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Title:  Composition and method for repairing neurological tissue

United States Patent:  6,241,981

Inventors:  Cobb; Mark A. (Newburgh, IN); Badylak; Stephen F. (West Lafayette, IN); Isom; Gary (West Lafayette, IN); Sharma; Archana (West Lafayette, IN)

Assignee:  Purdue Research Foundation (West Lafayette, IN)

Appl. No.:  242628

Filed:  February 19, 1999

PCT Filed:  September 16, 1997

PCT NO:  PCT/US97/16294

371 Date:  February 19, 1999

102(e) Date:  February 19, 1999

PCT PUB.NO.:  WO98/10775

PCT PUB. Date:  March 19, 1998

Abstract

The present invention relates to tissue graft constructs useful in promoting regrowth and healing of damaged or diseased nuerological related tissue structures. More particularly this invention is directed to a submucosa tissue graft construct and a method of inducing the formation of endogenous neurological structures at a site in need of endogenous neurological related tissue growth.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The submucosal tissue constructs of the present invention have been found to promote or induce the growth of neurological related tissues. In accordance with the present invention the term neurological related tissues includes neurons and glial cells, and dura mater, arachnoid and pia mater tissues. There is provided in accordance with this invention a method for utilizing compositions comprising warm-blooded vertebrate submucosal tissue to repair or to enhance the repair of damaged or diseased neurological related tissues in a warm-blooded vertebrate. The method comprises the step of contacting the site in need of repair with a composition comprising submucosal tissue.

Submucosal tissue suitable for use in accordance with the present invention comprises natural collagenous matrices that include highly conserved collagens, glycoproteins, proteoglycans, and glycosaminoglycans in their natural configuration and natural concentration. One source of submucosal tissue is the intestinal tissue of a warm-blooded vertebrate. Small intestinal tissue is a preferred source of submucosal tissue for use in this invention.

Submucosal tissue for use in this invention is derived from various warm-blooded vertebrate sources, including intestinal tissue harvested from animals raised for meat production, such as pigs, cattle and sheep or other warm-blooded vertebrates. This tissue can be used in either its natural configuration or in a comminuted or partially enzymatically digested fluidized form. Vertebrate submucosal tissue is a plentiful by-product of commercial meat production operations and is thus a low cost graft material, especially when the submucosal tissue is used in its native layer sheet configuration.

Suitable intestinal submucosal tissue typically comprises the tunica submucosa delaminated from both the tunica muscularis and at least the luminal portion of the tunica mucosa. In one embodiment of the present invention the intestinal submucosal tissue comprises the tunica submucosa and basilar portions of the tunica mucosa including the lamina muscularis mucosa and the stratum compactum. Those layers are known to vary in thickness and in definition dependent on the source vertebrate species.

The preparation of submucosal tissue for use in accordance with this invention is described in U.S. Pat. No. 4,902,508, the disclosure of which is expressly incorporated herein by reference. A segment of vertebrate intestine, preferably harvested from porcine, ovine or bovine species, but not excluding other species, is subjected to abrasion using a longitudinal wiping motion to remove the outer layers, comprising smooth muscle tissues, and the innermost layer, i.e., the luminal portion of the tunica mucosa. The submucosal tissue is rinsed with saline and optionally sterilized; it can be stored in a hydrated or dehydrated state. Lyophilized or air dried submucosa tissue can be rehydrated and used in accordance with this invention without significant loss of its biotropic and mechanical properties.

Submucosal tissue prepared from warm-blooded vertebrate organs typically has an abluminal and a luminal surface. The luminal surface is the submucosal surface facing the lumen of the organ source and typically adjacent to an inner mucosa layer in the organ source, whereas the abluminal surface is the submucosal surface facing away from the lumen of the organ source and typically in contact with smooth muscle tissue in the organ source.

The submucosal tissue graft compositions of the present invention can be preconditioned by stretching the material in a longitudinal or lateral direction as described in U.S. Pat. No. 5,275,826, the disclosure of which is expressly incorporated herein by reference. Multiple strips/pieces of submucosal tissue can also be fused together to form a unitary multi-layered submucosal tissue construct having a surface area greater than any to the individual strips/pieces of submucosal tissue. The process for forming large area/multilayered submucosal tissue constructs is described in U.S. patent application Ser. No. 08/418,515, the disclosure of which is expressly incorporated herein by reference. In summary, the process of forming large area sheets of submucosal tissue comprises overlapping at least a portion of one strip of submucosal tissue with at least a portion of another strip of submucosal tissue and applying pressure at least to said overlapped portions under conditions allowing dehydration of the submucosal tissue. Under these conditions the overlapped portions will become "fused" to form a unitary large sheet of tissue.

The large area graft constructs consist essentially of submucosal tissue, free of potentially compromising adhesives and chemical pretreatments, and they have a greater surface area and greater mechanical strength than the individual strips used to form the graft construct. The multilayered submucosal constructs can optionally be perforated as described in U.S. patent application Ser. No. 08/418,515, the disclosure of which is expressly incorporated herein by reference. The perforations of the submucosal tissue construct allow extracellular fluids to pass through the tissue graft material, decreasing fluid retention within the graft and enhancing the remodeling properties of the tissue grafts. The perforation of the submucosal tissue is especially beneficial for multi-laminate tissue graft constructs wherein the perforations also enhance the adhesive force between adjacent layers.

The submucosal tissue specified for use in accordance with this invention can also be in a fluidized form. Submucosal tissue can be fluidized by comminuting the tissue and optionally subjecting it to enzymatic digestion to form a substantially homogenous solution. The preparation of fluidized forms of submucosa tissue is described in U.S. Pat. No. 5,275,826, the disclosure of which is expressly incorporated herein by reference. Fluidized forms of submucosal tissue are prepared by comminuting submucosa tissue by tearing, cutting, grinding, or shearing the harvested submucosal tissue. Thus pieces of submucosal tissue can be comminuted by shearing in a high speed blender, or by grinding the submucosa in a frozen or freeze-dried state to produce a powder that can thereafter be hydrated with water or a buffered saline solution to form a submucosal fluid of liquid, gel-like or paste-like consistency. The fluidized submucosa formulation can further be treated with enzymes such as protease, including trypsin or pepsin at an acidic pH, for a period of time sufficient to solubilize all or a major portion of the submucosal tissue components and optionally filtered to provide a homogenous solution of partially solubilized submucosa.

The graft compositions of the present invention can be sterilized using conventional disinfection/sterilization techniques including glutaraldehyde tanning, formaldehyde tanning at acidic pH, propylene oxide treatment, ethylene oxide treatment, gas plasma sterilization, gamma radiation or electron beam treatment, and peracetic acid (PAA) disinfection. Sterilization techniques which do not adversely affect the mechanical strength, structure, and biotropic properties of the submucosal tissue are preferred. For instance, strong gamma radiation may cause loss of strength of the sheets of submucosal tissue. Preferred sterilization techniques include exposing the graft to peracetic acid, 1-4 Mrads gamma irradiation (more preferably 1-2.5 Mrads of gamma irradiation) or gas plasma sterilization. Typically, the submucosal tissue is subjected to two or more sterilization processes. After the submucosal tissue is treated in an initial disinfection step, for example by treatment with peracetic acid, the tissue may be wrapped in a plastic or foil wrap and sterilized again using electron beam or gamma irradiation sterilization techniques.

In accordance with one embodiment submucosal tissue is used as a tissue graft construct for the replacement or repair of damaged or diseased neurological related tissues. In particular the present submucosal tissue constructs have been found to promote the growth and proliferation of neurons. Accordingly, the present compositions can be used in a method of repairing damaged or diseased neurological related tissues in a warm-blooded vertebrate.

The submucosal tissue construct used in accordance with the present invention comprises intestinal submucosal tissue delaminated from both the tunica muscularis and at least the luminal portion of the tunica mucosa of warm-blooded vertebrate intestine, or a digest thereof. The construct can be combined with an added growth factor such as vascular endothelial growth factor, nerve growth factor or fibroblast growth factor or growth factor containing extracts of submucosal tissue. Alternatively, the tissue graft construct can comprise submucosal tissue in combination with peripheral neuronal tissue and optionally added growth factors.

In one embodiment, solid forms of submucosal tissue are combined with one or more growth factors by soaking the tissue in a buffered solution containing the growth factor. For example the submucosal tissue is soaked for 7-14 days at 4oC. in a PBS buffered solution containing about 5 to about 500 mg/ml, or more preferably 25 to about 100 mg/ml of the growth factor. Submucosal tissue readily bonds to proteins and will retain an association with a bioactive agent for several days. However, to enhance the uptake of the growth factors into the submucosal tissue, the tissue can be partially dehydrated before contacting the growth factor solution. For compositions comprising fluidized, solubilized or guanidine extracts of submucosal tissue, lyophilized powder or solutions of growth factors can be directly mixed with the submucosal tissue. For example, fluidized or solubilized submucosal tissue can be mixed with a growth factor and then packed within a tube of submucosal tissue (or other biodegradable tissue). The open end of the tube is sealed shut after filling the tube with the fluidized or solubilized submucosal tissue.

In accordance with the present invention, submucosal tissue of a warm-blooded vertebrate is used to manufacture a tissue graft construct useful for inducing the repair of neurological tissue in a warm-blooded vertebrate. The manufacture comprises the steps of combining submucosal tissue of a warm-blooded vertebrate, or a digest thereof, with an added growth factor selected from the group consisting of vascular endothelial growth factor, nerve growth factor and fibroblast growth factor.

In one embodiment the submucosal tissue is used to manufacture a graft construct that directs the in vivo growth of neurons along a predetermined path. The manufacture comprises the steps of forming a tube of biodegradable material, and filling the tube with fluidized submucosal tissue. The tube should be formed to have a diameter about 0.5 mm to about 4 mm for peripheral nerve applications, and about 1 mm to about 2 cm for central nerve applications. In one embodiment the tube is formed from submucosal tissue wherein the submucosal tissue is manipulated to define a cylinder having a diameter of the preferred size. Typically the submucosal tissue is prepared directly from intestinal tissue and thus has a generally cylindrical shape. The tissue can be manipulated to define a cylinder having the preferred diameter by suturing or otherwise securing the graft longitudinally and removing the excess tissue. For example, the graft construct can be prepared by selecting a sterile glass rod having an outer diameter equal to the desired diameter of the lumen of the formed graft construct. The glass rod is introduced into the graft lumen, redundant tissues is then gathered, and the desired lumen diameter is achieved by suturing along the length of the graft or by using other art recognized tissue securing techniques.

Alternatively, a tube of submucosal tissue can be formed by wrapping strips of submucosal tissue onto a mandrel wherein the wrapped submucosal tissue is overlapped leaving no portion of the underlying mandrel exposed. See U.S. Provisional Application Ser. No. 60/032,679, the disclosure of which is expressly incorporated herein. The submucosal tissue can be spirally wrapped onto a mandrel as a continuous piece of submucosal tissue, and multiple strips of submucosal tissue can be used to form the tubular constructs. The wrapped submucosal tissue is then compressed under dehydrating conditions and the tubular prosthesis is removed from the mandrel. The amount of overlap in a spirally wrapped construct in accordance with this embodiment ranges between 10 to 60% of the width of the previous strip and more preferably the overlapped portion is a 50% overlap.

Upon formation of the biodegradable tube, the tube is filled with fluidized or solubilized submucosal tissue and the tube is sealed at one or both ends of the tube using art recognized methods (including clamping, suturing, binding pastes, and compression under dehydrating conditions). Alternatively the tube can be sealed at one or both ends of the tube before being filled with fluidized/solubilized tissue. The tube can then be filled by injecting fluidized/solubilized tissue into the lumen through the use of a syringe.

The submucosal tissue graft constructs of the present invention are used to repair neurological related tissues and more particularly components of the central and peripheral nervous system. The method comprises contacting the site in need of repair with a composition comprising intestinal submucosal tissue delaminated from both the tunica muscularis and at least the luminal portion of the tunica mucosa of warm-blooded vertebrate intestine. The submucosal tissue can be used, for example, in sheet, strip, braid or loop form and surgically implanted at the site in need of repair. The submucosal tissue composition can also be administered in a fluidized form and injected into the warm-blooded vertebrate at the site in need of repair. Finally the composition may comprise fluidized submucosal tissue filled cylinder of submucosal tissue.

In one embodiment in accordance with the present invention, the submucosal tissue constructs are used to induce the formation of neurological related tissue between endogenous neurological tissue structures in a warm blooded vertebrate. The method comprises the steps of surgically implanting a tissue graft composition comprising submucosal tissue of a warm-blooded vertebrate, into the host to bridge the endogenous neurological tissue structures and induce endogenous neurological related tissue growth between the bridged neurological structures.

When submucosal tissue comprising the tunica submucosa and basilar portions of the tunica mucosa including the lamina muscularis mucosa and the stratum compactum, is used in non-fluidized form, it is preferred that it be implanted so that the stratum compactum contacts the surface tissues most prone to forming adhesions with the graft material.

In accordance with one embodiment damage to the spinal cord can be repaired by manually separating adjacent longitudinal neural fibers in the spinal cord, wherein the separations or incisions run parallel to the axons of the neurons of the spinal column and penetrate through the dura mater, arachnoid and pia mater layers. Strips of submucosal tissue are surgically implanted into the vertical separations or in natural sulci and thus are in direct contact with neurological related tissues and are held in place by those tissues. Alternatively, sheets of submucosa tissue can be used to wrap the exterior of the damaged area to promote repair of the damaged tissues. Optionally sutures can be utilized to secure the submucosal tissue in its desired location.

In applications where the spinal cord has been transected, the submucosal tissue can be positioned between the two severed ends to bridge the gap and serve as a framework that directs the growth of neurons of the two severed ends towards one another. Fluidized forms of submucosa can also be used in accordance with the present invention to repair damaged or diseased neurological related tissues. Advantageously the fluidized forms can be injected into the site in need of repair and thus can be used in a less invasive procedure to induce the proliferation of endogenous neurological related tissues.

In accordance with one embodiment a tissue graft composition, comprising submucosal tissue of a warm-blooded vertebrate, is administered to a warm-blooded vertebrate at a site in need of endogenous neurological related tissue growth in an amount effective to induce endogenous neurological related tissue growth at the site the composition is administered. The biotropic properties of submucosal tissue promote the growth of neurological tissue along "tracts" as defined by the path of the implanted submucosal tissue. Accordingly the growth of neurons, including neurons of central and peripheral nervous system, can be directed to a site in need of innervation. In one embodiment the growth of neurological tissue is "directed" through the use of a tissue graft construct comprising a tube filled with fluidized submucosal tissue. In this embodiment healthy neuronal tissue is inserted into one end of the tube and is placed in direct contact with the fluidized submucosal tissue contained within the tube. The opposite end of the tube is then placed at or near the site in need of innervation. The tube is fixed in place and provides an in vivo conduit for new neuronal growth and innervation of the desired site. The tube of fluidized submucosal tissue can also be utilized to repair transected nerves, wherein the two ends of the transected nerve are inserted into the tube to induce reattachment of the severed ends and restore nerve function.

Each of the following methods can be used in conjunction with the tissue graft constructs of the present invention to provide a conduit for directed de novo growth of neuronal tissues. In one embodiment submucosal tissue is prepared in the shape of a tube having a lumen and two open ends. In one embodiment, the tube of submucosal tissue is directly implanted into the host organism and the end of a damaged nerve fiber can be inserted into the lumen of the submucosal tissue tube. A syringe is then used to fill the tube with comminuted or solubilized submucosal tissue. Alternatively, submucosal tissue can be formed in the shape of a tube, filled with fluidized/solubilized submucosal tissue and sealed at each end. The sealed tube of submucosal tissue can then be stored until needed. In one embodiment the sealed tube of submucosal tissue is inserted into the host organism and fixed in place using techniques known to those skilled in the art. The inserted graft construct provides a conduit for new neuronal tissue growth. In one preferred embodiment, a slit or hole is cut into the tube of submucosal tissue and a damaged or resected end of a nerve tissue is inserted through the slit or hole and into the lumen of the tube.

The submucosal tissue used in accordance with the present invention can be used alone or combined with added growth factors such as vascular endothelial growth factor, nerve growth factor or acidic fibroblast growth factor. In addition peripheral nerve implants can be used in combination with submucosal tissue to enhance the repair of neuronal tissues. The term peripheral nerve implant as used herein refers to neuronal tissue harvested from the peripheral nervous system of a warm blooded vertebrate, and preferably autologous peripheral neuronal tissue. Additional components can be added to the neuronal tissue graft compositions that provide the compositions with structural support for applications involving the spinal cord, especially where portions of the spinal column are missing or need to be replaced. For example hydroxyapatite and/or other biocompatible calcium containing minerals can be combined with the graft composition, or metal posts or wires can also be used in combination with the submucosal tissue to give additional structural support for the replacement tissue.

The submucosal tissue graft constructs of the present invention can also be utilized to promote the growth and proliferation of other central nervous-associated and support tissues. Submucosal tissue enhances the repair of glial cells, and dura mater, arachnoid and pia mater tissues.

In one embodiment the submucosal tissue is used as a dural substitute, formed as a patch tissue graft shaped to cover a defect or hole formed in the endogenous dura mater. Presently available options for dura mater substitute materials have significant limitations: autologous materials are frequently inadequate in quantity and are obtained with the associated morbidity of additional incisions, and the handling characteristics of synthetic sheets are poor compared to biological materials. In addition, concern has been raised regarding long term risks of hemorrhage from tissue reaction to synthetic graft materials. Cadaveric dura is expensive, occasionally limited in supply, and has only fair handling characteristics. Of greater concern is its documented role as a vector in the transmission of the slow viruses such as Jakob-Creutzfeldt disease.

Submucosal tissue provides an excellent dural substitute since this material does not invoke an adverse immunologic response and induces proliferation of endogenous cells which invade and ultimately replace the graft with endogenous cells. An experiment was conducted utilizing rat hosts to confirm the utility of submucosal tissue compositions as dural substitutes.

Claim 1 of 23 Claims

What is claimed is:

1. A tissue graft construct for promoting the repair of damaged or diseased neurological related tissues in a warm-blooded vertebrate, said construct comprising intestinal submucosal tissue delaminated from both the tunica muscularis and at least the luminal portion of the tunica mucosa of warm-blooded vertebrate intestine, or a digest thereof and an added growth factor.

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