WO2010088678A2

WO2010088678A2 - Medical bead products

Info

Publication number: WO2010088678A2
Application number: PCT/US2010/022910
Authority: WO
Inventors: Umesh H. Patel; Adam Yestrepsky
Original assignee: Cook Biotech Incorporated
Priority date: 2009-02-02
Filing date: 2010-02-02
Publication date: 2010-08-05
Also published as: WO2010088678A3

Abstract

Described, in certain aspects, are medical bead products including an extracellular matrix (ECM) bead comprised of reconstituted biotropic ECM material. Such an ECM bead can have a generally homogeneous network of self- assembled collagen fibers, and comprise at least one bioactive agent retained in the ECM material, wherein the bioactive agent is selected from the group consisting of a proteoglycan, a growth factor, a glycoprotein, and a glycosaminoglycan. In some forms, the ECM bead further comprises at least one additional bioactive agent disposed thereon, for example, a pharmaceutical such as an antineoplastic agent. Preferably, the ECM material comprises submucosa, for example, porcine small intestinal submucosa (SIS).

Description

MEDICAL BEAD PRODUCTS

BACKGROUND

The present invention relates generally to medical technology, and in certain aspects, to bead-shaped medical products.

As further background, materials and other products containing collagen are widely used in medicine, for example, to replace, repair, augment, and/or otherwise treat wounded, diseased or otherwise damaged or defective tissue.

Suitable collagenous materials can be provided by collagenous extracellular matrix (ECM) materials. Such ECM materials can be provided, for example, by materials isolated from a suitable tissue source from a warm-blooded vertebrate, e.g., from the submucosal tissue of a mammal. Such isolated submucosal tissue, for example, small intestinal submucosa (SIS), can be processed so as to have bioremodelable properties and promote cellular invasion and ingrowth. As just one example, sheet-form SIS material has been suggested and used as a surgical graft for tissue support, for example, in hernia repair. Portions or all of the graft may include a multiple layer configuration to provide strength and/or reinforcement.

There remain needs for improved and/or alternative medical products and methods for manufacturing and using the same. The present invention is addressed to those needs.

SUMMARY

The present invention provides, in certain aspects, collagenous medical bead products having biotropic properties. Illustratively, a medical bead product of the invention comprises an extracellular matrix (ECM) bead comprised of reconstituted biotropic ECM material. Such an ECM bead has a generally homogeneous network of self-assembled collagen fibers, and comprises at least one bioactive agent retained in the ECM material, wherein the bioactive agent is selected from the group consisting of a proteoglycan, a growth factor, a glycoprotein, and a glycosaminoglycan. In certain aspects, at least one additional bioactive agent, for example, a pharmaceutical such as an antineoplastic agent, is disposed on (e.g., embedded within or otherwise incorporated into) the ECM bead. Preferably, the ECM material comprises submucosa, for example, porcine small intestinal submucosa (SIS).

In one particular embodiment, the invention provides a method of forming a medical bead product such as that described above. This method comprises providing a starting material, wherein the starting material includes solubilized ECM material including at least one retained bioactive agent selected from the group consisting of a proteoglycan, a growth factor, a glycoprotein, and a glycosaminoglycan. This method further comprises reconstituting the solubilized ECM material to form an ECM bead having a generally homogeneous network of self-assembled collagen fibers, and entraining the at least one bioactive agent. In certain aspects, such a reconstitution step includes introducing the starting material into a liquid medium, for example, into a buffered aqueous medium. For example, a droplet of solubilized ECM gel can be injected into a buffer bath to form a medical bead product in accordance with the present invention. Further, this method can comprise incorporating at least one additional bioactive agent into the ECM material before, during and/or after a reconstitution step. Illustratively, an exogenous growth factor can be added to the starting material before the reconstituted bead is formed, or alternatively, can be disposed on the reconstituted ECM bead after it is formed.

In another embodiment, the invention provides a method of treating a patient that includes grafting a patient with one or more medical bead products such as those described above. For example, medical bead products of the invention can be adapted to serve as substrates and/or scaffolds in the delivery of drugs, etc.. Illustratively, a particulate product (e.g., a particulate gel product) including a plurality of ECM beads coated with, impregnated with, or otherwise incorporating a chemotherapeutic agent can be implanted within the body to treat a tumor. In certain aspects, inventive medical bead products are implanted within the vascular system, e.g., within a vascular vessel, as an embolization device or aneurysm filling device as a method of treating an aneurysm.

Another embodiment of the present invention provides a medical kit comprising an ECM particulate product enclosed within a sealed package. The particulate product includes a plurality of ECM beads such as those described above, and can be provided in a hydrated, partially hydrated, or dehydrated state. In certain aspects, the medical kit includes suitable instrumentation for introducing the particulate product into the body of a patient, for example, a needle and syringe to inject a gel or otherwise flowable form of the product. The sealed package can be configured to maintain the ECM particulate product in a sterile condition when sterilely packaged therein, and can include indicia to communicate the contents of the package.

Yet another embodiment of the invention provides a method of forming a medical bead product. This particular method comprises the steps of: providing solubilized submucosal material including a proteoglycan, a glycosaminoglycan, and a growth factor; and subjecting the solubilized submucosal material to polymerization conditions to form a submucosa bead having a generally homogeneous network of self-assembled collagen fibers, wherein the submucosa bead retains the proteoglycan, the glycosaminoglycan, and the growth factor therein.

In another embodiment, the invention provides a medical bead product that includes an ECM bead comprised of reconstituted biotropic submucosa material, the ECM bead having a generally homogeneous network of self-assembled collagen fibers, and comprising at least one bioactive agent retained in the ECM material. The bioactive agent is selected from the group consisting of a proteoglycan, a growth factor, a glycoprotein, and a glycosaminoglycan.

Another embodiment of the invention provides a method of forming a medical bead product. This method comprises the steps of: (i) providing a starting material, wherein the starting material is comprised of a flowable extracellular matrix material including at least one retained bioactive agent, the at least one bioactive agent being selected from the group consisting of a proteoglycan, a growth factor, a glycoprotein, and a glycosaminoglycan; (ii) forming droplets comprised of the flowable extracellular matrix material; and (iii) subjecting the droplets to conditions effective to form solidified extracellular matrix beads entraining the at least one bioactive agent.

Other objects, embodiments, forms, features, advantages, aspects, and benefits of the present invention shall become apparent from the detailed description and drawings included herein. DETAILED DESCRIPTION

While the present invention may be embodied in many different forms, for the purpose of promoting an understanding of the principles of the present invention, reference will now be made to the embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modifications in the described embodiments and any further applications of the principles of the present invention as described herein are contemplated as would normally occur to one skilled in the art to which the invention relates.

As disclosed above, in certain aspects, the present invention provides unique and medically useful bead products. For example, a preferred bead product of the invention comprises an extracellular matrix (ECM) bead comprised of reconstituted remodelable, angiogenic ECM material. Such an ECM bead has a generally homogeneous network of self-assembled collagen fibers, and comprises at least one bioactive agent retained in the ECM material, wherein the bioactive agent is selected from the group consisting of a proteoglycan, a growth factor, a glycoprotein, and a glycosaminoglycan. In certain embodiments, the ECM bead further comprises at least one additional bioactive agent disposed thereon, for example, a chemotherapeutic agent. The invention also provides grafting methods utilizing such medical bead products. Particularly advantageous methods involve the use of such medical bead products to treat a tumor or damaged tissue (e.g., an aneurysm). The invention also provides methods of manufacturing such medical bead products and medical kits that include such medical bead products enclosed within sterile packaging.

Medical bead products of the invention should generally be biocompatible, and in advantageous embodiments of the invention, the bead products are comprised of a remodelable material. Particular advantage can be provided by medical bead products including a remodelable collagenous material. Such remodelable collagenous materials, whether reconstituted or naturally-derived, can be provided, for example, by collagenous materials isolated from a warm-blooded vertebrate, for example, a mammal such as a pig or a human. Such isolated collagenous material can be processed so as to have remodelable, angiogenic properties and promote cellular invasion and ingrowth. Remodelable materials may be used in this context to promote cellular growth within sites in which medical bead products of the invention are implanted or engrafted.

Suitable remodelable materials can be provided by collagenous extracellular matrix (ECM) materials possessing biotropic properties. For example, suitable collagenous materials include ECM materials such as those comprising submucosa, renal capsule membrane, dermal collagen, dura mater, pericardium, fascia lata, serosa, peritoneum or basement membrane layers, including liver basement membrane. Suitable submucosa materials for these purposes include, for instance, intestinal submucosa including small intestinal submucosa, stomach submucosa, urinary bladder submucosa, and uterine submucosa. Collagenous matrices comprising submucosa (potentially along with other associated tissues) useful in the present invention can be obtained by harvesting such tissue sources and delaminating the submucosa-containing matrix from smooth muscle layers, mucosal layers, and/or other layers occurring in the tissue source. For additional information as to some of the materials useful in the present invention, and their isolation and treatment, reference can be made, for example, to U.S. Patent Nos. 4,902,508, 5,554,389, 5,993,844, 6,206,931, and 6,099,567.

Submucosa-containing or other ECM tissue used in the invention is preferably highly purified, for example, as described in U.S. Patent No. 6,206,931 to Cook et al. Thus, preferred ECM material will exhibit an endotoxin level of less than about 12 endotoxin units (EU) per gram, more preferably less than about 5 EU per gram, and most preferably less than about 1 EU per gram. As additional preferences, the submucosa or other ECM material may have a bioburden of less than about 1 colony forming units (CFU) per gram, more preferably less than about 0.5 CFU per gram. Fungus levels are desirably similarly low, for example less than about 1 CFU per gram, more preferably less than about 0.5 CFU per gram. Nucleic acid levels are preferably less than about 5 μg/mg, more preferably less than about 2 μg/mg, and virus levels are preferably less than about 50 plaque forming units (PFU) per gram, more preferably less than about 5 PFU per gram. These and additional properties of submucosa or other ECM tissue taught in U.S. Patent No. 6,206,931 may be characteristic of any ECM tissue used in the present invention.

A typical layer thickness for an as-isolated submucosa or other ECM tissue layer, in instances where such a material layer is utilized in the invention, ranges from about 50 to about 250 microns when fully hydrated, more typically from about 50 to about 200 microns when fully hydrated. These layer thicknesses may vary with the type and age of the animal used as the tissue source. As well, these layer thicknesses may vary with the source of the tissue obtained from the animal source.

Submucosa or other ECM materials of the present invention can be derived from any suitable organ or other tissue source, usually sources containing connective tissues. The ECM materials processed for use in the invention will typically include abundant collagen, most commonly being constituted at least about 80% by weight collagen on a dry weight basis. Such naturally-derived ECM materials will for the most part include collagen fibers that are non-randomly oriented, for instance occurring as generally uniaxial or multi- axial but regularly oriented fibers. When processed to retain native bioactive factors, the ECM material can retain these factors interspersed as solids between, upon and/or within the collagen fibers. Particularly desirable naturally-derived ECM materials for use in the invention will include significant amounts of such interspersed, non- collagenous solids that are readily ascertainable under light microscopic examination with appropriate staining. Such non-collagenous solids can constitute a significant percentage of the dry weight of the ECM material in certain inventive embodiments, for example at least about 1%, at least about 3%, and at least about 5% by weight in various embodiments of the invention.

The submucosa or other ECM material used in the present invention may also exhibit an angiogenic character and thus be effective to induce angiogenesis in a host engrafted with the material. In this regard, angiogenesis is the process through which the body makes new blood vessels to generate increased blood supply to tissues. Thus, angiogenic materials, when contacted with host tissues, promote or encourage the formation of new blood vessels into the materials. Methods for measuring in vivo angiogenesis in response to biomaterial implantation have recently been developed. For example, one such method uses a subcutaneous implant model to determine the angiogenic character of a material. See, C. Heeschen et al., Nature Medicine 7 (2001), No. 7, 833-839. When combined with a fluorescence microangiography technique, this model can provide both quantitative and qualitative measures of angiogenesis into biomaterials. C. Johnson et al., Circulation Research 94 (2004), No. 2, 262-268.

ECM bead products of the invention may include one or more bioactive agents native to the source of the ECM tissue. For example, a submucosa or other remodelable ECM tissue material useful in some forms of the invention may retain one or more growth factors such as but not limited to basic fibroblast growth factor (FGF- 2), transforming growth factor beta (TGF-beta), epidermal growth factor (EGF), cartilage derived growth factor (CDGF) and/or platelet derived growth factor (PDGF). As well, submucosa or other ECM materials when used in the invention may retain other native bioactive components such as but not limited to proteins, glycoproteins, proteoglycans, and glycosaminoglycans. For example, an ECM material may retain heparin, heparin sulfate, hyaluronic acid, fibronectin, cytokines, and/or the like. Thus, generally speaking, a submucosa or other ECM material may retain one or more bioactive components that induce, directly or indirectly, a cellular response such as a change in cell morphology, proliferation, growth, protein or gene expression.

Further, in addition or as an alternative to the inclusion of such native bioactive components, non-native bioactive components such as those synthetically produced by recombinant technology or other methods (e.g., genetic material such as DNA), may be incorporated into an ECM material before, during, and/or after a bead formation step. Bioactive agents, when present in an ECM bead of the invention, may or may not be generally homogeneously dispersed in the bead matrix. Further, non-native bioactive components may be naturally-derived or recombinantly produced proteins that correspond to those natively occurring in an ECM tissue, but perhaps of a different species (e.g., human proteins applied to collagenous ECMs from other animals, such as pigs). Suitable non-native bioactive components useful in some forms of the invention may include one or more physiological compatible minerals, enzymes, genetic materials, hormones, and/or drug substances, just to name a few. Illustrative drug substances that may be incorporated into the ECM material include, for example, anti-clotting agents, e.g. heparin, antibiotics, anti-inflammatory agents, and anti-proliferative agents, e.g. taxol derivatives such as paclitaxel. In a particularly preferred embodiment, doxorubicin is disposed on the ECM material, although any suitable antineoplastic agent may be used in addition or as an alternative to doxorubicin. Such non-native bioactive components can be incorporated into and/or onto the ECM material in any suitable manner. For example, in some forms, a bioactive agent is mixed with a suitable flowable ECM starting material before a bead product is formed, and thus is incorporated into the formed bead. In other forms, a bioactive agent is incorporated into an already-formed bead in a suitable manner such as but not limited to by surface treatment (e.g., spraying) and/or impregnation (e.g., soaking).

Medical bead products of the invention can include xenograft material (i.e., cross-species material, such as tissue material from a non-human donor to a human recipient), allograft material (i.e., interspecies material, with tissue material from a donor of the same species as the recipient), and/or autograft material (i.e., where the donor and the recipient are the same individual). For example, in certain aspects of the invention, an implantable medical bead product includes ECM material, wherein the ECM material is xenogenic relative to the patient receiving the implant, and any added exogenous material(s) are from the same species (e.g. autologous or allogenic) as the patient receiving the implant. Illustratively, human patients may be treated with products including an xenogenic ECM material (e.g. porcine-, bovine- or ovine-derived material) that has been modified with exogenous human material(s) as described herein, those exogenous materials being naturally derived and/or recombinantly produced.

Certain method embodiments of the invention include reconstituting or otherwise re-assembling a suitable ECM starting material to form a medically useful bead product. One such method includes providing a starting material including solubilized submucosa, e.g., porcine small intestinal submucosa (SIS), and reconstituting this solubilized submucosa to form an ECM bead product. This ECM bead product has a generally homogenous network of self-assembled collagen fibers, and comprises at least one bioactive agent retained in the ECM material. In this context, "ECM bead" is meant to include any round or generally round ECM product including those with surface irregularities or deformations. Inventive medical bead products come in a variety of sizes, with bead diameters typically ranging from about 10 microns to about 600 microns, more typically from about 50 microns to about 500 microns. As well, bead products of the invention can be somewhat porous or essentially non-porous, and when porous, can exhibit varying degrees of porosity as desired for a particular application.

Suitable medical bead starting materials come in many different forms. In certain preferred aspects, such starting materials include flowable or otherwise conformable collagenous ECM materials that are at least partially solubilized or otherwise denatured or disassembled relative to their native collagenous structures. Illustratively, a suitable conformable ECM material may comprise an ECM material paste, a fluidized ECM material, and/or gelatinous ECM material. In some forms, an ECM graft material comprises a flowable composition comprising solubilized or suspended ECM material such as an ECM hydrolysate material.

Suitable flowable, remodelable ECM materials for use in this aspect of the invention can be prepared, for example, as described in U.S. Patent Nos. 5,275,826, 5,516,533, 6,206,931, and/or 6,444,229 or in International Publication No. WO2005020847 (Cook Biotech Incorporated) published March 10, 2005, which are each hereby incorporated by reference in their entirety. In this regard, a flowable ECM composition can be prepared from an isolated ECM material, for example, one of those listed above. Such an ECM material can be used to prepare a solubilized mixture including components of the material. This can be achieved by digestion of the ECM material in an acidic or basic medium and/or by contact with an appropriate enzyme or combination of enzymes.

For example, in certain embodiments, forming a suitable starting material includes first reducing ECM material to particulate form to aid in a digestion step. This can be achieved by tearing, cutting, grinding or shearing the isolated ECM material. Illustratively, shearing may be conducted in a fluid medium, and grinding may be conducted with the material in a frozen state. For example, the material can be contacted with liquid nitrogen to freeze it for purposes of facilitating grinding into powder form. Such techniques can involve freezing and pulverizing submucosa under liquid nitrogen in an industrial blender.

Such reduced ECM materials can be subjected to digestion using any suitable enzyme in an enzymatic digestion step. Suitable enzymes include, for example, serine proteases, aspartyl proteases, and matrix me tallopro teases. The concentration of the enzyme can be adjusted based on a number of factors including but not limited to the specific enzyme used, the amount of ECM material to be digested, the duration of the digestion, the temperature of the reaction, and the desired properties of the solubilized ECM material. In an illustrative embodiment, about 0.1% to about 0.2% of enzyme (pepsin, for example) can be used and the digestion can be conducted under cooled conditions for a period of time sufficient to substantially digest the ECM material. The digestion can be conducted at any suitable temperature, with temperatures ranging from 4° to 37° C being preferred. Likewise, any suitable duration of digestion can be used, such durations typically falling in the range of about 2 to 180 hours. The ratio of the concentration of ECM material (hydrated) to total enzyme usually ranges from about 25 to about 125 and more typically the ratio is about 50, and the digestion is conducted at approximately 4° C for approximately 24-72 hours. When an enzyme is used to aid in the digestion, the digestion will be performed at a pH at which the enzyme is active and more advantageously at a pH at which the enzyme is optimally active. Illustratively, pepsin exhibits optimal activity at pH's in the range of about 2 to 4.

Any enzymes or other disruptive agents used to solubilize or otherwise break down ECM materials are preferably removed or inactivated before or during a re-assembly step so as not to compromise formation of the medical bead and/or subsequent medical bead stability. Any disruptive agent, particularly enzymes, that remains present and active during any storage of the starting materials and/or bead products can potentially change the composition and/or other characteristics of these materials and products. Enzymes, such as pepsin, can be inactivated with protease inhibitors, a shift to neutral pH, a drop in temperature below 0° C, heat inactivation, through the removal of the enzyme by fractionation, and the like. A combination of these methods can be utilized to stop digestion of the ECM material at a predetermined endpoint. For example, the ECM starting material can be immediately frozen and later fractionated to limit digestion.

Illustratively, during preparation of a suitable starting material, the ECM material can be enzymatically digested for a sufficient time to produce a hydrolysate of ECM components. Accordingly, the ECM can be treated with one enzyme or with a mixture of enzymes to hydrolyze the structural components of the material and prepare a hydrolysate having multiple hydrolyzed components of reduced molecular weight. The length of digestion time can be varied depending on the application, and the digestion can be extended to completely solubilize the ECM material. In some modes of operation, the ECM material will be treated sufficiently to partially solubilize the material to produce a digest composition comprising hydrolyzed ECM components and non-hydrolyzed ECM components. The digest composition can then, in illustrative embodiments, be further processed to remove at least some of the non-hydrolyzed components. For example, the non- hydrolyzed components can be separated from the hydrolyzed portions by centrifugation, filtration, or other separation techniques known in the art.

In some aspects, ECM bead products are prepared from enzymatically digested vertebrate ECM material that has been fractionated under acidic conditions, for example including pH ranging from about 2 to less than 7, especially to remove low molecular weight components. In some forms, an ECM hydrolysate is fractionated by dialysis against a solution or other aqueous medium having an acidic pH, e.g., a pH ranging from about 2 to about 5, more desirably greater than 3 and less than 7. In addition to fractionating the hydrolysate under acidic conditions, the ECM hydrolysate can be fractionated under conditions of low ionic strength with minimal concentrations of salts such as those usually found in standard buffers such as NaCl, KCl, and PBS (e.g., Na₂HPO₄, KH₂PO₄, etc.) that can pass through the dialysis membrane and into the hydrolysate. Such fractionation conditions work to reduce the ionic strength of the ECM hydrolysate and can provide enhanced bead forming characteristics.

A bead product starting material produced by enzymatic digestion of the ECM material has a characteristic ratio of protein to carbohydrate. The ratio of protein to carbohydrate in the material can be determined by the enzyme utilized in the digestion step and/or by the duration of the digestion. The ratio may be similar to or may be substantially different from the protein to carbohydrate ratio of the native ECM tissue. For example, digestion of vertebrate ECM material with a protease such as pepsin, followed by dialysis, will form a fractionated ECM hydrolysate having a lower protein to carbohydrate ratio relative to the original ECM material.

In other forms, ECM bead products can be prepared from ECM material that has been enzymatically digested and fractionated under acidic conditions to form an ECM hydrolysate that has a protein to carbohydrate ratio different than that of the native ECM material. Such fractionation can be achieved entirely or at least in part by dialysis. The molecular weight cut-off of the ECM components to be included in the bead-forming material is selected based on the desired properties of the bead. Typically the molecular weight cut-off of the dialysis membrane (the molecular weight above which the membrane will prevent passage of molecules) is within in the range of about 2000 to about 10000 Dalton, and more preferably from about 3500 to about 5000 Dalton.

In forming some ECM bead starting materials,, apart from the potential removal of undigested ECM components after a digestion step and any controlled fractionation to remove low molecular weight components as discussed above, the ECM material is processed so as to avoid any substantial further physical separation of the ECM components. For example, when a more concentrated ECM hydrolysate material is desired, this can be accomplished by removing water from the system (e.g. by evaporation or lyophilization) as opposed to using conventional "salting out'Vcentrifugation techniques that would demonstrate significant selectivity in precipitating and isolating collagen, leaving behind amounts of other desired ECM components. Thus, in certain embodiments of the invention, solubilized ECM components of the ECM hydrolysate remain substantially unfractionated, or remain substantially unfractionated above a predetermined molecular weight cut-off such as that used in the dialysis membrane, e.g., above a given value in the range of about 2000 to 10000 Dalton, more preferably about 3500 to about 5000 Dalton.

As a potential step in the preparation of some starting ECM materials, vertebrate ECM material can be stored frozen (e.g. at about -20 to about -80° C) in either its solid, comminuted or enzymatically digested forms, or the materials can be stored, for example, after being hydrolyzed and fractionated. The ECM material can be stored in solvents that maintain the collagen in its native form and solubility. For example, one suitable storage solvent is 0.01 M acetic acid; however, other acids can be substituted, such as 0.01 N HCl. In one form, a fractionated ECM hydrolysate can be dried (by lyophilization, for example) and stored in a dehydrated/lyophilized state. The dried form can be rehydrated to prepare a flowable ECM composition (e.g., a gel) that can be used to form a medical bead in accordance with the present invention.

In some aspects, a fractionated ECM hydrolysate or other flowable ECM composition will exhibit the capacity to re-assemble upon adjusting the pH of a relatively more acidic aqueous medium containing it to about 5 to about 9, more preferably about 6.6 to about 8.0, and typically about 7.2 to about 7.8, thus inducing fibrillogenesis and matrix assembly. In one embodiment, the pH of a fractionated hydrolysate is adjusted by the addition of a buffer that does not leave a toxic residue, and has a physiological ion concentration and the capacity to hold physiological pH. Examples of suitable buffers include PBS, HEPES, and DMEM. Illustratively, the pH of the fractionated ECM hydrolysate can be raised by the addition of a buffered NaOH solution to 6.6 to 8.0, more preferably 7.2 to 7.8, to facilitate the formation of an ECM-containing bead. Any suitable concentration of NaOH solution can be used for these purposes, for example, including about 0.05 M to about 0.5 M NaOH. In accordance with an embodiment, the ECM hydrolysate is mixed with a buffer, and sufficient 0.25 N NaOH is added to the mixture to achieve the desired pH.

The ionic strength of a denatured or otherwise disassembled ECM material is believed to be important in maintaining the fibers of collagen in a state that allows for fibrillogenesis and matrix assembly upon neutralization of the ECM material. Accordingly, if needed, the salt concentration of the ECM material can be reduced prior to neutralization. The neutralized material can be caused or allowed to re-assemble at any suitable temperature, e.g. ranging from about 4° C to about 40° C. The temperature will typically affect the re-assembly times, which may range from about 5 to about 120 minutes at the higher formation temperatures and about 1 to about 8 hours at the lower formation temperatures. Typically, an ECM hydrolysate will be effective to self-assemble at elevated temperatures, for example at about 37° C. In this regard, preferred neutralized ECM hydrolysates will be effective to re-assemble in less than about ninety minutes at 37° C, for example approximately thirty to ninety minutes at 37° C.

In an illustrative embodiment, a particulate ECM material can be added to an ECM hydrolysate composition, which can then be used to form an ECM bead in accordance with the present invention. Such particulate ECM materials can be prepared by cutting, tearing, grinding or otherwise comminuting an ECM starting material. For example, a particulate ECM material having an average particle size of about 50 microns to about 500 microns may be included in a gellable ECM hydrolysate, more preferably about 100 microns to about 400 microns. The ECM particulate can be added in any suitable amount relative to the hydrolysate, with preferred ECM particulate to ECM hydrolysate weight ratios (based on dry solids) being about 0.1 : 1 to about 200: 1 , more preferably in the range of about 1 : 1 to about 100:1. The inclusion of such ECM particulates in the ultimate starting material can serve to provide additional material that can function to provide bioactivity to the bead (e.g. itself potentially including FGF-2 and/or other growth factors or bioactive substances as discussed herein) and/or serve as scaffolding material for tissue ingrowth.

In some forms, an ECM hydrolysate material to be used as a starting material in the invention will exhibit an injectable character and also incorporate an ECM particulate material. In these forms, the ECM particulate material can be included at a size and in an amount that effectively retains an injectable character to the hydrolysate composition.

In other forms, flowable ECM compositions to be used as starting materials in the invention may be disinfected by contacting an aqueous medium including ECM components with an oxidizing disinfectant. This mode of disinfection provides an improved ability to recover a disinfected ECM material that exhibits the capacity to form beneficial beads. In certain preparative methods, an aqueous medium containing ECM hydrolysate components can be disinfected by providing a peroxy disinfectant in the aqueous medium. This can be advantageously achieved using dialysis to deliver the peroxy disinfectant into and/or to remove the peroxy disinfectant from the aqueous medium containing the hydrolysate. In certain dinsinfection techniques, an aqueous medium containing the ECM hydrolysate is dialyzed against an aqueous medium containing the peroxy disinfectant to deliver the disinfectant into contact with the ECM hydrolysate, and then is dialyzed against an appropriate aqueous medium (e.g. an acidic aqueous medium) to at least substantially remove the peroxy disinfectant from the ECM hydrolysate. During this dialysis step, the peroxy compound passes through the dialysis membrane and into the ECM hydrolysate, and contacts ECM components for a sufficient period of time to disinfect the ECM components of the hydrolysate. In this regard, typical contact times will range from about 0.5 hours to about 8 hours and more typically from about 1 hour to about 4 hours. The period of contact will be sufficient to substantially disinfect the digest, including the removal of endotoxins and inactivation of virus material present. The removal of the peroxy disinfectant by dialysis may likewise be conducted over any suitable period of time, for example having a duration of about 4 to about 180 hours, more typically of about 24 to about 96 hours. In general, the disinfection step will desirably result in a disinfected ECM hydrolysate composition having sufficiently low levels of endotoxins, viral burdens, and other contaminant materials to render it suitable for use in forming medical bead products of the invention. Endotoxin levels below about 2 endotoxin units (EUs) per gram (dry weight) are preferred, more preferably below about 1 EU per gram, as are virus levels below 100 plaque forming units per gram (dry weight), more preferably below 1 plaque forming unit per gram.

An aqueous ECM hydrolysate composition can be a substantially homogeneous solution during a dialysis step for delivering an oxidizing disinfectant to the hydrolysate composition and/or during a dialysis step for removing an oxidizing disinfectant from the hydrolysate composition. Alternatively, an aqueous hydrolysate composition can include suspended ECM hydrolysate particles, optionally in combination with some dissolved ECM hydrolysate components, during any oxidizing disinfectant delivery and removal step. Dialysis processes in which at least some of the ECM hydrolysate components are dissolved during disinfectant delivery and/or removal steps are preferred, and those in which substantially all of the ECM hydrolysate components are dissolved are more preferred. A disinfection step can be conducted at any suitable temperature, and will typically be conducted between about 0° C and about 37° C, more typically between about 4° C and about 15° C. During this step, the concentration of the ECM hydrolysate solids in the aqueous medium can be in the range of about 2 mg/ml to about 200 mg/ml, and may vary somewhat through the course of the dialysis due to the migration of water through the membrane. In certain embodiments, a relatively unconcentrated digest is used, having a starting ECM solids level of about 5 mg/ml to about 15 mg/ml. In other embodiments, a relatively concentrated ECM hydrolysate is used at the start of the disinfection step, for example having a concentration of at least about 20 mg/ml and up to about 200 mg/ml, more preferably at least about 100 mg/ml and up to about 200 mg/ml. The use of concentrated ECM hydrolysates during this disinfection processing can result in an ultimate bead material composition having higher material strength than that obtained using similar processing with a lower concentration ECM hydrolysate. Accordingly, processes which involve the removal of amounts of water from the ECM hydrolysate resulting from the digestion prior to the disinfection processing step are preferred. For example, such processes may include removing only a portion of the water (e.g. about 10% to about 98% by weight of the water present) prior to the dialysis/disinfection step, or may include rendering the digest to a solid by drying the material by lyophilization or otherwise, reconstituting the dried material in an aqueous medium, and then treating that aqueous medium with the dialysis/disinfection step.

Disinfection of an aqueous medium containing an ECM hydrolysate can include adding a peroxy compound or other oxidizing disinfectant directly to the ECM hydrolysate, for example, being included in an aqueous medium used to rehydrate a dried ECM hydrolysate or being added directly to an aqueous ECM hydrolysate composition. The disinfectant can then be allowed to contact the ECM hydrolysate for a sufficient period of time under suitable conditions (e.g. as described above) to disinfect the hydrolysate, and then removed from contact with the hydrolysate. In one embodiment, the oxidizing disinfectant can then be removed using a dialysis procedure as discussed above. In other embodiments, the disinfectant can be partially or completely removed using other techniques such as chromatographic or ion exchange techniques, or can be partially or completely decomposed to physiologically acceptable components. For example, when using an oxidizing disinfectant containing hydrogen peroxide (e.g. hydrogen peroxide alone or a peracid such as peracetic acid), hydrogen peroxide can be allowed or caused to decompose to water and oxygen, for example, in some embodiments including the use of agents that promote the decomposition such as thermal energy or ionizing radiation, e.g. ultraviolet radiation.

In an alternative embodiment, an oxidizing disinfectant can be delivered into an aqueous medium containing an ECM hydrolysate by dialysis and processed sufficiently to disinfect the hydrolysate (e.g. as described above), and then removed using other techniques such as chromatographic or ion exchange techniques in whole or in part, or allowed or caused to decompose in whole or in part as discussed immediately above.

Peroxygen compounds that may be used in a disinfection step include, for example, hydrogen peroxide, organic peroxy compounds, and preferably peracids. Such disinfecting agents can be used in a liquid medium, preferably a solution, having a pH of about 1.5 to about 10.0, more desirably of about 2.0 to about 6.0. As to peracid compounds that can be used, these include peracetic acid, perpropioic acid, and/or perbenzoic acid. Peracetic acid is a preferred disinfecting agent when used in the present invention.

When used, peracetic acid is desirably diluted into about a 2% to about 50% by volume of alcohol solution, preferably ethanol. The concentration of the peracetic acid may range, for instance, from about 0.05% by volume to about 1.0% by volume. Most preferably, the concentration of the peracetic acid is from about 0.1% to about 0.3% by volume. When hydrogen peroxide is used, the concentration can range from about 0.05% to about 30% by volume. More desirably the hydrogen peroxide concentration is from about 1% to about 10% by volume, and most preferably from about 2% to about 5% by volume. The solution may or may not be buffered to a pH from about 5 to about 9, with more preferred pH's being from about 6 to about 7.5. These concentrations of hydrogen peroxide can be diluted in water or in an aqueous solution of about 2% to about 50% by volume of alcohol, most preferably ethanol. For additional information concerning preferred peroxy disinfecting agents useful in certain disinfecting embodiments of the present invention, reference can be made, for example, to U.S. Pat. No. 6,206,931.

In certain embodiments, ECM beads of the invention are prepared using suitable gel-form ECM materials. For example, fluidized ECM hydrolysates can be prepared in an aqueous medium, which can thereafter be effective to create a material in gel form. Such prepared aqueous mediums can have any suitable level of ECM hydrolysate therein. Typically, the ECM hydrolysate will be present in the aqueous medium at a concentration of about 2 mg/ml to about 200 mg/ml, more typically about 20 mg/ml to about 200 mg/ml, and in some embodiments about 30 mg/ml to about 120 mg/ml. In certain illustrative forms, the aqueous ECM hydrolysate composition will have an injectable character. Furthermore, ECM gel compositions can be prepared so that in addition to neutralization, heating to physiologic temperatures (such as 37° C) will substantially reduce the formation time of the bead product.

Upon selection of a suitable starting material, e.g., one including solubilized ECM material, this solubilized ECM material can be reconstituted, in certain embodiments, to form a medical bead product in accordance with the present invention. Reconstitution of the ECM material can be accomplished in any suitable manner. Illustratively, such a reconstitution or other re-assembly step can include introducing an amount of a flowable ECM material such as that described above into a liquid medium. Any suitable liquid medium and any suitable means for introducing the flowable ECM material into the liquid medium may be used in this regard. Also, the flowable ECM material can be in contact with the liquid medium for any amount of time sufficient to form an ECM bead in accordance with the present invention, e.g., one having a generally homogeneous network of self-assembled collagen fibers, and entraining at least one bioactive agent in the ECM material. In certain aspects, this contact time is varied to manipulate one or more characteristics of the ECM bead formed, for example, the bead's porosity.

In certain aspects, the liquid medium is effective to induce fibrillogenesis and thereafter facilitate and/or promote assembly of collagen fibers without having to add other materials to and/or otherwise manipulate the system, for example, without having to alter the temperature and/or pH of the system. (In this context, the term "system" refers to at least the combination including the starting material and the liquid medium.) In these embodiments, the contact time between the flowable ECM material and the liquid medium can be from a fraction of a second to several days. A sufficient contact time to form an ECM bead in accordance with the present invention can depend on a number of factors including but not limited to one or more properties of the ECM starting material and/or the liquid medium used, as well as the extent or degree of matrix assembly desired. In this regard, different combinations of such factors can be developed through routine experimentation so as to provide an ECM bead having optimal characteristics for a particular application.

In one illustrative embodiment, an amount of solubilized ECM material (e.g., porcine SIS is introduced into a buffered aqueous medium to form an ECM bead in accordance with the present invention. Any suitable buffered aqueous medium may be utilized in this regard, and advantageously, a buffered aqueous medium will be selected so as not to leave a toxic residue on or within the reconstituted product formed, and to have a physiological ion concentration and the capacity to hold physiological pH. Suitable buffered aqueous mediums for such purposes may include any of the buffers previously disclosed for preparing a suitable starting ECM gel material, e.g., PBS, HEPES, and DMEM. Also, other suitable media can be used and, if desired, rinsed or otherwise processed to remove any undesired residues from the beads. In certain embodiments, a flowable ECM material is extruded into a buffered aqueous medium.

In another illustrative embodiment, an ECM gel is introduced into a buffer bath to form a reconstituted ECM bead in accordance with the present invention. Such an ECM gel can be formed, for example, from solubilized ECM material as generally described above. In this regard, portions of the ECM gel will have already undergone a certain amount of fibrillogenesis. Accordingly, introducing such a gel into a buffer bath or other suitable liquid medium will further the fibrillogenesis and matrix assembly, leading to a reconstituted or otherwise reassembled ECM bead in accordance with the present invention. Illustratively, droplets of submucosa gel can be injected into a buffer bath to produce such beads. In this regard, a number of factors including but not limited to the type of injection used, the injection speed, the characteristics of the buffer bath, and/or the like, can be manipulated to alter one or more characteristics of the reconstituted ECM beads formed. In certain aspects, a two-layer medical bead product can be formed by injecting or otherwise introducing a solubilized ECM material into a liquid medium via a double-walled coaxial injection nozzle.

In other forms, a liquid medium (at least as initially provided) is not configured to induce re-assembly of the collagen (or at least not to the extent of the liquid mediums described above). In such forms, re-assembly of the collagen fibers is induced and carried out by further manipulating the system, for example, by adding other materials to the system and/or altering certain properties of the system such as but not limited to its temperature, pH, and/or the like. For example, it should be noted that solubilized ECM material will typically be effective to self- assemble at elevated temperatures, for example, at about 37° C. Accordingly, in certain embodiments, an ECM bead can be formed by dispersing solubilized ECM material in a liquid medium and sufficiently raising the temperature of the system to form a medical bead product in accordance with the present invention. In this regard, reconstitution times can be varied by adjusting the temperature of the liquid medium containing the dispersed ECM droplets. In certain aspects, the solubilized ECM material is dispersed in the liquid medium in the form of droplets to form an emulsion, wherein conventional methods can be used to prepare the emulsion by stirring, vibrating, or otherwise agitating the solubilized ECM material in the liquid medium. The size of dispersed droplets can be adjusted by controlling the degree of agitation. In some of these aspects, solubilized ECM material is reconstituted under gravitational force of less than one gravity, preferably about zero gravity.

One illustrative method of the invention includes dispersing a neutral solubilized ECM material in a suitable water-immiscible liquid medium in the form of numerous droplets to form an emulsion. Thereafter, the droplets are solidified or substantially solidified by suitably raising the temperature of the emulsion. Such medical bead products may further be crosslinked by any of the methods herein disclosed. Another method of the invention includes dispersing acidic solubilized ECM in a suitable water-immiscible liquid medium in the form of numerous droplets to form an emulsion. Such droplets can then be solidified or otherwise assembled into bead products by addition of a suitable water-miscible liquid medium and an alkali to the emulsion. These and other suitable bead formation steps can also include spray drying, extrusion, electrostatic droplet formation steps, and/or the like.

As previously mentioned, ECM bead products of the present invention can be formed in such a way that allows them to retain one or more native bioactive substances (such as those described above) in their ECM material. In certain embodiments, one or more additional bioactive agents can be incorporated into and/or onto the ECM material before, during, and/or after a bead formation step. For example, any of the non-native bioactive agents previously described (e.g., proteins, carbohydrates, growth factors, therapeutics, nucleic acids, cells, pharmaceuticals, and the like) can be added to the starting material before the ECM bead is formed, or alternatively, can be disposed on the ECM bead after it is formed. This may be accomplished, for example, by forming a dry mixture of a powdered ECM hydrolysate with the additional component(s), and then gelling the mixture before forming a bead, or by incorporating the additional component(s) into an aqueous, composition of the ECM hydrolysate before, during (e.g. with), or after addition of the neutralization agent to cause or allow re-assembly. The additional component(s) can also be added to the formed ECM bead, e.g., by infusing or mixing the component(s) into the bead and/or coating them onto the bead.

In certain preferred aspects, medical bead products of the invention are adapted to serve as substrates and/or scaffolds in the delivery of drugs, etc. to patients. For example, an antineoplastic agent such as but not limited to doxorubicin can be added to a medical bead of the invention. Such drugs or other bioactive agents can be bound or otherwise engaged to the matrix in any suitable fashion, e.g., stored on the collagen fibers of the network or within the pores of the matrix. In certain aspects, incorporation of a drug into a medical bead product of the invention includes suitably contacted the ECM bead with the drug. This can be achieved by spraying, soaking, or otherwise contacting the ECM material with an aqueous solution of the drug or other bioactive agent for a period of time sufficient to incorporate a desired amount of the drug. This contact time may vary, for example, from a few seconds to several days, depending upon the circumstances. Different degrees of porosity can affect the type and/or rate of cell infiltration (tissue ingrowth) into the ECM bead. It should be noted that remodelable ECM materials having a relatively more open matrix structure (i.e., higher porosity) are capable of exhibiting different material properties than those having a relatively more closed or collapsed matrix structure. For example, an ECM material having a relatively more open matrix structure is generally softer and more readily compliant to an implant site than one having a relatively more closed matrix structure. Also, the rate and amount of tissue growth in and/or around a remodelable material can be influenced by a number of factors, including the amount of open space available in the material's matrix structure for the infusion and support of a patient's cell building components, such as fibroblasts. Therefore, an open matrix structure can provide for quicker, and potentially more, growth of patient tissue in and/or around the remodelable material, which in turn, can lead to quicker remodeling of the material by patient tissue. In certain aspects, a medical bead product of the invention is provided having a suitable degree of porosity to promote cellular invasion and/or ingrowth, yet is sufficiently stiff and compression resistant to suit a particular application, for example, to fill and protect a wound, to augment tissue, just to name a few.

In addition to or as an alternative to the incorporation of any non-native bioactive components as described above, other forms of manipulation and/or processing can be performed on medical bead products of the invention. For example, in certain aspects, medical bead products are subjected to drying conditions, for example, to lyophilization conditions, e.g., freeze-drying. Freeze- drying the material can include, for example, freezing the material, including any hydrate contained therein, and thereafter placing the material under vacuum. When sufficient vacuum is applied, as is known in the art, the frozen hydrate will generally sublime, i.e., turn directly from a solid to a gas. The resulting water vapor can then be removed from the material, thereby drying the material. Alternatively, subjecting the material to lyophilization conditions can include, in certain aspects, applying sufficient vacuum to the material to cause evaporative cooling, which simultaneously freezes and dries the material. Utilizing evaporative cooling can eliminate having to "pre-freeze" the material.

Also, it is advantageous when performing any form of drying operations in accordance with the invention, to do so under relatively mild temperature exposure conditions that minimize deleterious effects upon components of the medical bead products, for example, reconstituted material structures and potentially bioactive substances present, e.g., in the case of ECM-containing materials. Thus, drying operations conducted with no or substantially no duration of exposure to temperatures above human body temperature or slightly higher, say, no higher than about 38°C, will preferably be used in some forms of the present invention. These include, for example, forced air drying at less than about 38°C, or with no active heating - at about room temperature (about 25⁰C) or with cooling. Relatively low temperature conditions also, of course, include lyophilization conditions.

In other embodiments, a medical bead product of the invention, once formed, can be incorporated into a liquid or solid matrix. For example, a plurality of medical bead products of the present invention can be incorporated into a liquid carrier to form an injectable or otherwise flowable suspension of the beads, e.g., a form suitable for delivery by a needle, catheter, or other suitable device.

In some aspects of the invention, reconstituted or otherwise re-assembled ECM beads are subjected to a crosslinking process. Suitable crosslinking techniques for this aspect of the invention include but are not limited to photo- crosslinking, chemical crosslinking, and protein crosslinking induced by dehydration or other means. Nonetheless, because certain crosslinking techniques, certain crosslinking agents, and/or certain degrees of crosslinking can destroy the remodelable properties of a remodelable material, where preservation of remodelable properties is desired, any crosslinking of the remodelable ECM material can be performed to an extent or in a fashion that allows the material to retain at least a portion of its remodelable properties. Chemical crosslinkers that may be used include for example aldehydes such as glutaraldehydes, diimides such as carbodiimides, e.g., l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, ribose or other sugars, acyl-azide, sulfo-N-hydroxysuccinamide, or polyepoxide compounds, including for example polyglycidyl ethers such as ethyleneglycol diglycidyl ether, available under the trade name DENACOL EX810 from Nagese Chemical Co., Osaka, Japan, and glycerol poly glycerol ether available under the trade name DENACOL EX 313 also from Nagese Chemical Co. Typically, when used, polyglycerol ethers or other polyepoxide compounds will have from 2 to about 10 epoxide groups per molecule.

Additionally, in certain embodiments, the present invention provides medical graft products comprising one or more pieces of an ECM sheet material that are formed into a wadded up or otherwise randomly compacted condition. These products can take a variety of shapes including bead and bead- like constructs and other constructs including generally rounded features. Also provided are methods for forming such products, for example, methods that include randomly compacting one or more pieces of an ECM sheet material (e.g., a single or multilayer ECM sheet material) into a ball-like shape and in such a way that the ECM material generally retains this shape. Illustratively, a suitable formation process can include providing an ECM sheet material in a partially or otherwise completely wetted or hydrated form, and then forcibly manipulating the sheet such that a compacted, at least somewhat more dried construct is formed. Constructs of various sizes can be formed. In some instances, the size and other features of a construct being formed can be altered by adjusting the amount of material being used, the extent of heating and/or drying applied (if any), the amount of compression imparted to the piece(s) of material and/or other process conditions as discussed elsewhere herein. In some forms, wadding up or otherwise randomly compacting a piece of ECM sheet material includes placing the piece between, and in contact with, two or more surfaces of a compacting device (e.g., a device including opposing planar or non-planar plates, rollers, kneaders and/or other suitably- shaped surfaces for compacting a material) and then causing the surfaces to move relative to one another such that the piece of material is forced by the surfaces into a wadded up or otherwise randomized or generally randomized condition. As an ECM sheet material is compacted, portions of the sheet can fold, twist and/or roll over one another in a variety of fashions. The movement of any of the device surfaces relative to another surface may or may not be random. Before, after or while a sheet material is compacted between such surfaces, the material may be subjected to any number of additional processing steps. Heating, drying, wetting and/or other steps may be employed at various times during formation of a compacted construct. Additional material pieces and/or other substances (e.g., drugs, bioactive agents, etc.) may also be added to the piece of material before, after or while the construct is being formed.

In one embodiment, one or more hydrated sheets of ECM material are placed in a space occurring between two opposing metallic or non- metallic plates, and in such a way that the plates are impinging somewhat upon the sheet(s) of

ECM material. Thereafter, these plates are caused to move relative to one another (e.g., over one another in a circular, oval, etc. fashion) such that the plates force the hydrated ECM sheet material into a wadded up condition. With some compacting devices, one or more of the surfaces will be adjustable, and if desired, the orientation of a surface relative to another surface can be adjusted at various intervals during a formation process. Illustratively, as a piece of ECM material is being compacted between two opposing plates, the plates can be moved progressively closer to one another. Optionally, additional processing steps such as a drying step (e.g., a vacuum) may be applied during all or part of a wadding process. Some devices are known for wadding up or otherwise randomly compacting paper and other sheet-form and non-sheet form materials. Such devices may be utilized, and if necessary adapted, to compact ECM materials in accordance with the present invention.

As one or more pieces of an ECM material are compacted, surfaces of the material will be forced into contact with one another. It is known that dehydration of ECM material portions in forced contact with one another effectively bonds the material portions to one another, even in the absence of other agents for achieving a bond, although such agents can be used while also taking advantage at least in part of the dehydration-induced bonding. With sufficient compression and dehydration, the ECM material portions can be caused to form a compacted, generally unitary ECM structure. When drying is included as part of a formation process, any suitable drying technique can be used as is known in the art, for example, air drying and lyophilization.

In some embodiments, an inventive construct will include one or more threads or other elongate collagenous pieces that are wadded-up or otherwise randomly or non-randomly compacted to form a relatively more compact three- dimensional collagenous structure. In a preferred method for forming such a structure, a hydrated ECM strand will be rolled and/ or folded up or otherwise compacted and subsequently dried to form a dried, compacted ECM construct. Initially such collagenous pieces can exhibit a variety of shapes and configurations including in some forms one or more filaments, single and multi- strand threads, strips, cords, strings, bands, ropes, etc. of material. Further, such elongate collagenous pieces can optionally be subjected to one or more physical, chemical, biological and/or other alterations or manipulations as discussed herein prior to being transformed into an alternate from. Illustratively, in certain forms, opposite ends of an elongate ECM thread will be tied together or otherwise connected to form a loop of ECM material and/or one or more knots can be tied in such a thread prior to the thread being wadded up or placed into a ball, etc. When a loop is formed, it can prevent the free ends of the elongate piece from subsequently coming loose in the dried, finished construct.

Whether using a single piece or multiple collagenous pieces, e.g., 2 to 20 or more ECM material threads, as the elongate piece(s) are wadded up into a three- dimension structure, there will be several locations throughout the three- dimensional construct in which a collagenous piece contacts itself and/or one or more other collagenous pieces. When an inventive construct includes multiple such regions in which a hydrated collagenous surface contacts another hydrated collagenous surface, these surfaces will desirably be of a character so as to form an attachment to one another by virtue of being dried while in contact with one another. In some forms, dehydration of such collagenous surfaces in contact with one another will adhere the surfaces together, at least temporarily. In this regard, by adhering together like-material surfaces in a wadded-up construct, it is possible to start with one or more elongate collagenous pieces and form unique mesh or mesh-like three-dimensional constructs. Optionally, compressive forces may be applied to the wadded up structure during a drying or pre-drying step. Using varying levels of compression and dehydration, two previously unconnected collagenous regions can be bonded to one another to a considerable degree, even in the absence of other agents for achieving a bond although such agents can be used while also taking advantage at least in part on the dehydration-induced bonding. Vacuum pressing operations, and the closely bonded nature that they can characteristically impart to the collagen-containing materials, are highly advantageous and preferred in some embodiments. Additionally or alternatively, an adhesive, glue or other bonding agent may be used in achieving a bond between such contacting surfaces. Suitable bonding agents may include, for example, collagen gels or pastes, gelatin, or other agents including reactive monomers or polymers, for example cyanoacrylate adhesives. A variety of dehydration-induced bonding methods can be used in this regard. The term "dehydrating conditions" can include any mechanical or environmental condition which promotes or induces the removal of water from the collagenous material. To promote dehydration of a compressed ECM construct, for example, a mechanical surface compressing the matrix structure can be water permeable. Dehydration of an ECM material can optionally be further enhanced by applying blotting material, heating the matrix structure or blowing air, or other inert gas, across the exterior of a surface compressing the material. One particularly useful method of dehydration bonding collagenous surfaces is lyophilization, e.g. freeze-drying or evaporative cooling conditions.

In one specific illustrative embodiment, a single strand of ECM material is provided, and the free ends of the strand are tied together to form a loop. The material may be pre-hydrated and/or hydrated as needed during processing. The loop of material is then rolled and/or otherwise compacted to form a hydrated, ball- like structure in which the ECM strand contacts itself in multiple locations throughout the three-dimensional construct. The ball-like structure can be compacted until a desirable ECM material density is achieved. In some forms, the hydrated thread will be wadded up and compressed until a rather dense construct is formed. With or without being further compressed, the hydrated construct can then be subjected to one or more drying conditions such as a lyophilization process to produce a dried, ball or ball-like ECM construct. Illustratively, all or part of the construct can be crimped or otherwise compressed into various shapes during and/or after a drying step. A dried, compressed construct can take a cylindrical shape, for example, for loading into a catheter for deployment into a vascular or other bodily location where such a plug or fill-type device could provide benefit to the patient. A crimped construct of this sort will generally try to revert back to its prior shape upon rehydration. The dried construct can be used in a variety of applications as discussed herein including occupying a bodily cavity or space, occluding blood flow, filling an aneurysm, etc. Multiple of these devices can be loaded and deployed into the same location if desired. Some of the constructs will exhibit a degree of compressibility and thus will be able to conform somewhat to the shape of the vessel or other bodily structure it is placed into. The construct or any portion thereof may be treated with a radiopaque marker so that its location can be viewed during and after deployment.

The present invention also provides a medical kit that includes an ECM particulate product enclosed within a sealed package. The particulate product includes a plurality of ECM beads such as those described above. In certain aspects, the medical kit includes suitable instrumentation for introducing the particulate product into the body of a patient, for example, a syringe to inject a gel or otherwise flowable form of the product.

The sealed package can be configured to maintain the ECM particulate product in a sterile condition when sterilely packaged therein. Sterilization of the medical kit may be achieved, for example, by irradiation, ethylene oxide gas, or any other suitable sterilization technique, and the materials and other properties of the medical packaging will be selected accordingly. Also, ECM particulate products of the invention can be contained in a sterile packaging in any suitable state. Suitable states include, for example, a hydrated or dehydrated state. The particulate products can be dehydrated by any means known in the art (e.g., lyophilization or air dried). If a particulate product of the present invention is stored in a dehydrated state, it is preferred that it retains all of its biological and mechanical properties (e.g., shape, density, flexibility, etc.) upon rehydration.

The materials and other properties of the packaging will be selected accordingly. For example, the package can include indicia to communicate the contents of the package to a person and/or a machine, computer, or other electronic device. Such indicia may include the dimensions of, the type of materials used to form, and/or the physical state of, the contents of the package. In certain embodiments, the particulate product is packaged for sale with instructions for use. For example, in a particularly preferred embodiment, a medical kit includes at least one particulate product sealed within a sterile package, wherein the packaging can have visible indicia identifying the at least one particulate product as having physical characteristics as disclosed herein, and/or can contain or otherwise be associated with printed materials identifying the contents as having such physical characteristics and including information concerning its use as a tissue graft product. The packaging could also include visible indicia relating to the dimensions of the reconstituted ECM beads of the at least one particulate product, and/or relating to the treatment site(s) for which the at least one particulate product is configured.

The ECM bead products disclosed herein find wide use in the field of medicine, and in this regard, can be used and adapted to provide a variety of devices and objects suitable for application to and/or implantation within a patient. The present invention also provides, in certain aspects, various methods for using these products, for example, to replace, augment, repair, and/or otherwise suitably treat diseased or otherwise damaged or defective tissue of a patient. Illustratively, bead products of the invention can be configured as implantable devices suitable for tissue grafting, bulking tissue, providing hemostasis, and/or providing occlusion in a passageway or other open space within the body of a patient (e.g., as an embolization device, etc.). Inventive bead products can also provide wound healing products suitable for cutaneous, intracutaneous, and/or subcutaneous wound treatment, e.g., as burn treatment product. As well, bead products of the invention find use as precursor materials for forming a variety of other medical devices, or components thereof. In certain embodiments, by injecting or otherwise implanting reconstituted ECM beads having biotropic properties into the locale of a tissue defect or a wound in need of healing, one can readily take advantage of these biotropic properties in providing treatment. In certain other embodiments, a particulate product including a plurality of such medical beads carried by a suitable liquid medium is implanted within a patient' s body. For example, a particulate gel product can be implanted within the vascular system, perhaps within, on, or around a vascular vessel in need of treatment. Illustratively, when a particulate product having a suitable viscosity is injected into the lumen of an aneurysm, the product will generally stay in the lumen and provide therapeutic benefit to the aneurysm. The particulate product is so made to either partially or fully cause occlusion of the vessel, to cause emboli formation, or to pack (or fill) an aneurysm lumen. As an embolization or aneurysm lumen filling device, particulate products of the invention are particularly advantageous in that they promote healing of the occluded area and healing of the aneurysm. In preferred aspects, a particulate gel product including one or more drugs is injected into, on, or around a tumor as part of a chemoembolization procedure or method.

Medical bead products of the invention can be made radiopaque by a variety of conventional procedures. In this regard, any radiopaque substance, including but not limited to, tantalum such as tantalum powder, can be incorporated into the beads. Other radiopaque materials comprise bismuth, iodine, and barium, as well as other conventional markers.

As used in the specification and claims, following long-standing patent law practice, the terms "a" and "an," when used in conjunction with the word "comprising" or "including" means one or more.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Further, any theory, mechanism of operation, proof, or finding stated herein is meant to further enhance understanding of the present invention, and is not intended to limit the present invention in any way to such theory, mechanism of operation, proof, or finding. While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only selected embodiments have been shown and described and that all equivalents, changes, and modifications that come within the spirit of the inventions as defined herein or by the following claims are desired to be protected.

Claims

CLAIMSWhat is claimed is:

1. A medical bead product, comprising: an extracellular matrix bead comprised of reconstituted biotropic extracellular matrix material, the extracellular matrix bead having a generally homogeneous network of self-assembled collagen fibers, and comprising at least one bioactive agent retained in the extracellular matrix material, the bioactive agent selected from the group consisting of a proteoglycan, a growth factor, a glycoprotein, and a glycosaminoglycan.

2. The medical bead product of claim 1, wherein said collagen fibers comprise collagen from said extracellular matrix material.

3. The medical bead product of claim 1, wherein said at least one bioactive agent is a proteoglycan, a growth factor, and a glycosaminoglycan.

4. The medical bead product of claim 1, wherein said extracellular matrix material comprises submucosa.

5. The medical bead product of claim 4, wherein said submucosa comprises porcine submucosa.

6. The medical bead product of claim 4, wherein said submucosa comprises small intestine submucosa, urinary bladder submucosa, or stomach submucosa.

7. The medical bead product of claim 1, wherein extracellular matrix material comprises serosa, pericardium, dura mater, peritoneum, or dermal collagen.

8. The medical bead product of claim 1, wherein said at least one bioactive agent is native to said extracellular matrix material.

9. The medical bead product of claim 1, wherein said extracellular matrix bead further comprises at least one additional bioactive agent selected from the group consisting of a growth factor, a protein, a proteoglycan, a glycosaminoglycan, a physiologically compatible mineral, an antibiotic, a chemotherapeutic agent, a pharmaceutical, an enzyme, a hormone, or genetic material.

10. The medical bead product of claim 9, wherein said at least one additional bioactive agent is non-native to said extracellular matrix material.

11. The medical bead product of claim 9, wherein said at least one additional bioactive agent comprises a pharmaceutical.

12. The medical bead product of claim 11, wherein said pharmaceutical comprises an antineoplastic agent.

13. The medical bead product of claim 12, wherein said antineoplastic agent comprises doxorubicin.

14. The medical bead product of claim 1, wherein said extracellular matrix bead has a diameter of 50 microns to 500 microns.

15. The medical bead product of claim 1, wherein said extracellular matrix bead is crosslinked with a crosslinking agent.

16. The medical bead product of claim 1, wherein the extracellular matrix bead further comprises a lyophilized component.

17. The medical bead product of claim 1 adapted for implantation within a patient as a chemoembolic device.

18. A method of forming a medical bead product, comprising: providing a starting material including solubilized extracellular matrix material including at least one retained bioactive agent, said bioactive agent selected from the group consisting of a proteoglycan, a growth factor, a glycoprotein, and a glycosaminoglycan; and reconstituting said solubilized extracellular matrix material to form an extracellular matrix bead having a generally homogeneous network of self-assembled collagen fibers, said extracellular matrix bead entraining said at least one bioactive agent.

19. The method of claim 18, wherein said solubilized extracellular matrix material is in gel form.

20. The method of claim 18, wherein said reconstituting includes introducing said solubilized extracellular matrix material into a liquid medium.

21. The method of claim 20, wherein the liquid medium is a buffered aqueous medium.

22. The method of claim 18, wherein said at least one bioactive agent comprises a growth factor.

23. The method of claim 22, wherein said growth factor is TGF-beta.

24. The method of claim 18, wherein said at least one bioactive agent is a proteoglycan, a glycosaminoglycan, and a growth factor.

25. The method of claim 18, wherein said extracellular matrix material comprises submucosa.

26. The medical product of claim 18, wherein said extracellular matrix material comprises porcine submucosa.

27. The method of claim 18, wherein said starting material also includes at least one additional bioactive agent selected from the group consisting of a growth factor, a protein, a proteoglycan, a glycosaminoglycan, a physiologically compatible mineral, an antibiotic, a chemotherapeutic agent, a pharmaceutical, an enzyme, a hormone, or genetic material, said reconstituted extracellular matrix bead entraining said at least one additional bioactive agent.

28. The method of claim 18, further comprising disposing on said reconstituted extracellular matrix bead at least one additional bioactive agent selected from the group consisting of a growth factor, a protein, a proteoglycan, a glycosaminoglycan, a physiologically compatible mineral, an antibiotic, a chemotherapeutic agent, a pharmaceutical, an enzyme, a hormone, or genetic material.

29. The method of claim 28, wherein said at least one additional bioactive agent comprises a pharmaceutical.

30. The method of claim 18, further comprising subjecting said medical product to lyophilization conditions.

31. The method of claim 18, further comprising subjecting said medical product to sterilization conditions.

32. A method of treating a patient, including grafting a patient with at least one medical bead product, said medical bead product comprising an extracellular matrix bead comprised of reconstituted biotropic extracellular matrix material, said extracellular matrix bead having a generally homogeneous network of self-assembled collagen fibers, and comprising at least one bioactive agent retained in the extracellular matrix material, said bioactive agent selected from the group consisting of a proteoglycan, a growth factor, a glycoprotein, and a glycosaminoglycan.

33. The method of claim 32, wherein said grafting comprises implanting said at least one medical bead product within said patient.

34. The method of claim 33, wherein said at least one medical bead product is implanted with the vascular system of said patient.

35. The method of claim 33, wherein said at least one medical bead product is implanted to treat a tumor.

36. The method of claim 32, wherein said extracellular matrix bead further comprises a chemotherapeutic agent.

37. A medical product, comprising an extracellular matrix particulate product enclosed within a sealed package, said particulate product including a plurality of extracellular matrix beads comprised of reconstituted biotropic extracellular matrix material, wherein each of said plurality of extracellular matrix beads has a generally homogeneous network of self-assembled collagen fibers, and comprises at least one bioactive agent retained in the extracellular matrix material, said bioactive agent selected from the group consisting of a proteoglycan, a growth factor, a glycoprotein, and a glycosaminoglycan..

38. The medical product of claim 37, further comprising instrumentation to introduce said particulate product to the body of a patient.

39. The medical product of claim 37, wherein said sealed package is configured to maintain said extracellular matrix particulate product in a sterile condition when sterilely packaged therein.

40. The medical product of claim 37, wherein said sealed package includes indicia to communicate the contents of said package.

41. A method of forming a medical bead product, comprising: providing solubilized submucosal material including a proteoglycan, a glycosaminoglycan, and a growth factor; and subjecting said solubilized submucosal material to polymerization conditions to form a submucosa bead having a generally homogeneous network of self-assembled collagen fibers, said submucosa bead retaining said proteoglycan, said glycosaminoglycan, and said growth factor therein.

42. A medical bead product, comprising: an extracellular matrix bead comprised of reconstituted biotropic submucosa material, the extracellular matrix bead having a generally homogeneous network of self-assembled collagen fibers, and comprising at least one bioactive agent retained in the extracellular matrix material, the bioactive agent selected from the group consisting of a proteoglycan, a growth factor, a glycoprotein, and a glycosaminoglycan.

43. A method of forming a medical bead product, comprising: providing a starting material including a flowable extracellular matrix material comprising at least one retained bioactive agent, said bioactive agent selected from the group consisting of a proteoglycan, a growth factor, a glycoprotein, and a glycosaminoglycan; forming droplets comprised of said flowable extracellular matrix material; and subjecting said droplets to conditions effective to form solidified extracellular matrix beads entraining the at least one bioactive agent.