stimulating factor (G-CSF), and granulocyte-macrophage colony stimulating factor (GM-CSF). There are many structurally and evolutionarily related proteins that make up large families of growth factors, and there are numerous growth factor families, e.g., the neurotrophins (NGF, BDNF, and NT3). The neurotrophins are a family of molecules that promote the growth and survival of nervous tissue. Examples of neurotrophins include, but are not limited to, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4 (NT-4). See U.S. Pat. Nos. 5,843,914 to Johnson, Jr. et al.; 5,488,099 to Persson et al.; 5,438,121 to Barde et al.; 5,235,043 to Collins et al.; and 6,005,081 to Burton et al.

For example, nerve growth factor (NGF) can be added to the keratin matrix composition in an amount effective to promote the regeneration, growth and survival of nervous tissue. The NGF is provided in concentrations ranging from 0.1 ng/mL to 1000 ng/mL. More preferably, NGF is provided in concentrations ranging from 1 ng/mL to 100 ng/mL, and most preferably 10 ng/mL to 100 ng/mL. See U.S. Pat. No. 6,063,757 to Urso.

Devices and Methods of Use.

As used herein, "support structure," "conduit," "scaffold," etc., is any suitable structure into which a damaged nerve may be placed, and can support or contain the keratin matrix material during nerve regeneration. In general, the structure is formed of a physiologically acceptable material. As shown in FIGS. 1A and 1B (see Original Patent), in some embodiments the support structure 10 has an elongate opening 11 formed therein. While FIGS. 1A and 1B show a conduit structure in the shape of a tube having a single longitudinal opening, any suitable shape, including square, hexagonal, triangular, etc., with any number of openings (such as fibrils as described below) may be used. Other examples of embodiments suitable to carry out the present invention will be apparent to those skilled in the art. For example, the support structure can be in the shape of a gutter, with or without an additional top piece. The gutter support structure may also have a top piece, placed in such a way as to "sandwich" the damaged nerve between the two pieces.

The material from which the support structure is formed can be bioabsorbable or inert (that is, non-bioabsorbable). Any bioabsorbable material may be used, including but not limited to natural materials such as collagen, laminin, alginate and combinations thereof, etc., as well as synthetic materials such as poly(lactide), poly(glycolide), poly(caproic acid), combinations thereof, etc. Materials may be polymeric or non-polymeric. Examples of suitable support structures include, but are not limited to, the artificial neural tubes described in U.S. Pat. Nos. 6,589,257 and 6,090,117 to Shimizu, the guide tubes described in U.S. Pat. No. 5,656,605 to Hansson et al., the tubular prostheses described in U.S. Pat. No. 4,662,884 to Stensaas, the elastomeric devices described in U.S. Pat. No. 5,468,253 to Bezwada et al., and the biopolymer rods with oriented fibrils (which fibrils then form a plurality of elongate openings or tubes containing the matrix described herein) as described in U.S. Pat. No. 6,461,629 to Tranquillo et al.

Other options for configuration of the support structure include having a longitudinal slit to facilitate the positioning of the structure around a damaged nerve, such as described in U.S. Pat. No. 4,662,884 to Stensaas. The interior wall portion of the support structure may optionally be patterned to facilitate or guide regeneration, as described in U.S. Pat. No. 6,676,675 to Mallapragada et al. The elongate opening may optionally contain guiding filaments dispersed within the matrix and extending along the logitudinal dimension of the support structure, as described in U.S. Pat. No. 5,656,605 to Hansson et al. The support structure may optionally include one, two or more electrodes connected to or otherwise operatively associated therewith to aid in applying an electric field to the nerve to facilitate regeneration.

The support structure may be packaged in sterile form in a sterile aseptic container. The sterile matrix composition may be provided in the support structure as packaged, in hydrated or dehydrated form (for subsequent hydration with a suitable solution such as sterile physiologically acceptable saline solution once opened for use), or the matrix packaged separately (in hydrated or dehydrated form, in a vial, syringe, or any other suitable container) for administration into the support structure before or during the time of use.

In some embodiments, the support structure is positioned around the damaged region of the nerve, and matrix is added as necessary. This may be carried out by any suitable technique, such as by opening the structure (e.g., along a longitudinal slit) and then enclosing it around the damaged portion of the nerve, by inserting each stump (proximal, distal) of a severed nerve into opposite ends of the support structure opening, etc. Sutures, surgical adhesives, staples, clasps, prongs formed on the inner surface of the support structure at each end thereof, or any other suitable technique may be used to secure the nerve in place. FIG. 1B shows a support structure embodiment having one or more fastening prongs 12 on the inner wall thereof at both end portions thereof to facilitate securing the structure onto the nerves, which prongs can be in any suitable shape (e.g., dimples, whiskers, pointed, blunt, etc.) formed by any suitable technique such as molding, microstamping, printing, lithography, crimping or partially punching, etc., depending upon the particular material from which the support structure is formed.

Surgical procedures can otherwise be carried out in accordance with known techniques, including but not limited to those described in U.S. Pat. Nos. 6,589,257 and 6,090,117 to Shimizu, U.S. Pat. No. 5,656,605 to Hansson et al., U.S. Pat. No. 4,662,884 to Stensaas, U.S. Pat. No. 5,468,253 to Bezwada et al., and U.S. Pat. No. 6,676,675 to Mallapragada et al.

Claim 1 of 29 Claims

1. A method for promoting the growth of a peripheral nerve in a mammal, comprising: encasing a damaged region of said nerve in a support structure having an elongate opening therein; and administering a physiologically acceptable matrix composition into said opening, said matrix composition comprising a soluble keratose, kerateine, or a mixture thereof, in an amount effective to promote the growth of said nerve at said damaged region.
 

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