Compositions to reverse vascular calcifications and methods and devices for using the same

Abstract

Calcium-binding compositions and methods of treating vascular calcifications using the calcium-binding compositions are disclosed herein. The calcium-binding compositions include a water-soluble biopolymer, such as collagen, and a calcium-binder. The methods of treating vascular calcifications include delivering a calcium-binding composition including a water- soluble biopolymer, such as collagen, and a calcium-binder to a treatment site.

Description

Medtronic Ref. No. A0011118WOO 1COMPOSITIONS TO REVERSE VASCULAR CALCIFICATIONS AND METHODS AND DEVICES FOR USING THE SAMEFIELD

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 736,824, filed December 20, 2024, and to U.S. Provisional Patent Application Serial No. 63 / 825,136, filed June 17, 2025, the disclosures of each of which are incorporated herein by reference.

[0002] This disclosure generally relates to compositions, methods, and devices for treating vascular calcifications and, more specifically, to compositions, methods, and devices for localized delivery of calcium-binding compositions within the cardiovascular system in order to remove calcium from vascular calcifications.BACKGROUND

[0003] Vascular calcification (VC), such as in peripheral artery disease (PAD), is an accumulation of calcium phosphate salts in the vascular tissue and presents therapeutic challenges in up to 70 % of patients with PAD. Such challenges include achieving percutaneous access to treatment sites, inability to inflate angioplasty balloons used for treatment, and increased risk of uncontrolled vessel dissection.SUMMARY

[0004] This disclosure generally relates to calcium-binding compositions and methods of treating vascular calcifications with such calcium-binding compositions by delivering such calcium- binding compositions within the cardiovascular system in order to remove calcium from vascular calcifications.

[0005] In one example, aspects of this disclosure relate to a calcium-binding composition including a water-soluble biopolymer and a calcium-binder.

[0006] In another example, aspects of this disclosure relate to a method for treating a vascular calcification. The method includes delivering to a treatment site a calcium-binding composition including a water-soluble biopolymer and a calcium-binder. The calcium-binding composition removes calcium from the vascular calcification.Medtronic Ref. No. A0011118WOO 1

[0007] The calcium-binding compositions described herein include calcium-binders, which may be described as inorganic or organic compounds that dissolve or bind ionized calcium (Ca++) in aqueous solutions, such as bodily fluids or analogues or models thereof. Calcium-binders, such as chelating agents and weak acids, may bind calcium ions from a vascular calcification, resulting in, or contributing to, removal (e.g., dissolution) of the vascular calcification. Calcium removed from the vascular calcification by calcium-binders is hindered, or prevented, from interacting with phosphate, proteins, lipids or other substances in the body. The removed calcium may then be excreted.

[0008] As described further herein, reaction between calcium-binders and healthier tissue, such as non-calcified vessel wall tissue, may result in damage to the healthier tissue. For example, repeated or prolonged use of acidic calcium-binders (e.g., citric acid) may result in damage to vessel walls. To mitigate the risk of damage, calcium-binding compositions disclosed herein include water-soluble biopolymers (e.g., collagen). As described in the present disclosure, water- soluble biopolymers such as collagen may advantageously incorporate calcium-binders and release them over time at a treatment site within the cardiovascular system. Without wishing to be bound by theory, calcium-binding compositions incorporating calcium-binders and water- soluble biopolymers may advantageously extend the residence time of the calcium-binders in the vasculature and mitigate the potential reactivity of the calcium-binders with the vasculature.

[0009] The term “vascular calcification” as used herein refers to the accumulation of calcium phosphate salts in the vascular tissue, such as intimal calcifications (e.g., resulting in a plaque lesion) and medial calcifications. As used herein, the term “treating a vascular calcification” refers to at least partially removing a vascular calcification at the target site, such as by demineralizing, dissolving, decalcifying, reducing in volume, reducing in density, or reducing in size a vascular calcification. The term “plaque lesion” as used herein refers to an accumulation of materials, such as calcium salts, connective tissue material, elastic tissue material, cellular debris, and cholesterol crystals, forming an irregular deposit in and on a vascular wall.

[0010] The term “substantially” as used here has the same meaning as “significantly,” and can be understood to modify the term that follows by at least 90 %, at least 95 %, or at least 98 %. The term “substantially free” of a particular compound means that the compositions of the present invention contain less than 1,000 parts per million (ppm) of the recited compound. The term “essentially free” of a particular compound means that the compositions of the present invention contain less than 100 parts per million (ppm) of the recited compound. The term “completelyMedtronic Ref. No. A0011118WOO 1 free” of a particular compound means that the compositions of the present invention contain less than 20 parts per billion (ppb) of the recited compound. In the context of the aforementioned phrases, the compositions of the present invention contain less than the aforementioned amount of the compound whether the compound itself is present in unreacted form or has been reacted with one or more other materials.

[0011] All numbers are assumed to be modified by the term “about” and in certain embodiments, preferably, by the term “exactly.” As used herein in connection with a measured quantity, the term “about” refers to that variation in the measured quantity as would be expected by the skilled artisan, and is understood to have the same meaning as “approximately” and to cover a typical margin of error, such as ±5 % of the stated value. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0012] Terms such as “a,” “an,” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration.

[0013] The terms “a,” “an,” and “the” are used interchangeably with the term “at least one.” The phrases “at least one of’ and “comprises at least one of’ followed by a list refers to any one of the items in the list and any combination of two or more items in the list.

[0014] As used here, the term “or” is generally employed in its usual sense including “and / or” unless the content clearly dictates otherwise. The term “and / or” means one or all of the listed elements or a combination of any two or more of the listed elements.

[0015] The recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc. or 10 or less includes 10, 9.4, 7.6, 5, 4.3, 2.9, 1.62, 0.3, etc.). Where a range of values is “up to” or “at least” a particular value, that value is included within the range.

[0016] As used here, “have,” “having,” “include,” “including,” “comprise,” “comprising,” or the like are used in their open-ended sense, and generally mean “including, but not limited to.” It will be understood that “consisting essentially of,” “consisting of,” and the like are subsumed in “comprising” and the like.

[0017] The term “polymer” and “polymeric material” include, but are not limited to homopolymers, copolymers, blends of two or more homopolymers, blends of two or more copolymers, blends of one or more homopolymers and one or more copolymers that have anyMedtronic Ref. No. A0011118WOO 1 geometric configuration such as a linear configuration, branched configuration, graft configuration, star configuration, isotactic symmetry, syndiotactic symmetry, atactic symmetry, or any combination thereof. Copolymers are polymers polymerized from two or more monomers and include block copolymers, alternating copolymers, periodic copolymers, statistical copolymers, stereoblock copolymers, gradient copolymers, and the like.

[0018] The words “preferred” and “preferably” refer to embodiments that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure, including the claims.

[0019] The details of one or more aspects of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the techniques described in this disclosure will be apparent from the description and drawings, and from the claims.

[0020] The above summary of the present disclosure is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the disclosure, guidance is provided through lists of examples, which examples may be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive or exhaustive list. Thus, the scope of the present disclosure should not be limited to the specific illustrative structures described herein, but rather extends at least to the structures described by the language of the claims, and the equivalents of those structures. Any of the elements that are positively recited in this specification as alternatives may be explicitly included in the claims or excluded from the claims, in any combination as desired. Although various theories and possible mechanisms may have been discussed herein, in no event should such discussions serve to limit the claimable subject matter.BRIEF DESCRIPTION OF DRAWINGS

[0021] FIG. 1 A is a cross-sectional axial illustration of a vessel having an intimal vascular calcification.

[0022] FIG. IB is a cross-sectional axial illustration of a vessel having a medial calcification.Medtronic Ref. No. A0011118WOO 1

[0023] FIG. 2 is a flow diagram showing aspects of a method of treating a vascular calcification, such as the vascular calcifications shown in FIGS. 1 A and IB.

[0024] FIGS. 3 A and 3B are graphs showing the citric acid release kinetics of illustrative hydrogel calcium-binding compositions.

[0025] FIGS. 4 A and 4B show microCT images of slices of ex-vivo calcified arteries before (FIG. 4A) and after (FIG. 4B) exposure to illustrative hydrogel calcium-binding compositions.

[0026] FIG. 5 shows images of ex-vivo calcified arteries after exposure to illustrative film calcium-binding compositions.

[0027] FIGS. 6A-6C show images of ex-vivo arterial tissue samples after immersion for 24 hours in illustrative hydrogel calcium-binding compositions.

[0028] FIG. 7 shows a chart of the histopathology scores of the ex-vivo calcified arterial tissue samples of FIGS. 6A-6C after immersion for 24 hours in the illustrative hydrogel calcium- binding compositions.

[0029] FIGS. 8A-8F show images of cultured smooth muscle cells after treatment with illustrative hydrogel calcium-binding compositions.

[0030] FIGS. 9 A and 9B show microCT images of reference slices of ex-vivo calcified arteries before (FIG. 9A) and after (FIG. 9B) exposure to deionized water.

[0031] FIGS.10A and 10B show microCT images of slices of ex-vivo calcified arteries before (FIG. 10A) and after (FIG. 10B) exposure to illustrative hydrogel calcium-binding compositions.DETAILED DESCRIPTION

[0032] The present disclosure provides calcium-binding compositions and methods of treating vascular calcifications using such compositions. The calcium-binding compositions of the present disclosure include a water-soluble biopolymer and a calcium-binder. The calcium-binding compositions may further include at least one additive, such as osteoclast promotors, delivery fluids, microbubbles, microspheres, nanoparticles, anti-restenotic agents, plasticizers, photoinitiators, anti-inflammatory agents, biochemical targeting agents, vessel restoration agents, or any combination thereof.

[0033] Treating vascular calcifications using calcium-binding compositions includes delivering a calcium-binding composition to a treatment site, wherein the calcium-binding composition removes calcium from the vascular calcification.Medtronic Ref. No. A0011118WOO 1Vascular Calcifications

[0034] Vascular calcifications are generally divided into two types: intimal calcifications and medial calcifications. Intimal calcifications and medial calcifications are independent pathological states driven by distinct molecular mechanisms. The vasculature above the knee is more prone to intimal calcifications, while vasculature below the knee is more prone to medial calcifications. Current clinical practice for VC treatment focuses on mechanically removing calcium deposits. However, heavy calcification may result in, or contribute to, a higher rate of acute procedural failure due to vessel recoil or dissection.

[0035] A cross-sectional axial illustration of a vessel 10 having an intimal vascular calcification is shown in FIG. 1 A. The vessel 10 includes adventitia 12 (i.e., the vessel’s outer layer of fibrous connective tissue), intima 16 (i.e., the vessel’s innermost layer), media 14 (i.e., the layer of the vessel between the adventitia 12 and the intima 16), and a central lumen 18. Intimal vascular calcification generally develops in a systemic inflammatory milieu and is influenced by traditional risk factors for cardiovascular disease. Intimal vascular calcification typically involves the intima of large and medium sized elastic arteries. Calcium salts 22 (e.g., hydroxyapatite) accumulate within the intima 16, which may cause accumulation of other materials, resulting in a plaque lesion 20, such as an atherosclerotic plaque lesion, as shown in FIG. 1 A. The plaque lesion 20 may include a fibrous plaque cap 24, formed from the original intimal tissue, which is gradually replaced by a more fibrous, collagen rich layer. Materials accumulate under the plaque cap 24, such as calcium salt 22, lipids 26, plaque 28, macrophages 30, and a necrotic core 32. The plaque cap 24 may form over vascular smooth muscle cells 11 (VSMC), under and around which material may accumulate.

[0036] A cross-sectional axial illustration of the vessel 10 having a medial calcification 40 is shown in FIG. IB. Medial calcifications may occur in an otherwise apparently healthy vascular media (i.e., the layer of the vessel between the vessel’s adventitia and the intima), often as a result of changes in calcium metabolism. Medial calcifications typically affect medium-sized and small muscular arteries, and may be associated with Type 2 diabetes and chronic kidney disease (CKD). As shown in FIG. IB, the medial calcification 40 forms where calcium salts 22 (e.g., hydroxyapatite) accumulate in the media 14 of the vessel 10, which includes VSMC 11 and medial elastic lamellae 13. Accumulation of the calcium salts 22 in the VSCM 11 and the medial elastic lamellae 13 results in, or contributes to, stiffening of the vessel wall, which in turn results in, or contributes to, increased blood pressure and increased risk of cardiovascular mortality.Medtronic Ref. No. A0011118WOO 1Water-Soluble Biopolymers

[0037] The calcium-binding compositions of the present disclosure include a water-soluble biopolymer (e.g., at least one water-soluble biopolymer, one, two, three, or more water-soluble biopolymers, etc.). Water-soluble biopolymers may be described as polymers that can be produced by the cells of living organisms and that are soluble in water, such as at a temperature of 0° C to 60° C or preferably 20° C to 40° C, at a concentration of at least 1 g / L or at least 0.1 % wt / vol, and at a pH of 1 to 4 or 6 to 8.

[0038] The calcium-binding composition may include a wide variety of suitable water-soluble biopolymers. Suitable water-soluble biopolymers may include biosourced (i.e., natural) biopolymers (i.e., biopolymers produced by the cells of living organisms), synthesized biopolymers (i.e., human-made biopolymers analogous to natural biopolymers and obtained by abiotic chemical routes), or any combination thereof. Suitable water-soluble biopolymers may include biosynthetic biopolymers (i.e., recombinant biopolymers). Suitable water-soluble biopolymers may be selected based on factors such as affinity with acidic substances and in vivo degradation characteristics (e.g., desired degradation time scale, little or no release of degradation residuals / byproducts that cause inflammation or embolization, etc.). In some embodiments, suitable water-soluble biopolymers include proteins or polypeptides (e.g., albumin, globin, collagen, collagen derivatives, partially hydrolyzed proteins, gelatins, etc.). In some embodiments, suitable water-soluble biopolymers include polysaccharides (e.g., alginate, chitosan, chitosan derivatives, hyaluronic acid, starch, cellulose, dextran, chondroitin sulfate, sodium hyaluronate, etc.). In some embodiments, suitable water-soluble biopolymers include cellulose derivatives (e.g., oxidized cellulose, carboxymethylcellulose, maltodextrin, dextran, dextran derivatives, etc.).

[0039] In at least one embodiment, the water-soluble biopolymer includes collagen. Collagen may be described a polypeptide having amino acids bound together to form a triple helix of elongated fibrils (i.e., a collagen helix), which forms the structural and mechanical scaffold of bone, skin, tendons, cornea, blood vessel walls, and other connective tissues. As set forth in the present disclosure, collagen advantageously has a high affinity with acidic substances, is stable in acidic conditions, is biodegradable in a biological environment, and has degradation residuals and byproducts that are generally well-tolerated by the body. As additionally set forth in the present disclosure, collagen morphology can be modified to afford a variety of degradation characteristics (e.g., degradation time scale) and mechanical properties. In some embodiments,Medtronic Ref. No. A0011118WOO 1 the water-soluble biopolymer includes collagen, animal-derived collagen (e.g., porcine collagen, fish collagen, human collagen, bovine collagen, etc.), synthetic collagen, biosynthetic collagen (i.e., recombinant collagen), at least partially denatured collagen, at least partially oxidized collagen, at least partially crosslinked collagen, at least partially hydrolyzed collagen (e.g., gelatins), or any combination thereof.

[0040] In some embodiments, the water-soluble biopolymer includes, native collagen, which may be described as collagen having its native triple helix structure, such as pepsin-soluble type I collagen extracted from porcine skin. In its native form, type I collagen may only be degraded by collagenases and metalloproteinases.

[0041] The calcium-binding compositions of the present disclosure may include a wide variety of suitable forms. Suitable forms of the calcium-binding composition include, for example, aqueous solutions, hydrogels, films, sponges, foams, or any combination thereof. Suitable forms of the calcium-binding composition may be selected based on factors such as desired delivery technique (e.g., applying, coating, releasing, injecting, etc., as described further here with regard to the illustrative method 100).

[0042] In some embodiments, the calcium-binding composition is in the form of an aqueous solution, such as a viscous aqueous solution. In some embodiments, the calcium-binding composition is in the form of a hydrogel. In some embodiments, the calcium -binding composition is in the form of a film. In some embodiments, the calcium-binding composition is in the form of a sponge or a foam.

[0043] In one or more embodiments, the form of the calcium-binding composition is primarily determined by the form of the water-soluble polymer. The water-soluble polymer may include a wide variety of suitable forms. Suitable forms of the water-soluble polymer include, for example, viscous liquids, hydrogels, films, sponges, foams, powders, or any combination thereof. Suitable forms of the water-soluble polymer may be selected based on factors such as desired delivery technique (e.g., applying, coating, releasing, injecting, etc., as described further here with regard to the illustrative method 100). In some embodiments, the water-soluble biopolymer is in the form of an aqueous fluid, such as a viscous fluid. In some embodiments, the water-soluble biopolymer is in the form of a hydrogel. In some embodiments, the water-soluble biopolymer is in the form of a film. In some embodiments, the water-soluble biopolymer is in the form of a sponge. In some embodiments, the water-soluble biopolymer is in the form of a wet foam. In such embodiments, the wet foam’s porous structure may advantageously allow blood circulationMedtronic Ref. No. A0011118WOO 1 and prevent occlusion while the water-soluble biopolymer is at the target site. In some embodiments, the water-soluble biopolymer is in the form of a powder. In some embodiments, the water-soluble biopolymer includes a hydrogel, or is in the form of a hydrogel.

[0044] The calcium-binding composition may include a wide variety of suitable amounts of the water-soluble biopolymer based on the total weight of the calcium-binding composition. Suitable amounts of water-soluble biopolymer may be selected based on factors, such as stability of the calcium-binding composition in physiological conditions, stability of the calcium-binding composition during sterilization and storage, desired release profile of the calcium-binder, desired rheological properties of the calcium-binding composition, desired ductility of the calcium-binding composition (e.g., during manufacturing, application, etc.), and control of particulate matter released during degradation or dissolution, as a few examples.

[0045] Suitable amounts of water-soluble biopolymer may be selected based on the desired form of the calcium-binding composition (e.g., aqueous solutions, hydrogels, films, sponges, etc.). In some embodiments, the calcium-binding may be in the form of a “wet” system (e.g., a viscous fluid, a hydrogel, etc.). In some such embodiments, the calcium-binding composition includes 0.1 wt-% to 20 wt-% or 5 wt-% to 10 wt-% of water-soluble biopolymer based on the total weight of the calcium-binding composition, or based on the total weight of the wet system. In some embodiments where the calcium-binding composition includes a wet system, the calcium-binding composition includes 0.1 wt-% or greater, 0.5 wt-% or greater, 1 wt-% or greater, 5 wt-% or greater, 10 wt-% or greater, 15 wt-% or greater, or 20 wt-% or greater of water-soluble biopolymer based on the total weight of the calcium-binding composition, or based on the total weight of the wet system. In some embodiments where the calcium-binding composition includes a wet system, the calcium-binding composition includes 25 wt-% or less, 20 wt-% or less, 15 wt- % or less, 10 wt-% or less, 5 wt-% or less, 3 wt-% or less, 1 wt-% or less, 0.5 wt-% or less, or 0.1 wt-% or less of water-soluble biopolymer based on the total weight of the calcium-binding composition, or based on the total weight of the wet system.

[0046] In some embodiments, the calcium-binding composition may be in the form of a “dry” system (e.g., a film, a sponge, etc.). In some such embodiments, the calcium-binding composition includes 5 wt-% to 75 wt-% or 15 wt-% to 40 wt-% of water-soluble polymer based on the total weight of the calcium-binding composition, or based on the total weight of the dry system. In some embodiments where the calcium-binding composition includes a dry system, the calcium- binding composition includes 5 wt-% or greater, 10 wt-% or greater, 15 wt-% or greater, 20 wt-%Medtronic Ref. No. A0011118WOO 1 or greater, 25 wt-% or greater, 30 wt-% or greater, 35 wt-% or greater, 40 wt-% or greater, 45 wt- % or greater, 50 wt-% or greater, 55 wt-% or greater, 60 wt-% or greater, 65 wt-% or greater, 70 wt-% or greater, or 75 wt-% or greater of water-soluble polymer based on the total weight of the calcium-binding composition, or based on the total weight of the dry system. In some embodiments where the calcium-binding composition includes a dry system, the calcium-binding composition includes 75 wt-% or less, 70 wt-% or less, 65 wt-% or less, 60 wt-% or less, 55 wt- % or less, 50 wt-% or less, 45 wt-% or less, 40 wt-% or less, 35 wt-% or less, 30 wt-% or less, 25 wt-% or less, 20 wt-% or less, 15 wt-% or less, 10 wt-% or less, or 5 wt-% or less of water- soluble polymer based on the total weight of the calcium-binding composition, or based on the total weight of the dry system.

[0047] In some embodiments, such as embodiments where the calcium-binding composition includes a film, the calcium-binding composition includes 1 milligram per square centimeter (mg / cm2) to 20 mg / cm2or 3 mg / cm2to 9.6 mg / cm2of water-soluble polymer based on the total area of the film. In some embodiments, such as embodiments where the calcium-binding composition includes a film, the calcium-binding composition includes 1 mg / cm2or greater, 3 mg / cm2or greater, 5 mg / cm2or greater, 7 mg / cm2or greater, 9.6 mg / cm2or greater, 10 mg / cm2or greater, 12 mg / cm2or greater, 15 mg / cm2or greater, 17 mg / cm2or greater, or 20 mg / cm2or greater of water-soluble polymer based on the total area of the film. In some embodiments, such as embodiments where the calcium-binding composition includes a film, the calcium-binding composition includes 20 mg / cm2or less, 17 mg / cm2or less, 15 mg / cm2or less, 12 mg / cm2or less, 10 mg / cm2or less, 9.6 mg / cm2or less, 7 mg / cm2or less, 5 mg / cm2or less, 3 mg / cm2or less, or 1 mg / cm2or less of water-soluble polymer based on the total area of the film.

[0048] In some embodiments, such as embodiments where the calcium-binding composition includes a foam or sponge, the calcium-binding composition includes 1 mg per cubic cm (mg / cc) to 60 mg / cc, 1.5 mg / cc to 3 mg / cc, or 5 mg / cc to 50 mg / cc of water-soluble polymer based on the total volume of the foam or sponge. In some embodiments, such as embodiments where the calcium-binding composition includes a foam or sponge, the calcium-binding composition includes 1 mg / cc or greater, 1.5 mg / cc or greater, 2 mg / cc or greater, 3 mg / cc or greater, 5 mg / cc or greater, 10 mg / cc or greater, 20 mg / cc or greater, 30 mg / cc or greater, 40 mg / cc or greater, or 50 mg / cc or greater of water-soluble polymer based on the total volume of the foam or sponge. In some embodiments, such as embodiments where the calcium-binding composition includes a foam or sponge, the calcium-binding composition includes 50 mg / cc or less, 40 mg / cc or less, 30Medtronic Ref. No. A0011118WOO 1 mg / cc or less, 20 mg / cc or less, 10 mg / cc or less, 5 mg / cc or less, 3 mg / cc or less, 2 mg / cc or less, 1.5 mg / cc or less, or 1 mg / cc or less of water-soluble polymer based on the total volume of the foam or sponge.

[0049] In one or more embodiments, the water-soluble biopolymer includes denatured collagen, or at least partially denatured collagen. The denaturation temperature of pepsin-soluble type I collagen is 37° C, above which the triple-helical configuration may be irreversibly lost. Cooling denatured collagen (e.g., a solution of denatured collagen) may form crosslinked 3-dimensional networks (i.e., “hydrogels”). Such denatured collagen hydrogels may be described as having similar viscoelastic properties to a solid (i.e., without flow) compared with viscous solutions containing native collagen. Denatured collagen may be degraded by a wide variety of proteases (e.g., trypsin, pepsin, pronase, etc.). In embodiments of the calcium-binding composition including denatured collagen, the degree of denaturing and amount of denatured collagen based on the weight of the calcium-binding composition may each be selected based on factors such as the desired viscoelastic properties of the calcium-binding composition or the desired degradation or dissolution characteristics of the calcium-binding composition. For example, a greater degree of denaturing, or a greater amount of denatured collagen may contribute to, or result in, higher elasticity or slower degradation or dissolution in vivo. As another example, factors for selecting the degree of denaturing and amount of denatured collagen based on the weight of the calcium- binding composition may include the desired release profile of the calcium-binder.

[0050] In at least one embodiment, the water-soluble biopolymer includes chemically- functionalized collagen. Collagen may be functionalized chemically, for example, by the modification of chemical groups of the collagen, such as primary amine groups, carboxyl groups, hydroxyl groups, thiol groups, glycoside groups, or any combination thereof. In embodiments of the calcium-binding composition including chemically-functionalized collagen, the degree of functionalization and amount of chemically-functionalized collagen (e.g., based on the weight of the calcium-binding composition) may each be selected based on factors such as the desired viscoelastic properties of the calcium-binding composition or the desired degradation or dissolution characteristics of the calcium-binding composition. For example, a greater degree of chemical functionalization, or a greater amount of chemically-functionalized collagen may contribute to, or result in, higher elasticity or slower degradation or dissolution in vivo. As another example, factors for selecting the degree of chemical functionalization and amount ofMedtronic Ref. No. A0011118WOO 1 chemically-functionalized collagen based on the weight of the calcium-binding composition may include the desired release profile of the calcium-binder.

[0051] In some embodiments, the water-soluble biopolymer includes crosslinked collagen, or at least partially crosslinked collagen. Crosslinked collagen may be formed using a wide variety of suitable techniques. Suitable techniques for forming crosslinked collagen include, for example, crosslinking using oxidizing collagen, crosslinking using acrylamide collagen, crosslinking using an additive composition, crosslinking using heat treatment, and crosslinking using a dehyrothermal treatment, as examples. Crosslinked collagen may advantageously be relatively more stable (e.g., relatively slower to dissolve) in body fluids at 37° C (i.e., in vivo). For example, while materials (e.g., films, sponges, hydrogels, etc.) including collagen (e.g., native collagen or denatured collagen) without crosslinking may dissolve within a few minutes in body fluids at 37° C, crosslinked collagen may take hours, days, or even weeks to dissolve in body fluids at 37° C, depending upon the amount of crosslinking, and the amount of crosslinked collagen as a portion of the composition. In other words, crosslinked collagen in calcium-binding compositions may result in, or contribute to, slower degradation or dissolution (e.g., of the collagen at the treatment site). In particular, degradation by collagenases may be slowed down by crosslinked collagen.

[0052] In some embodiments, the water-soluble biopolymer includes oxidized collagen, or at least partially oxidized collagen. Oxidized collagen may be formed, for example, from type I collagen by oxidation of hydroxylysine and the glycoside groups of the sugar moi eties in the collagen with periodic acid, thereby affording aldehyde groups, which may react with the primary amine groups of the collagen to form chemical crosslinks within the oxidized collagen. Such oxidized collagen may advantageously be used to form dry systems (e.g., powders, films, foams, sponges, and combinations thereof), which may advantageously be relatively more stable (e.g., relatively slower to dissolve) in body fluids at 37° C (i.e., in vivo).

[0053] In one or more embodiments, the water-soluble biopolymer includes acrylamide collagen (i.e., acrylamide-modified collagen), which may be formed, for example, by reacting primary amine groups of collagen (e.g., type I collagen) with N-acryloxysuccinimide, thereby affording acrylamide groups. The formed acrylamide groups may react with each other in the presence of a reactive species (e.g., free radicals), thereby crosslinking the collagen. Reactive species may be released, for example, as a result of the absorption of ultraviolet light by a water-soluble photoinitiator. Acrylamide collagen may advantageously afford crosslinked collagen in a low pHMedtronic Ref. No. A0011118WOO 1 composition, such as in low pH compositions described herein. In embodiments of the calcium- binding composition including acrylamide collagen, the amount of acrylamide collagen based on the weight of the calcium-binding composition may be selected based on factors such as the desired viscoelastic properties of the calcium-binding composition or the desired degradation or dissolution characteristics of the calcium-binding composition. For example, a greater amount of acrylamide collagen may contribute to, or result in, higher elasticity or slower degradation or dissolution in vivo. As another example, factors for selecting the amount of acrylamide collagen based on the weight of the calcium-binding composition may include the desired release profile of the calcium-binder. Acrylamide collagen may advantageously be used to form dry systems (e.g., powders, films, foams, sponges, and combinations thereof), which may advantageously be relatively more stable (e.g., relatively slower to dissolve) in body fluids at 37° C (i.e., in vivo).

[0054] In at least one embodiment, the water-soluble biopolymer includes collagen (e.g., native collagen, denatured collagen, etc.) crosslinked using an additive molecule. Thusly crosslinked collagen may be formed, for example, by reacting collagen with aldehyde functionalized polysaccharides (e.g., dextran aldehyde, alginate aldehyde, maltodextrin aldehyde, etc.). The aldehyde groups of functionalized polysaccharides may react with the primary amine groups in the collagen. Collagen crosslinked using an additive molecule may advantageously be used to form dry systems (e.g., powders, films, foams, sponges, and combinations thereof) or wet systems (e.g., viscous fluids, hydrogels, etc.) which may advantageously be relatively more stable (e.g., relatively slower to dissolve) in body fluids at 37° C (i.e., in vivo). Collagen crosslinked using an additive molecule may advantageously be crosslinked to a relatively greater extent, for example, compared to collagen crosslinked via other techniques (e.g., collagen acrylamide, oxidized collagen, etc.). In some embodiments, the calcium-binding composition includes 0.1 wt-% to 10 wt-% or 1 wt-% to 3 wt-% of aldehyde functionalized polysaccharides based on the total weight of the calcium-binding composition, or based on the total weight of the water-soluble biopolymer. In some embodiments, the calcium-binding composition includes 0.1 wt-% or greater, 0.5 wt-% or greater, 1 wt-% or greater, 3 wt-% or greater, 5 wt-% or greater, 7 wt-% or greater, or 10 wt-% or greater of aldehyde functionalized polysaccharides based on the total weight of the calcium-binding composition, or based on the total weight of the water-soluble biopolymer. In some embodiments, the calcium -binding composition includes 10 wt-% or less, 7 wt-% or less, 5 wt-% or less, 3 wt-% or less, 1 wt-% or less, 0.5 wt-% or less, or 0.1 wt-% or lessMedtronic Ref. No. A0011118WOO 1 of aldehyde functionalized polysaccharides based on the total weight of the calcium-binding composition, or based on the total weight of the water-soluble biopolymer.

[0055] In some embodiments, the water-soluble biopolymer includes dry native collagen or denatured collagen crosslinked using dehyrothermal treatment (DHT). For example, native (or otherwise unmodified / functionalized) collagen may be crosslinked by applying a thermal treatment under vacuum when the collagen is in a dry state. DHT collagen may advantageously be used to form dry systems (e.g., powders, films, foams, sponges, and combinations thereof), which may advantageously be relatively more stable (e.g., relatively slower to dissolve) in body fluids at 37° C (i.e., in vivo). Dehyrothermal treatment of the collagen in a dry state can be performed at a wide variety of suitable air temperatures. Suitable air temperatures may be, or include, 60° C to 180° C or 100° C to 140° C, as examples. Suitable air temperatures may be selected based on factors, such as air pressure during the DHT, where suitable air temperatures may be lower under lower air pressure conditions. For example, DHT at 100° C to 110° C and standard atmospheric pressure and DHT at 40° C to 50° C and less than 10 millibars ambient pressure may result in similar collagen structuring. In some embodiments, freeze-drying collagen, for example, at less than 10 microbars. Under suitable DHT conditions, collagen rearranges with a structure reinforced by hydrogen bonds, which are not present in native collagen, such as native collagen extracted and purified from porcine dermis. DHT collagen in calcium-binding compositions may result in, or contribute to, slower degradation or dissolution (e.g., of the collagen at the treatment site). In particular, degradation by collagenases may be slowed down by DHT collagen.

[0056] In some embodiments, the water-soluble biopolymer may be free of telopeptides, substantially free of telopeptides, essentially free of telopeptides, or completely free of telopeptides. For example, during purification of collagen, telopeptides may be removed by pepsin. Removal of telopeptides may result in, or contribute to, reduced risk of collagen sensitization. In other words, removal of telopeptides may advantageously improve biocompatibility of water-soluble biopolymers, such as collagen, compared to water-soluble biopolymers that have not had telopeptides removed.

[0057] In one or more embodiments, the water-soluble biopolymer includes a combination of two or more collagens (e.g., three or more collagens, four or more collagens, etc.). For example, the water-soluble biopolymer may include one or more native collagens (e.g., unmodified, nonfunctionalized, etc.) and one or more modified / functionalized collagens (e.g., crosslinkedMedtronic Ref. No. A0011118WOO 1 collagen, acrylamide-modified collagen, etc.). Water-soluble biopolymers including a combination of two or more collagens may include a wide variety of suitable ratios of collagens. Suitable ratios of collagens may include, for example, between 20: 1 and 1 :20 or between 10: 1 and 1 :10 native collagen to modified / functionalized collagen. As further examples, suitable ratios of collagens may include 1 :20 or greater, 1 : 15 or greater, 1 : 10 or greater, 1 :7 or greater, 1 :5 or greater, 1 :3 or greater, 1 :2 or greater, 1 : 1 or greater, 2: 1 or greater, 3 : 1 or greater, 5 : 1 or greater, 7: 1 or greater, 10: 1 or greater, 15: 1 or greater, or 20: 1 or greater, and / or 20: 1 or less, 15:1 or less, 10: 1 or less, 7: 1 or less, 5: 1 or less, 3: 1 or less, 2: 1 or less, 1 : 1 or less, 1 :2 or less, 2: 1 or less, 3: 1 or less, 5: 1 or less, 7: 1 or less, 10: 1 or less, 15: 1 or less, or 20: 1 or less. It will be understood in view of this disclosure that any suitable ratio of collagens may be used, and the disclosure is not limited in this regard. It will further be understood in view of this disclosure that suitable ratio of collagens may be selected and affected based on factors such as those described herein.Calcium-binder

[0058] The calcium-binding compositions of the present disclosure include a calcium-binder (e.g., at least one calcium-binder, one, two, three, or more calcium-binders, etc.). Calcium- binders may be described as inorganic or organic compounds that dissolve or bind ionized calcium in aqueous solutions, such as bodily fluids or analogues or models thereof.

[0059] The calcium-binding composition may include a wide variety of suitable calcium-binders. Suitable calcium-binders may be selected based on factors such as calcium-binding capacity, water solubility, biocompatibility, or acidity. In some embodiments, suitable calcium-binders may include chelating agents and weak acids. Chelating agents may be described as chemical compounds that react with metal ions to form a stable complex that is soluble in water, such as at a temperature of 0° C to 60° C or preferably 20° C to 40° C, at a concentration of at least 1 g / L or at least 0.1 % wt / vol, and at a pH of 1 to 4 or 6 to 8. A weak acid may be described as an acid that dissociates incompletely in a solution, releasing only some of its hydrogen atoms into the solution. In some embodiments, the calcium-binder includes ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), N-(2- hydroxyethyl) ethylene diamine-N,N',N'-triacetic acid (HEDTA), ethylene diaminetetraacetic acid (EDTA), dimercaptosuccinic acid (DMSA), 2,3 -dimercaptopropanesulfonic acid (DMPS), thiamine tetrahydrofurfuryl disulfide (TTFD), sodium tripolyphosphate (STP), sodium thiosulfate (NaTS), alpha lipoic acid (ALA), citric acid (CA), ascorbic acid (AA), N-acetylneuraminic acidMedtronic Ref. No. A0011118WOO 1(NANA), or any combination thereof. In some embodiments, the calcium-binder includes a particulate calcium-binder. For example, the calcium-binder may be in the form of microparticles, nanoparticles, or nanocrystals of the calcium-binder. In some embodiments, the calcium-binder includes nanocrystalline EDTA or EGTA.

[0060] The calcium-binding composition may include a wide variety of suitable amounts of the calcium-binder based on the total weight of the calcium-binding composition. Suitable amounts of the calcium-binder may be selected based on factors, such as, for example, balancing the strength of desirable therapeutic effects of the calcium-binding composition (e.g., the level, or amount, of demineralization, decalcification, etc.) against the strength of undesirable side-effects of the calcium-binding composition (e.g., the risk, or amount of damage that may be caused to the vascular wall) of the calcium-binding composition. As further examples, suitable amounts of the calcium-binder may be selected based on factors such as desired rheological properties of the calcium-binding composition, biocompatibility, material compatibility (e.g., with the water- soluble biopolymer), and desired ductility of the calcium-binding composition (e.g., during manufacturing, application, etc.). In some embodiments, the calcium-binding composition includes 1 wt-% to 30 wt-% or 3 wt-% to 10 wt-% of calcium-binder based on the total weight of the calcium-binding composition. In some embodiments, the calcium-binding composition includes 1 wt-% or greater, 3 wt-% or greater, 5 wt-% or greater, 10 wt-% or greater, 15 wt-% or greater, 20 wt-% or greater, 25 wt-% or greater, 30 wt-% or greater, or 35 wt-% or greater of calcium-binder based on the total weight of the calcium-binding composition. In some embodiments, the calcium-binding composition includes 40 wt-% or less, 35 wt-% or less, 30 wt- % or less, 25 wt-% or less, 20 wt-% or less, 15 wt-% or less, 10 wt-% or less, 5 wt-% or less, 3 wt-% or less, or 1 wt-% or less of calcium-binder based on the total weight of the calcium- binding composition.

[0061] The calcium-binding composition may have a wide variety of suitable pH values. In some embodiments, the calcium-binding composition has a pH value of 1 to 3.5 or 2 to 3. In some embodiments, the calcium -binding composition has a pH value of 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, or 1 or less. In some embodiments, the calcium-binding composition has a pH value of 6 to 8. Suitable calcium -binding composition pH values may be selected based on factors, such as the calcium-binder (or calcium-binders) included in the calcium-binding composition. In some embodiments, the calcium-binding composition has a pH value of 2 to 4 and includes 1 wt-% to 20 wt-% of citric acid. In some embodiments, the calcium-Medtronic Ref. No. A0011118WOO 1 binding composition has a pH value of 1 to 2 and includes 15 wt-% to 20 wt-% of citric acid. In some embodiments, the calcium -binding composition has a pH value of 2 to 3 and includes 10 wt-% to 15 wt-% of citric acid. In some embodiments, the calcium-binding composition has a pH value of 3 to 4 and includes 1 wt-% to 5 wt-% of citric acid. In some embodiments, the calcium- binding composition has a pH value of 1 to 3 and includes 25 wt-% to 35 wt-% of citric acid. In some embodiments, the calcium-binding composition has a pH value of 6 to 8 and includes 1 wt- % to 10 wt-% EDTA. In some embodiments, the calcium -binding composition has a pH value of 2 to 4 and includes 1 wt-% to 10 wt-% ascorbic acid. In some embodiments, the calcium -binding composition has a pH value of 2 to 4 and includes 1 wt-% to 10 wt-% acetic acid. In some embodiments, the calcium-binding composition has a pH value of 2 to 4 and includes 1 wt-% to 5 wt-% N-acetylneuraminic acid.Additives

[0062] In one or more embodiments, the calcium-binding composition includes an additive (e.g., at least one additive, one, two, three, or more additives, etc.). Likewise, the methods described herein may include applying (e.g., delivering, coating, releasing, etc.) at least one additive. A wide variety of suitable additives may be used. Suitable additives may include, for example, plasticizers, photoinitiators, osteoclast promotors, delivery fluids, microbubbles (e.g., ligand- functionalized microbubbles, microbubbles with phospholipid monolayers, microbubbles with phospholipid bilayers, etc.), microspheres, nanoparticles, anti-inflammatory agents, vessel restoration agents, biochemical targeting agents, anti -thrombotic agents, anti-restenotic agents (e.g., anti -proliferative agents, cytotoxic agents, and other compositions that can reduce or prevent the recurrence of stenosis), or any combination thereof.

[0063] In some embodiments, the additive includes a plasticizer (e.g., at least one plasticizer). Plasticizers may be described as materials that improve flexibility of the calcium-binding composition (e.g., in some embodiments where the calcium-binding composition includes a film). A wide variety of suitable plasticizers may be used. Suitable plasticizers include, for example, glycerol, polyethylene glycol, sorbitol, and any combination thereof.

[0064] In some embodiments, the additive includes a biochemical targeting agent (e.g., at least one biochemical targeting agent). Biochemical targeting agents may be described as materials (e.g., proteins, chemical agents, etc.) that can be used to selectively target particular tissue regions or biochemical pathways. A wide variety of suitable biochemical targeting agents may beMedtronic Ref. No. A0011118WOO 1 used. Suitable biochemical targeting agents include, for example, antibodies, ligands, biomolecules for targeting particular receptors, or any combination thereof.

[0065] In some embodiments, the additive includes a photoinitiator (e.g., at least one photoinitiator). Photoinitiators may be described as materials that release reactive species (e.g., free radicals) as a result of absorbing electromagnetic radiation (e.g., UV light). As described above with respect to acrylamide collagen, photoinitiators may advantageously be used to promote reaction of acrylamide groups of acrylamide collagen by releasing reactive species. A wide variety of suitable photoinitiators may be used. Suitable photoinitiators include, for example, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

[0066] In some embodiments, the additive includes an osteoclast promotor (e.g., at least one osteoclast promotor). An osteoclast promotor is a material (e.g., a compound, composition, etc.) capable of promoting osteoclast activity. Osteoclasts are cells that break down old or damaged bone tissue. A wide variety of suitable osteoclast promotors may be used, such as, for example, chemoattractants for osteoclasts, compounds that switch cell phenotype towards osteoclast development, cytokines, vascular endothelial growth factor, or other compositions suitable to promote osteoclast activity at the treatment site. As further examples, suitable osteoclast promotors may include M-CSF (macrophage-colony stimulating factor), RANKL (receptor activator of nuclear factor-K ligand), IL-6 (Interleukin 6), Prostaglandin E2, and the like. It will be understood in view of the present disclosure that a wide variety of suitable osteoclast promotors may be used, and the disclosure is not limited in this regard.

[0067] In some embodiments, the additive includes a cell disruptor (e.g., at least one cell disruptor). Cell disruptors may be described as compounds or compositions capable of disrupting cell structures, such as by inducing osmotic lysis or apoptosis. The calcium-binding composition may include a wide variety of suitable cell disruptors. In some embodiments, the cell disruptor includes alcohols (e.g., ethanol, 2-propanol), acetone, dimethyl sulfoxide (DMSO), ethers, glucose solution, surfactant (e.g., sodium dodecyl sulfate), or any combination thereof. Suitable cell disruptors may be selected based on factors, such as, for example, material compatibility between the cell disruptor and other constituents of the calcium-binding composition. In some embodiments, suitable cell disruptors are selected based on capability of inducing osmotic lysis in cells at the treatment site. Osmotic lysis in cells at the treatment may advantageously improve penetration of the calcium-binder into the treatment site, thereby increasing interaction between the calcium-binder and calcifications at the treatment site. For example, exposure of cells to aMedtronic Ref. No. A0011118WOO 1 glucose solution (e.g., a 10 wt-% glucose solution) may result in, or contribute to, a hypotonic extracellular environment, which may cause the cells to take on water, swell, and subsequently burst. As another example, organic solvents like alcohols, may disrupt cells by permeating the cell walls and membranes. As yet another example, EDTA may disrupt cells by chelating cations of the cell, resulting in holes in the cell’s walls. As still another example, surfactants may disrupt the interface between hydrophobic and hydrophilic systems. As still yet another example, chaotropic agents, such as urea, may allow certain hydrophobic compounds to dissolve in aqueous solutions by disrupting the structure of water to result in a less hydrophilic environment and weakening the hydrophobic interactions among solute molecules. Similarly, suitable cell disruptors may be selected based on capability of inducing cell apoptosis, or programmed cell death. For example, DMSO may induce apoptosis.

[0068] The calcium-binding composition may include a wide variety of suitable amounts of cell disruptor based on the total weight of the calcium-binding composition. Suitable amounts of cell disruptor may be selected based on factors, such as, for example, desired strength of cell disruptor effects (e.g., osmotic lysis, cell apoptosis, etc.). In some embodiments, the calcium- binding composition includes 1 wt-% to 10 wt-% of cell disruptor based on the total weight of the calcium-binding composition. In some embodiments, the calcium-binding composition includes 0.5 wt-% or greater, 1 wt-% or greater, 2 wt-% or greater, 5 wt-% or greater, 8 wt-% or greater, or 10 wt-% or greater of cell disruptor based on the total weight of the calcium-binding composition. In some embodiments, the calcium-binding composition includes 15 wt-% or less, 10 wt-% or less, 8 wt-% or less, 5 wt-% or less, 2 wt-% or less, or 1 wt-% or less of cell disruptor based on the total weight of the calcium-binding composition.

[0069] In some embodiments, the additive includes an anti-inflammatory agent (e.g., at least one anti-inflammatory agent). Anti-inflammatory agents may be described as materials capable of reducing inflammation (e.g., at the target site). Anti-inflammatory agents may advantageously promote healing of the target site, such as healing of a lesion. A wide variety of suitable antiinflammatory agents may be used. Suitable anti-inflammatory agents include, for example, dexamethasone, geraniol, D-(-)-salicin, salicylic acid, curcumin, resveratrol, pigallocatechin gallate, capsaicin, lovastatin, anti-inflammatory cytokines, or any combination thereof. The calcium-binding composition may include a wide variety of suitable amounts of antiinflammatory agent. In some embodiments, the calcium-binding composition includes 0.5 wt-% to 10 wt-% of anti-inflammatory agent based on the total weight of the calcium -bindingMedtronic Ref. No. A0011118WOO 1 composition. In some embodiments, the calcium-binding composition includes 0.1 wt-% or greater, 0.5 wt-% or greater, 1 wt-% or greater, 5 wt-% or greater, or 10 wt-% or greater of antiinflammatory agent based on the total weight of the calcium-binding composition. In some embodiments, the calcium-binding composition includes 10 wt-% or less, 5 wt-% or less, 1 wt-% or less, or 0.5 wt-% or less of anti-inflammatory agent based on the total weight of the calcium- binding composition.

[0070] In some embodiments, the additive includes a vessel restoration agent (e.g., at least one vessel restoration agent). Vessel restoration agents may be described as materials that promote the endothelization of the blood vessel wall (i.e., at the target site) to promote or maintain blood flow by counteracting the constricting effect of plaque buildup. A wide variety of suitable vessel restoration agents may be used. Suitable vessel restoration agents include, for example, vascular endothelial growth factor, nitric oxide catalyst selenocystamine (SeCA), imatinib, bivalirudin, flavonoids, statins, and ACE inhibitors (angiotensin-converting enzyme inhibitors).

[0071] In some embodiments, the additive includes microbubbles, microspheres, or the like, which may generally be described as spherical particles, typically ranging in diameter from 1 micrometers (um) to 20 um and, in the case of microbubbles, having a gas core surrounded by an outer shell (e.g., lipid, lipopolymer, polymer, protein, polyelectrolyte etc.). Microbubbles, microspheres, and the like may advantageously improve penetration of the calcium-binding composition (and the calcium-binder, in particular) into the target site. For example, sonication of microbubbles, microspheres, and the like in the calcium-binding composition (as further described herein with regard to the illustrative method 100) may advantageously result in, or contribute to, improved porosity at the treatment site, such as improved porosity in a plaque lesion. The additive may include a wide variety of suitable microbubbles, microspheres, or the like. For example, the additive may include sulfur hexafluoride lipid-type A microspheres (available, e.g., from Bracco Diagnostics, Inc. of Princeton, NJ, under the trade name LUMASON). As another example, the additive may include lipid-encapsulated microbubbles with a gas core (available, e.g., from Revvity, Inc. of Waltham, MA, under the trade name VESSELVUE and from Lantheus Medical Imaging of North Billerica, MA, under the trade name DEFINITY). As yet another example, the additive may include perflutren protein-type A microspheres (available, e.g., from GE Healthcare Technology Systems, Inc. of Chicago, IL, under the trade name OPTISON).Medtronic Ref. No. A0011118WOO 1

[0072] The calcium-binding composition may include a wide variety of suitable amounts of microbubbles, microspheres, or the like based on, e.g., a treatment dose of the calcium-binding composition. Suitable amounts of microbubbles, microspheres, or the like may be selected based on factors, such as, desired strength of microbubbles, microspheres, or the like. In some embodiments, a treatment dose includes 0.1 milliliter (ml) to 1 ml of microbubbles, microspheres, or the like.

[0073] In some embodiments, the additive includes nanoparticles, which may be described as particles of less than one micron in size that can be used in pore formation, in navigation, or to provide therapy and may optionally be loaded or conjugated with additives (e.g., acids). Nanoparticles may advantageously improve penetration of the calcium-binding composition (and the calcium-binder, in particular) into the target site. For example, sonication of nanoparticles in the calcium-binding composition (as further described herein with regard to the illustrative method 100) may advantageously result in, or contribute to, improved porosity at the treatment site, such as improved porosity in a plaque lesion. The additive may include a wide variety of suitable nanoparticles. For example, the nanoparticles may include proteins (e.g., antibodies), peptides, pure drug particles, viral vectors, nucleic acids, lipid or polymer-based vectors, or any combination thereof. In some embodiments, the nanoparticles may be bound to proteins, peptides, or nucleic acids.

[0074] In some embodiments, the additive includes ligand-functionalized microbubbles. Ligand- functionalized microbubbles may be described as microbubbles having shells functionalized with ligands thereon to target specific biomarkers associated with vascular calcification. Biomarkers associated with vascular calcification include, for example, fibroblast growth factor 23 (FGF-23), uncarb oxy lated MGP (matrix Gia protein), Smad 11 / 5 / 8 expression, bone morphogenetic proteins, carbonic anhydrase II (CA II), serum calciprotein particles (CPPs), P-selectin, MP2, Runx2, Wnt / beta-catenin.Methods

[0075] Methods of treating vascular calcifications using the calcium-binding compositions of the present disclosure are described herein.

[0076] FIG. 2 is a flow diagram showing aspects of an illustrative method 100 disclosed herein. The steps may be conducted in any order. In some embodiments, multiple steps may be conducted at the same time. Steps shown in dashed boxes are optional steps. Each optional stepMedtronic Ref. No. A0011118WOO 1 may be performed in a method that includes none or one or more of any additional optional steps (if multiple optional steps are included). For example, a first optional step may be performed in conjunction with one or more additional optional steps; or performed not in conjunction with an additional optional step.

[0077] A flow diagram showing aspects of the illustrative method 100 of treating a vascular calcification disclosed herein is shown in FIG. 2. The method 100 includes delivering (e.g., applying, coating, releasing, injecting, etc.) a calcium-binding composition to a treatment site at step 110. Delivering the calcium-binding composition at step 110 includes delivering a water- soluble biopolymer to the treatment site at step 112 and delivering a calcium-binder to the treatment site at step 114. Delivering the calcium-binding composition at step 110 optionally includes delivering an additive to the treatment site at step 116. In some embodiments, the method 100 optionally includes delivering (e.g., applying, coating, releasing, injecting, etc.) an additive to the treatment site at step 120. In some embodiments, the method 100 optionally includes promoting uptake of the calcium-binding composition into the treatment site at step 130. Promoting uptake of the calcium -binding composition into the treatment site at step 130 may include optional sonoporation at step 132 to increase permeability of biological barriers at or near the treatment site, optionally delivering electric current across the treatment site at step 134 to open pores in the treatment site, and optionally penetrating the treatment site at step 136 to create openings in the treatment site.

[0078] The method 100 results in removal of calcium from the vascular calcification by the calcium-binding composition. A wide variety of suitable techniques (e.g., methods, devices, systems, etc.) may be used to determine (e.g., measure, detect, monitor) a level, or amount, of vascular calcification. Suitable techniques may include, for example, imaging tests such as ultrasound, magnetic resonance angiography (MRA), and computed tomographic (CT) angiography.

[0079] In some embodiments, delivering the water-soluble biopolymer to the treatment site at step 112 and delivering the calcium-binder to the treatment site at step 114 occur concurrently. In some embodiments, the calcium-binder and the water-soluble biopolymer are delivered together (e.g., as a mixture or solution). Delivering the water-soluble biopolymer to the treatment site at step 112 and delivering the calcium-binder to the treatment site at step 114 may optionally occur separately. In such embodiments, the calcium-binding composition may be described as mixed in vivo. In some embodiments, delivering the calcium-binding composition to the treatment site atMedtronic Ref. No. A0011118WOO 1 step 110 includes delivering the water-soluble biopolymer to the treatment site (step 112) and subsequently delivering the calcium-binder to the treatment site (step 114).

[0080] Delivering the calcium -binding composition to the treatment site at step 110 may include using a wide variety of suitable devices, such as a fluid delivery device. Suitable fluid delivery devices may include, for example, a perforated balloon catheter (available, e.g., from Atrium Medical Corporation of Hudson, NH, under the trade name CLEARWAY RX Local Therapeutic Infusion Catheter), a needleless high-pressure balloon injection system (as further described in U.S. Patent No. 7,975,703, entitled “Device and method for needle-less interstitial injection of fluid for ablation of cardiac tissue” and granted July 12, 2011, which is incorporated herein by reference in its entirety), a cartridge dispenser comprising a catheter (available, e.g., from Medtronic, Inc. of Minneapolis, MN under the trade name VENASEAL and from Merit Medical Systems of South Jordan, UT under the trade name CLARIVEIN IC), or any combination thereof. In some embodiments, the fluid delivery device includes a balloon system, such as a double balloon system to occlude blood flow at the target site (available, e.g., from Advanced Catheter Therapies, Inc. of Chattanooga, TN, under the trade name PRESSANA Occlusion Perfusion Catheter).

[0081] Delivering the calcium-binding composition to the treatment site at step 110 may include delivering the calcium-binding composition to a wide variety of suitable treatment sites. Suitable treatment sites generally include vascular calcification (i.e., medial calcification, intimal calcification, or both) and may be selected based on factors such as navigability at the treatment site (e.g., navigability of the vessel lumen adjacent to the target vascular calcification), navigability to the treatment site (e.g., navigability of vessel lumens leading to the target vascular calcification), and extent of calcification, as examples. In some embodiments, the target site includes a vascular wall or a calcified vascular wall. In some embodiments, the treatment site includes a medial vascular calcification. In some embodiments, the treatment site includes an intimal vascular calcification. In some embodiments, the treatment site includes a plaque lesion. In some embodiments, the treatment site includes a total occlusion (e.g., a chronic total occlusion). In some embodiments, delivering the calcium-binding composition to the treatment site at step 110 includes delivering the calcium -binding composition to portions of a blood vessel proximate to (e.g., adjacent to) and upstream of the vascular calcification or portions of a blood vessel proximate to (e.g., adjacent to) and downstream of the vascular calcification.Medtronic Ref. No. A0011118WOO 1

[0082] The calcium -binding composition may be delivered to the treatment site at step 110 using a wide variety of suitable techniques (e.g., method, device, system, etc.). Suitable delivery techniques may be selected based on factors such as navigability at the treatment site (e.g., navigability of the vessel lumen adjacent to the target vascular calcification), navigability to the treatment site (e.g., navigability of vessel lumens leading to the target vascular calcification), and the form of the calcium-binding composition (e.g., aqueous solution, hydrogel, film, sponge, etc.), as examples.

[0083] In some embodiments, the calcium-binding composition is delivered to the treatment site at step 110 by releasing (e.g., injecting, dispensing, extruding, etc.) the calcium-binding composition at the target site, such as into a blood vessel at or upstream of the treatment site. Releasing the calcium-binding composition at the target site may be suitable, for example, in embodiments where the calcium-binding composition is in an injectable, dispensable, or extrudable form (e.g., in the form of an aqueous solution, a hydrogel, etc.). In some embodiments, the calcium -binding composition is delivered to the treatment site at step 110 by coating or applying the calcium-binding composition onto a surface of the treatment site, such as onto a plaque cap of a plaque lesion or onto a surface of the blood vessel’s intima proximate to (e.g., adjacent to) a calcification (e.g., a medial calcification). Coating or applying the calcium- binding composition onto a surface of the treatment site may be suitable, for example, in embodiments where the calcium-binding composition is in the form of a hydrogel or a film. In some embodiments, the calcium-binding composition is delivered to the treatment site at step 110 by disposing the calcium-binding composition adjacent to or in direct contact with a surface of the treatment site, such as a plaque cap of a plaque lesion or a surface of the blood vessel’s intima proximate to (e.g., adjacent to) a medial calcification. Disposing the calcium-binding composition adjacent to or in direct contact with a surface of the treatment site may be suitable, for example, in embodiments where the calcium-binding composition is in the form of a film or a sponge.

[0084] In one or more embodiments, the calcium-binding composition is delivered to the treatment site at step 110 by disposing or compressing the calcium-binding composition in contact with a surface of the treatment site, such as a plaque cap of a plaque lesion or a surface of the blood vessel’s intima proximate to (e.g., adjacent to) a medial calcification using a balloon, a stent, a plug, a filter, or any combination thereof. Disposing, or compressing, the calcium- binding composition in contact with a surface of the treatment site using a balloon, a stent, aMedtronic Ref. No. A0011118WOO 1 plug, a filter, or the like may be suitable, for example, in embodiments where the calcium- binding composition is in the form of a film, a sponge, or a hydrogel. In some embodiments, the calcium-binding composition may be applied to an expandable device, such as a balloon or an expandable stent, which may be coated with the calcium-binding composition and then expanded at the treatment site to thereby affect contact between the calcium-binding composition and the treatment site. The expandable device may be coated with the calcium-binding composition, for example, by spraying, dipping, needle deposition, and the like. In some embodiments, the expandable device may be expanded at the treatment site after the calcium-binding composition has been delivered (i.e., after step 110) to help maintain contact between the calcium-binding composition and the treatment site. In other words, the expandable device may be used to keep the calcium-binding composition in place after the calcium-binding composition has been delivered to the treatment site. Using expandable devices to deliver the calcium-binding composition or to keep the calcium-binding composition in place at the treatment site may advantageously facilitate reaction between the calcium-binder and the vascular calcification, and thereby improve calcium removal.

[0085] In illustrative methods of the present disclosure utilizing balloons, a wide variety of suitable balloons (e.g., angioplasty balloons, balloon catheters, balloon systems, coated balloons, etc.) may be used. Suitable balloons may include, for example, NANOCROSS Elite PTA catheter, FORTREX HP PTA balloon catheter, EVERCROSS PTA catheter, RAPIDCROSS RX PTA catheter, PACIFIC Plus PTA catheter, and ADMIRAL XTREME PTA balloon catheter, each available from Medtronic, Inc. of Minneapolis, MN.

[0086] In illustrative methods of the present disclosure utilizing stents, a wide variety of suitable stents (e.g., self-expandable stents, balloon-expandable stents, bioresorbable stents, etc.) may be used. Suitable stents may include, for example, COMPLETE SE Stent System, VISI-PRO balloon-expandable peripheral stent system, and EVERFLEX self-expanding peripheral stent system, each available from Medtronic, Inc. of Minneapolis, MN. In some embodiments, resorbable polymer stents are used (available, e.g., from Reva Medical of San Diego, CA, under the trade name MOTIV or from Abbot of Green Oaks, IL, under the trade name ESPIRIT BTK resorbable scaffold).

[0087] The method 100 optionally includes promoting uptake of the calcium -binding composition into the treatment site at step 130. Improving uptake of the calcium-binding composition into the treatment site may advantageously increase interaction between theMedtronic Ref. No. A0011118WOO 1 calcium-binder and calcium salts of the vascular calcification, or improve transport / efficiency of the calcium-binder, thereby improving removal of the vascular calcification.

[0088] In one or more embodiment, promoting uptake of the calcium-binding composition into the treatment site at step 130 includes sonoporation at or near the treatment site at step 132. Sonoporation may be described as delivery of ultrasonic energy to the treatment site (i.e., sonicating the treatment site) to thereby create pores in the vessel wall via acoustic radiation forces, hyperthermia, or effects of cavitation. Opening pores in the intimal wall may thereby improve uptake of the calcium-binding composition, particularly the calcium-binder, into the treatment site by providing openings for the calcium-binding composition to travel through. Sonication may be delivered to the treatment site endovascularly, externally to the body, or both. In some embodiments, sonoporation at step 132 includes delivering microbubbles or nanoparticles to the treatment site (e.g., injecting, dispensing, extruding, etc. microbubbles / nanoparticles concurrent to delivering the calcium-binding composition to the treatment site at step 110, as part of delivering the calcium -binding composition to the treatment site at step 110, or as an independent step) and sonicating the microbubbles or nanoparticles at a suitable amplitude and frequency to induce cavitation, thereby opening pores in the vessel wall. In some embodiments, sonoporation at step 132 includes delivering ultrasonic energy to the calcium-binding composition at a suitable frequency and amplitude to generate cavitation bubbles in the calcium-binding composition.

[0089] In at least one embodiment, promoting uptake of the calcium-binding composition into the treatment site at step 130 includes delivering electrical current across the treatment site at step 134. Delivering electrical current across the treatment site may temporarily open pores in the treatment site (e.g., in the vascular wall), which may be described as electroporation. Opening pores in the treatment site may advantageously improve uptake of the calcium-binding composition, particularly the calcium-binder, into the treatment site by providing openings for the calcium-binding composition to travel through.

[0090] In some embodiments, promoting uptake of the calcium-binding composition into the treatment site at step 130 includes application of local hyperthermia, for example, using high- frequency ultrasound, radiofrequency, light energy, or any combination thereof.

[0091] In one or more embodiments, promoting uptake of the calcium-binding composition into the treatment site at step 130 includes penetrating the treatment site at step 136 to thereby create openings in the treatment site for the calcium-binder to travel into the treatment site. In someMedtronic Ref. No. A0011118WOO 1 embodiments, penetrating the treatment site at step 136 may include penetrating the treatment site with penetrating protrusions (e.g., microprotrusions, microneedles, pins, spikes, etc.) (available, e.g., from Reflow Medical, Inc. of San Clemente, CA, under the trade name SPUR Retrievable Scaffold Therapy or from Mercator MedSystems, Inc. of San Leandro, CA, under the trade name BULLFROG).

[0092] It should be understood that various aspects disclosed herein may be combined in different combinations than the combinations specifically presented in the description and accompanying drawings. It should also be understood that, depending on the example, certain acts or events of any of the processes or methods described herein may be performed in a different sequence, may be added, merged, or left out altogether (e.g., all described acts or events may not be necessary to carry out the techniques). In addition, while certain aspects of this disclosure are described as being performed by a single module or unit for purposes of clarity, it should be understood that the techniques of this disclosure may be performed by a combination of units or modules associated with, for example, a medical device.

[0093] In one or more examples, the described techniques may be implemented in hardware, software, firmware, or any combination thereof. If implemented in software, the functions may be stored as one or more instructions or code on a computer-readable medium and executed by a hardware-based processing unit. Computer-readable media may include computer-readable storage media, which corresponds to a tangible medium such as data storage media (e.g., RAM, ROM, EEPROM, flash memory, or any other medium that can be used to store desired program code in the form of instructions or data structures and that can be accessed by a computer).

[0094] Instructions may be executed by one or more processors, such as one or more digital signal processors (DSPs), general purpose microprocessors, application specific integrated circuits (ASICs), field programmable logic arrays (FPGAs), or other equivalent integrated or discrete logic circuitry.ILLUSTRATIVE ASPECTS

[0095] Aspect 1 is a calcium-binding composition comprising: a water-soluble biopolymer; and a calcium-binder. Aspect 2 is a method for treating a vascular calcification, the method comprising: delivering to a treatment site a calcium-binding composition comprising: a water-soluble biopolymer; and a calcium-binder; wherein the calcium-binding composition removes calcium from the vascular calcification.Medtronic Ref. No. A0011118WOO 1

[0096] Aspect 3 is the composition according to aspect 1 or the method according to aspect 2, wherein the water-soluble biopolymer comprises collagen, porcine collagen, biosynthetic collagen, at least partially denatured collagen, at least partially oxidized collagen, at least partially crosslinked collagen, dehyrothermal treated collagen, freeze-dried collagen, methacrylate collagen, or any combination thereof. Aspect 4 is the method or the composition according to any one of aspects 1 through 3, wherein the calcium-binding composition comprises 0.1 wt-% to 70 wt-% of the water-soluble biopolymer based on the weight of the calcium-binding composition. Aspect 5 is the method or the composition according to any one of aspects 1 through 4, wherein the calcium-binding composition comprises 1 wt-% to 30 wt-% of the calcium-binder based on the weight of the calcium-binding composition.

[0097] Aspect 6 is the method or the composition according to any one of aspects 1 through 5, wherein the calcium-binding composition has a pH value of 1 to 4. Aspect 7 is the method or the composition according to any one of aspects 1 through 6, wherein the calcium-binding composition has a pH value of 6 to 8.

[0098] Aspect 8 is the method or the composition according to any one of aspects 1 through 7, wherein the water-soluble biopolymer is substantially free of telopeptides. Aspect 9 is the method or the composition according to any one of aspects 1 through 8, wherein the water-soluble biopolymer in the form of an aqueous solution, a viscous fluid, a hydrogel, a film, a powder, a sponge, or any combination thereof.

[0099] Aspect 10 is the method or the composition according to any one of aspects 1 through 9, wherein the calcium-binding composition further comprises at least one additive. Aspect 11 is the method or the composition according to aspect 10, wherein the at least one additive comprises an osteoclast promotor, the osteoclast promotor comprising an osteoclast chemoattractant, a compound that switches cell phenotype towards osteoclast development, a cytokine, vascular endothelial growth factor, M-CSF (macrophage-colony stimulating factor), RANKL (receptor activator of nuclear factor-K ligand), IL-6 (Interleukin 6), Prostaglandin E2, or any combination thereof. Aspect 12 is the method or the composition according to aspect 10 or 11, wherein the at least one additive comprises a cell disruptor, the cell disruptor optionally comprising dimethyl sulfoxide, EDTA, an alcohol, an ether, glucose, a surfactant, or any combination thereof. Aspect 13 is the method or the composition according to any one of aspects 10 through 12, wherein the at least one additive comprises ligand-functionalized microbubbles configured to target fibroblast growth factor 23, uncarb oxy lated matrix Gia protein, Smad 11 / 5 / 8 expression, boneMedtronic Ref. No. A0011118WOO 1 morphogenetic proteins, carbonic anhydrase II, serum calciprotein particles, P-selectin, MP2, Runx2, Wnt / beta-catenin, or any combination thereof. Aspect 14 is the method or the composition according to any one of aspects 10 through 13, wherein the at least one additive comprises microbubbles optionally comprising phospholipid monolayers or phospholipid bilayers. Aspect 15 is the method or the composition according to any one of aspects 10 through 14, wherein the at least one additive comprises nanoparticles comprising proteins, peptides, nucleic acids, or any combination thereof. Aspect 16 is the method or the composition according to any one of aspects 10 through 15, wherein the at least one additive comprises nanoparticles bound to proteins, peptides, nucleic acids, or any combination thereof. Aspect 17 is the method or the composition according to any one of aspects 10 through 16, wherein the at least one additive comprises an anti-inflammatory agent, a vessel restoration agent, an anti-restenotic agent, or any combination thereof.

[0100] Aspect 18 is the method or the composition according to any one of aspects 1 through 17, wherein the calcium-binder comprises EDTA, citric acid, ascorbic acid, acetic acid, N- acetylneuraminic acid, or any combination thereof. Aspect 19 is the method or the composition according to any one of aspects 1 through 18, wherein the calcium-binder comprises citric acid, wherein the calcium-binding composition has a pH value of 1 to 4, and wherein the calcium- binding composition comprises 1 wt-% to 30 wt-% of the citric acid based on the weight of the calcium-binding composition. Aspect 20 is the method or the composition according to any one of aspects 1 through 19, wherein the water-soluble biopolymer comprises a biopolymer selected from the group consisting of collagen, alginate, chitosan, and hyaluronic acid.

[0101] Aspect 21 is the method according to any one of aspects 2 through 20, further comprising positioning a stent proximate to the treatment site and optionally expanding the stent to compress the calcium-binding composition in contact with a surface of the treatment site. Aspect 22 is the method according to any one of aspects 2 through 21, further comprising compressing, using a stent, a balloon, or both, the calcium-binding composition in contact with a surface of the treatment site.

[0102] Aspect 23 is the method according to any one of aspects 2 through 22, wherein the calcium-binding composition further comprises nanoparticles, microbubbles, or both, and wherein the method further comprises sonicating the nanoparticles, microbubbles, or both at the treatment site. Aspect 24 is the method according to any one of aspects 2 through 23, further comprising: delivering to the treatment site nanoparticles, microbubbles, or both; and sonicatingMedtronic Ref. No. A0011118WOO 1 the nanoparticles, microbubbles, or both at the treatment site. Aspect 25 is the method according to any one of aspects 2 through 24, further comprising penetrating, optionally using penetrating protrusions, the treatment site to thereby create openings in the treatment site for the calcium- binder to travel into the treatment site. Aspect 26 is the method according to any one of aspects 2 through 25, further comprising delivering an electric current across the treatment site to thereby open pores in the treatment site for the calcium-binder to travel into the treatment site.

[0103] Aspect 27 is the method according to any one of aspects 2 through 26, wherein delivering to the treatment site the calcium-binding composition comprises delivering the water-soluble biopolymer to the treatment site and subsequently delivering the calcium-binder to the treatment site. Aspect 28 is the method according to any one of aspects 2 through 27, further comprising delivering at least one additive to the treatment site. Aspect 29 is the method according to any one of aspects 2 through 28, wherein delivering to the treatment site the calcium-binding composition comprises positioning the calcium-binding composition against the treatment site using a balloon, a stent, a plug, a filter, or any combination thereof. Aspect 30 is the method according to any one of aspects 2 through 29, wherein delivering to the treatment site the calcium-binding composition comprises applying the calcium-binding composition to the treatment site using a perforated balloon catheter, a needleless high-pressure balloon injection system, a cartridge dispenser comprising a catheter, a balloon system, or any combination thereof.

[0104] Aspect 31 is the method according to any one of aspects 2 through 30, wherein the treatment site is a vascular wall or a calcified vascular wall. Aspect 32 is the method according to any one of aspects 2 through 31, wherein the water-soluble biopolymer is in the form of an aqueous solution, a viscous fluid, a hydrogel, a powder, or a film, and wherein delivering the calcium-binding composition to the treatment site comprises applying the aqueous solution, the hydrogel, or the film to the treatment site. Aspect 33 is the method according to any one of aspects 2 through 32, wherein the water-soluble biopolymer is in the form of a sponge, and wherein delivering to the treatment site the calcium-binding composition comprises positioning the sponge proximate to the treatment site. Aspect 34 is the method according to any one of aspects 2 through 33, wherein the vascular calcification comprises a plaque lesion, and wherein the method optionally further comprises compressing, using a stent, a balloon, or both, the calcium -binding composition in contact with a surface of the plaque lesion. Aspect 35 is the method according to any one of aspects 2 through 34, wherein the vascular calcificationMedtronic Ref. No. A0011118WOO 1 comprises medial calcification and wherein the method optionally further comprises compressing, using a stent, a balloon, or both, the calcium-binding composition in contact with a surface of the target site proximate the medial calcification. Aspect 36 is the method according to any one of aspects 2 through 35, wherein the vascular calcification comprises intimal calcification and wherein the method optionally further comprises compressing, using a stent, a balloon, or both, the calcium-binding composition in contact with a surface of the intimal calcification.EXAMPLES

[0105] These Examples are merely for illustrative purposes and are not meant to be overly- limiting on the scope of the appended claims. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values set forth in the specific Examples are reported as precisely as possible. Any numerical value, however, inherently contains certain uncertainty necessarily resulting from the standard deviation found in their respective testing measurements. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0106] Unless otherwise noted, all parts, percentages, ratios, etc. in the Examples and the rest of the specification are by weight.

[0107] The following abbreviations may be used in the following examples or other places in this disclosure: ppm = parts per million; ppb = parts per billion; ml = milliliter; L = liter; LPM = liters per minute; m = meter; mm = millimeter; cm = centimeter; um = micrometer; kg = kilogram, g = gram, ug = microgram; mg = milligram; min = minute; s = second; h = hour; °C = degrees Celsius, wt-% = weight percent; and DI water = deionized water.

[0108] Table l is a materials table giving a list of components used in the Examples and their associated source (e.g., vendor), abbreviation, and chemical abstract service (CAS) number.Table 1. Materials used in the Examples.Medtronic Ref. No. A0011118WOO 1Example 1A: Preparing hydrogel calcium-binding compositions.

[0109] Method 1. Illustrative hydrogels with calcium-binder were prepared from the gelation of a denatured collagen solution. Briefly the collagen was solubilized in hot distilled water to denature the native structure of the collagen (i.e., the helicoidal structure). The solubilization conditions were determined in order to prevent the hydrolysis of polypeptides chains constituting the collagen. A calcium-binder quantity was then added to the denatured solution. After homogenization, the solution was casted and cooled into silicon molds. Like a gelatin solution, the denatured collagen solution formed a hydrogel by cooling. The concentrations of collagen in the hydrogels of Method 1 were 1.0 wt-% to 10 wt-%. The concentrations of calcium-binder in the hydrogels of Method 1 were 5.0 wt-% to 20 wt-%. The pH values of the hydrogels of Method 1 were 1.5 to 3.0.

[0110] Method 2. Collagen fibers were modified to add acrylamide groups on the backbone of the collagen. Briefly, the collagen was solubilized in hot distilled water. Then, N- acryloxysuccinimide was added to the solution and the pH value was adjusted to 7.0. After the reaction between collagen and N-acryloxysuccinimide, the solution was cooled at 20° C to gel the denatured collagen. The gel was washed several times with distilled water to remove excess N-acryloxysuccinimide and reaction byproducts. The gel was freeze-dried to obtain fibers of acrylamide collagen. Using the fibers of acrylamide collagen, hydrogels with calcium-binderMedtronic Ref. No. A0011118WOO 1 were prepared. The acrylamide collagen was briefly solubilized in hot distilled water and a calcium-binder quantity was added to the solution. Then, a photoinitiator (LAP), was added to the solution. After the complete solubilization of the LAP, the solution was casted and cooled at 20° C to gel the denatured collagen. After the gelation of the solution, the hydrogel was exposed to UV light to form chemical crosslinks between the acrylamide groups. The concentrations of acrylamide collagen in the hydrogels of Method 2 were 1.0 wt-% to 10 wt-%. The concentration of calcium-binder in the hydrogels of Method 2 were 5.0 wt-% to 20 wt-%. The concentrations of LAP in the hydrogels of Method 2 were 0.01 wt-% to 0.3 wt-%. The pH values of in the hydrogels of Method 2 were 1.5 to 3.0.[OHl] Method 3. Unmodified collagen and the acrylamide collagen of Method 2 were mixed. The acrylamide collagen was briefly solubilized in hot distilled water and then the unmodified collagen fibers were added. After solubilization and denaturation of the unmodified collagen, the calcium-binder and the photoinitiator (LAP) were added into the solution. The solution was casted and cooled at 20° C to gel the denatured collagen. After the gelation of the solution, the hydrogel was exposed to UV light to form chemical crosslinks between the acrylamide groups. The concentrations of unmodified collagen in the hydrogels of Method 3 were 0.5 wt-% to 10 wt- %. The concentrations of acrylamide collagen in the hydrogels of Method 3 were 0.1 wt-% to 2.0 wt-%. The concentrations of calcium-binder in the hydrogels of Method 3 were 5.0 wt-% to 20 wt-%. The concentrations of LAP in the hydrogels of Method 3 were 0.01 wt-% to 0.3 wt-%. The pH values of the hydrogels of Method 3 were 1.5 to 3.0.Example IB: Evaluating the calcium-binder release kinetics of hydrogels.

[0112] Formulations of illustrative hydrogels prepared according to the Methods described in Example IB are shown in Table 2.Table 2. Formulations tested in Example IB.Medtronic Ref. No. A0011118WOO 1

[0113] A cylindrical portion of hydrogel with a thickness of 1 cm was put into saline solution (0.9 % NaCl) at 37° C. To assess the release kinetics of citric acid from the hydrogel into the solution over the time, various samples were collected after immersion of the hydrogel. The quantity of citric acid into the samples was determined by acid-base titration. Graphs of the results of the hydrogel release kinetics evaluations are shown in FIGS. 3 A and 3B.

[0114] A graph of the measured citric acid release kinetics for Examples A, B, and C of Table 2 is shown in FIG 3 A. A graph of the measured citric acid release kinetics for Example A (prepared according to Method 1), Example F, and Example H of Table 2 is shown in FIG 3B. Hydrogels having only unmodified denatured collagen (Example A) were observed to dissolve relatively rapidly in saline solution at 37° C, leading to a nearly immediate release of almost all the quantity of citric acid in the hydrogel. Hydrogels having acrylamide collagen and having been photocrosslinked with UV light (Examples B and C) were observed to last longer at 37° C and, consequently, the release of citric acid was observed to be slower. Example C, having less acrylamide collagen compared to Example B was observed to exhibit faster release of the citric acid.Example 2A: The ability of calcium-binders to dissolve natural hydroxyapatite (bone).

[0115] Various solutions of calcium-binders were tested to determine the effect of the various calcium-binders on natural hydroxyapatite (bovine bones). Bone samples of approximately 2 grams (g) each were immersed in 250 ml of aqueous solutions of various calcium-binders. The calcium content of the bone samples was determined before and after testing by Inductively Coupled Plasma Optical Emission spectroscopy (ICP). To prepare samples for ICP, each sample was calcined at 700° C for 10-12 hours to remove the bone organic matrix. All bone samples were weighed with an analytical balance and visually inspected before and after exposure. 25 ml of each reaction solution was collected after 24 hours. These solutions were analyzed to determine the calcium concentration using ICP, the results of which are shown in Table 3.Medtronic Ref. No. A0011118WOO 1Table 3. Calcium-binders tested in Example 2A and results.Example 2B: The ability of collagen-based hydrogels to dissolve natural hydroxyapatite (bone).

[0116] Various calcium-binding compositions prepared in the form of collagen-based hydrogels in accordance with illustrative methods and compositions described in the present disclosure were tested to determine the effect of the various calcium-binding compositions on natural hydroxyapatite (bovine bones). Hydrogel calcium-binding compositions including unmodified collagen were prepared according to Table 4. Bovine bone samples of approximately 0.4 g were immersed for 24 hours in 5 ml of a respective hydrogel calcium-binding composition.Table 4. Compositions tested in Example 2B and results after 1 hour.Medtronic Ref. No. A0011118WOO 1

[0117] The bone samples were characterized to evaluate bone mass before and after exposure. Change in mass of the bone samples is shown in Table 4. After exposure, the reaction solutions were characterized to evaluate the concentration of calcium in solution using ICP analysis. Calcium concentration of the reaction solutions after exposure are shown in Table 4.

[0118] After exposure, all bone samples except for the reference (water only) were observed to be discolored, to be rougher on the surface, and to have portions of granular material in the structure. All bone samples except for the reference (water only) showed a decrease in weight ranging from approximately 6 % to 19 %. The hydrogels having 5 wt-% citric acid, 10 wt-% citric acid, and 8 wt-% EDTA were observed to provide the best results both in terms of reduction in bone sample mass and content of calcium in reaction solutions.Example 2C: Determination of the ability of collagen-based films to dissolve synthetic hydroxyapatite (powder)

[0119] Various calcium-binding compositions prepared in the form of collagen-based films in accordance with illustrative methods and compositions described in the present disclosure were tested to determine the effect of the various film calcium-binding compositions on synthetic hydroxyapatite (powder). Film calcium-binding compositions including unmodified collagen were prepared according to Table 5. Hydroxyapatite samples were obtained by immersing 100 mg of hydroxyapatite and the respective film in 3 ml of water. After 24 hours of exposure, the samples were diluted to 8 ml.Medtronic Ref. No. A0011118WOO 1Table 5. Compositions tested in Example 2C and results.

[0120] The reaction solutions were characterized to evaluate the concentration of calcium in solution using ICP analysis. Based on the results shown in Table 5, several film calcium-binding compositions were observed to be very effective in dissolving synthetic hydroxyapatite.Example 2D: Determination of the ability of collagen-based hydrogels with citric acid to dissolve synthetic hydroxyapatite (powder)

[0121] Various calcium-binding compositions prepared in the form of collagen-based hydrogels in accordance with illustrative methods and compositions described in the present disclosure and having citric acid concentrations up to 30 wt-% were tested to determine the effect of the various calcium-binding compositions on synthetic hydroxyapatite (powder). Hydrogel calcium-binding compositions (Formulations 1-17), as well as comparative aqueous citric acid solutions (C.A. 1 and 2), were prepared according to Table 6. The hydrogel calcium-binding compositions included various concentrations of acrylamide modified collagen (ADC), pepsin-soluble type I collagen (PSC), citric acid, and Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, a photoinitiator). 1 gram of each hydrogel calcium-binding composition was coated onto Nylon 12 (PA 12) plates. Hydroxyapatite samples were obtained by immersing 30 mg of hydroxyapatiteMedtronic Ref. No. A0011118WOO 1 and the respective hydrogel calcium-binding composition or the respective comparative aqueous citric acid solution in 50 ml of water for 24 hours at 37° C.Table 6. Compositions tested in Example 2D and results after 1 hour.

[0122] The reaction solutions were characterized to evaluate the concentration of calcium in solution (“Avg. Ca (ug / g)” in Table 6) using ICP analysis. Based on the results shown in Table 6, several hydrogel calcium -binding compositions having citric acid concentrations up to 30 wt-% were observed to dissolve calcium at similar rates compared to aqueous solutions having comparable concentrations of citric acid.Medtronic Ref. No. A0011118WOO 1

[0123] Regression analysis of the results shown in Table 6 yielded the following regression formula:[Ca2+] = 65 + 17.9[C.A.] - 0.4[C.A.]2

[0124] Wherein [Ca2+] is the calcium concentration in micrograms per g and [C.A.] is the citric acid concentration in wt-%. Based on the regression analysis, it was observed that the calcium- dissolution process of the hydrogel calcium-binding compositions may be described by a quadratic model as a function of citric acid concentration. Further based on the regression analysis, it was observed that a citric acid concentration of about 22 wt-% may be expected to afford the most effective calcium dissolution.Example 3A: Determination of the ability of calcium-binder solutions to dissolve calcification on highly calcified human ex-vivo arteries.

[0125] Various solutions of calcium-binders were tested to determine the effect of the various calcium-binders on calcified human ex-vivo arteries. Calcified arterial tissues evaluated were harvested from cadavers after each cadaver had been used for an unrelated study. Ex-vivo calcified artery samples of approximately 1 g were immersed for 24 hours in 20 ml aqueous solutions of various calcium-binders at room temperature. After 1 hour of exposure and after 24 hours of exposure, the reaction solutions were characterized to evaluate the concentration of calcium in solution using micro-CT analysis. Additionally, before exposure and after 24 hours of exposure, the ex-vivo artery samples were analyzed using MicroCT imaging to evaluate the change in density of hyaluronic acid (HA). The calcium-binder solutions tested, the calcium concentrations of the reaction solutions after 1 hour and after 24 hours, and the change in density of the ex-vivo artery samples are shown in Table 7.Table 7. Calcium-binder solutions tested in Example 3 A and results.Medtronic Ref. No. A0011118WOO 1

[0126] Based on the results in Table 7, it was observed that vascular calcifications were adequately dissolved by the calcium-binder solutions.

[0127] After exposure for 1 hour and 24 hours, the ex-vivo calcified artery samples were visually inspected. Based on the visual inspection, it was observed that the tissues appeared to be degraded.Example 3B: Determination of the effect of collagen-based hydrogels on highly calcified human ex-vivo arteries.

[0128] Various calcium-binding compositions prepared in the form of hydrogels in accordance with illustrative methods and compositions described in the present disclosure were tested to determine the effect of the various calcium-binding compositions on calcified human ex-vivo arteries. Ex-vivo calcified artery samples of approximately 1 g were immersed for 1 hour in 20 ml of respective hydrogel calcium-binding compositions of unmodified collagen (1.5 wt-%) and various calcium-binders (10 wt-%). After exposure, the reaction solutions were characterized to evaluate the concentration of calcium in solution using micro-CT analysis. Calcium-binders used in each hydrogel calcium-binding composition and calcium concentration of the reaction solutions after 1 hour are shown in Table 8.Table 8. Compositions tested in Example 3B and results.Medtronic Ref. No. A0011118WOO 1

[0129] All hydrogel calcium-binding composition samples were observed to dissolve vessel calcifications compared with the reference water samples. The citric acid hydrogels (samples 1-3 of Table 8) and EDTA hydrogels (samples 7-9 of Table 8) were observed to partially dissolve vascular calcifications after just 1 hour of treatment.

[0130] After exposure, the ex-vivo calcified artery samples were visually inspected. Based on the visual inspection, it was observed that the tissues exposed to the hydrogel calcium-binding compositions appeared to be swollen, discolored, and partially degraded.

[0131] After exposure, three ex-vivo calcified artery samples exposed to Hydrogel 1 (1.5 wt-% collagen and 10 wt-% citric acid) were also analyzed using MicroCT imaging, which provides high resolution X-ray images of tissue samples, allowing detailed analysis of small features such as microcalcifications. MicroCT images of the ex-vivo calcified arteries before and after exposure to the hydrogel are shown in FIG. 4A (before exposure) and FIG. 4B (after exposure). In FIGS. 4A and 4B, tissues appear light gray and calcified portions appear white. As shown in FIGS. 4A and 4B, calcified portions of the artery samples observed before the exposure were observed to be gone after the exposure.Medtronic Ref. No. A0011118WOO 1Example 3C: Determination of the effect of collagen-based films on highly calcified human ex-vivo arteries.

[0132] A calcium-binding composition prepared in the form of films in accordance with illustrative methods and compositions described in the present disclosure was tested to determine the effect of the various calcium-binding compositions on calcified human ex-vivo arteries. Three cylindrical pieces of artery were each treated with an angioplasty balloon equipped with a VISIPRO balloon-expandable peripheral stent system (available from Medtronic, Inc. of Minneapolis, MN). A sample of the film calcium-binding composition including unmodified collagen had previously been applied to the surface of each stent in order to completely cover the area. After deployment of the stent by inflating the balloon to 8 atm, the films were adherent to respective vascular walls. The films and stents were left to react for 24 hours under a flow of water at 37° C and pumped at 70 revolutions per minute (RPM). The film composition was prepared according to Table 9.Table 9. Film composition tested in Example 3C.

[0133] After 24 hours under the flow of water, each film was observed to have completely dissolved. After exposure, the ex-vivo calcified arteries were visually inspected. Images of the ex-vivo calcified arteries after exposure with the film are shown in FIG. 5. In visual inspection of the artery samples after exposure, a change in color to red-brown (seen as daker portions in FIG. 5) was observed in the treated area compared with the untreated area.Medtronic Ref. No. A0011118WOO 1Example 3D: Determination of the effect of collagen-based hydrogels with citric acid on highly calcified human ex-vivo arteries

[0134] Various calcium-binding compositions prepared in the form of collagen-based hydrogels in accordance with illustrative methods and compositions described herein and having citric acid concentrations up to 30 wt-% were tested to determine the effect of the various calcium -binding compositions on calcified human ex-vivo arteries. Hydrogel calcium-binding compositions were prepared according to Table 10. The hydrogel calcium-binding compositions included various concentrations of acrylamide modified collagen (ADC), pepsin-soluble type I collagen (PSC), citric acid, and Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, a photoinitiator). 1 gram of each hydrogel calcium-binding composition was coated onto Nylon 12 (PA 12) plates. Ex-vivo calcified artery samples of approximately 1 g were placed in close contact with a PA 12 plate coated with a respective hydrogel calcium-binding composition and immersed in deionized water for 24 hours at 37° C. Reference ex-vivo calcified artery samples were placed in contact with an un-coated PA 12 plate and immersed in deionized water for 24 hours at 37° C.

[0135] Table 10. Compositions tested in Example 3D and results.

[0136] After exposure, the reaction solutions were characterized to evaluate the concentration of calcium in solution (“Avg. Ca (ug / g)” in Table 10) using ICP analysis. Based on the results shown in Table 10, it was observed that each of formulations 14, 18, and 19 were able to dissolve vascular calcification in human tissues. It was further observed that greater concentrations of citric acid were associated with greater dissolution of vascular calcification, with formulation 14 (having 30 wt-% citric acid) showing greater dissolution of vascular calcification than the other formulations tested.

[0137] After exposure, each ex-vivo calcified artery sample were also analyzed using MicroCT imaging. MicroCT images of the reference ex-vivo calcified arteries before and after exposure to the DI water are shown in FIG. 9A (before) and FIG. 9B (after). In FIGS. 9A and 9B, tissuesMedtronic Ref. No. A0011118WOO 1 appear light gray and calcified portions appear white. As can be seen in FIGS. 9 A and 9B, no significant differences in calcification density (approximately 5 %) were observed following the reference exposure to deionized water.

[0138] MicroCT images of the ex-vivo calcified arteries before and after exposure to the collagen-based hydrogel according to Formulation ID 14, having 30 wt-% citric acid are shown in FIG. 10A (before) and FIG. 10B (after). In FIGS. 10A and 10B, tissues appear light gray and calcified portions appear white. As can be seen in FIGS. 10A and 10B, it was observed that the density of calcification was significantly reduced (by approximately 65 % to 70 %) following exposure to the collagen-based hydrogel according to Formulation ID 14, having 30 wt-% citric acid.Example 4: Histopathological evaluation of mineralization within the tunica media and of tissue degradation / autolysis.

[0139] One artery from the same donor was cut into nine equal portions and these were then immersed in three different solutions for 24 hours. Group A was immersed in deionized water. Group B was immersed in citric acid in water (120 mg / ml). Group C was immersed in a hydrogel in accordance with illustrative methods and compositions described in the present disclosure, having 10 % unmodified collagen and 120 mg / ml citric acid. All samples were in previously- labeled 50 ml conical tubes containing 10 % formalin. Each tube contained three sections of previously cut cadaveric tissue. One cross section and two longitudinal sections were taken from each of the samples. There were 9 cassettes in total.

[0140] To evaluate tunica media mineralization, a histological key was used. An observation that no mineralization is present corresponds to a score of 0 (none). A score of 1 (minimal) corresponds to mineralization affecting less than 10 % of the tunica media. A score of 2 (mild) corresponds to mineralization affecting 10-25 % of the tunica media. A score of 3 (moderate) corresponds to mineralization affecting 25-50 % of the tunica media. A score of 4 (severe) corresponds to mineralization affecting greater than 50 % of the tunica media.

[0141] Similarly, to evaluate tissue degradation, a histological key was used. A score of 0 (none) corresponds to observations of no hypereosinophilic staining globally, no loss of nuclear differential staining, clear cytoplasmic features, clear cellular borders, visible smooth muscle within the tunica media, endothelial present in small-medium caliber vessels, and adipocytes clearly defined. A score of 1 (minimal) corresponds to observations of minimal hypereosinophilicMedtronic Ref. No. A0011118WOO 1 staining globally, minimal loss of nuclear differential staining, cytoplasmic features can be clearly identified, cellular borders can be defined, smooth muscle exhibits minimal shrinkage with minimal hypereosinophilic cytoplasm, endothelium present in small-medium caliber vessels, and adipocytes minimally imperceptible. A score of 2 (mild) corresponds to mild hypereosinophilic staining globally, mild loss of nuclear differential staining with few pyknotic / fading nuclei, cytoplasmic features mildly imperceptible, cellular borders mildly faint to imperceptible, smooth muscle cytoplasm exhibits mild fragmentation, hypereosinophilic staining and loss of differential nuclear staining, endothelium imperceptible in small-medium caliber vessels, and adipocytes inapparent. A score of 3 (moderate) corresponds to moderate hypereosinophilic staining globally, loss of nuclear differential staining with moderate pyknotic / fading nuclei, cytoplasmic features moderately imperceptible to inapparent, cellular borders moderately imperceptible to inapparent, smooth muscle exhibits moderate cytoplasmic vacuolization with multifocal loss of nuclear differential staining throughout, endothelium inapparent in small-medium caliber vessels, and adipocytes inapparent. A score of 4 (severe) corresponds to marked hypereosinophilic staining globally, faint to absent nuclear differential staining, cytoplasmic features inapparent, cellular borders inapparent, smooth muscle is hypereosinophilic and nuclear differential staining is faint to absent, endothelium inapparent in small-medium caliber vessels, and adipocytes inapparent.

[0142] The tissue samples were visually inspected after immersion for 24 hours. Images of the tissue samples after immersion for 24 hours are shown in FIG. 6A (Group A, water), FIG. 6B (Group B, citric acid), and FIG. 6C (Group C, hydrogel). A chart of the histopathology scores of the tissue samples after immersion for 24 hours is shown in FIG. 7.

[0143] Histopathology of Group A (DI Water) was evaluated for three portions of the tissue sample: 23-181 A, 23-181B, and 23-181C. In each of the Group A portions, mild tissue degradation / autolysis was observed, which was considered to align with the type of tissue sampled.

[0144] For portion 23-181 A, mineralization was observed to be located predominantly within the mid tunica media in a linear pattern and rarely within the fibrous connective tissue of the tunica adventitia. Mineralization was observed to be deeply basophilic (i.e., darkly stained by contrast dye) and finely to coarsely granular with clumping. For portion 23-181B, mineralization was observed to be located predominantly within the mid tunica media in a linear distribution and rarely within small-caliber vessels of the tunica adventitia. Mineralization was observed to rangeMedtronic Ref. No. A0011118WOO 1 from feathered to finely stippled and mildly basophilic to deeply basophilic with coarse granules and clusters. For portion 23-181C, mineralization was observed to be located within the mid tunica media in a linear distribution. Mineralization was observed to be deeply basophilic (i.e., darkly stained by contrast dye) and finely to coarsely granular with clumping.

[0145] Histopathology of Group B (Citric Acid) was evaluated for three portions of the tissue sample: 23-182A, 23-182B, and 23-182C. In each of the Group B portions, significant tissue degradation / autolysis was observed when compared to the DI water control group (Group A).

[0146] For portion 23-182A, mineralization was observed to be located in the luminal- to midtunica media. Mineralization was observed to be feathered to finely stippled and mild to moderately basophilic. For portion 23-182B, no mineralization was observed. For portion 23- 182C, mineralization was observed to be located within the mid tunica media in a linear distribution. Mineralization was observed to be feathered to finely stippled and mild to moderately basophilic.

[0147] Histopathology of Group C (Hydrogel) was evaluated for three portions of the tissue sample: 23-183A, 23-183B, and 23-183C. In each of the Group C portions, moderate tissue degradation / autolysis was observed when compared to the DI water control group (Group A). For portion 23-183 A, mineralization was observed to be located within the mid tunica media in a linear distribution. Mineralization was observed to be finely stippled and minimally basophilic. For portion 23-183B, mineralization was observed to be located within the tunica intima. Mineralization was observed to be coarsely granular. For portion 23-183C, mineralization was observed to be located within the mid tunica media. Mineralization was observed to be feathered to finely stippled and mildly to moderately basophilic.

[0148] In all sections and groups there was observable tissue degradation / autolysis. The tissue degradation / autolysis in Group B (aqueous citric acid) was observed to be the most significant with global changes throughout the entire tissue section. Group B was observed to exhibit complete loss of differential nuclear staining with smudgy, hypereosinophilic cell cytoplasm and a distinct loss of intact cell borders.Example 5: Evaluation of treatments of calcified vascular smooth muscle cell cultures with hydrogel formulations.

[0149] Human Aortic Smooth muscle cells were plated at a concentration of 50,000 cells / well and incubated overnight. After 24 hours, the regular growth media was removed and all wellsMedtronic Ref. No. A0011118WOO 1 except the untreated control (regular growth media) received high phosphorous media. Treatments both liquid (10 % volume of media in well) and hydrogel calcium-binding composition samples in accordance with illustrative methods and compositions described in the present disclosure were added to the media (200 - 250 microliters (ul) per well). The hydrogel compositions are shown in Table 11, in which “Collagen A” refers to porcine collagen, “Collagen B” refers to oxidized porcine collagen, and “Crosslink” refers to whether the hydrogel underwent high-temperature crosslinking, as described in the present disclosure. The plate was incubated for 4 days and stained with Alizarin Red Stain to indicate calcium deposits.Table 11. Hydrogel compositions tested in Example 5.Medtronic Ref. No. A0011118WOO 1

[0150] Images of cultured smooth muscle cells after treatment with the hydrogel calcium-binding composition samples of Table 11 are shown in FIGS. 8A-8F, wherein the darkest gray or black shows plate visible between cells (e.g., due to poor cell growth), wherein the medium gray shows red-stained (mineralized) cells, and wherein the lightest gray or white shows non-mineralized cells. Samples 1, 5, 6, 8, 9, and 18 were observed to exhibit an excellent ability to inhibit calcification while preserving cell viability at the same time. In particular, hydrogel calcium- binding composition samples having citric acid concentration of up to 5.5 % were observed to inhibit calcification while preserving cell viability at the same time. Relative to the hydrogel calcium-binding composition samples, comparative aqueous solutions having a citric acid concentration of no more than 1 % were observed to provide similar results in terms of cell viability under comparable conditions.

Claims

Medtronic Ref. No. A0011118WOO 1CLAIMSWhat is claimed is:

1. A calcium-binding composition comprising: a water-soluble biopolymer; and a calcium-binder.

2. The composition according to claim 1, wherein the water-soluble biopolymer comprises collagen, porcine collagen, biosynthetic collagen, at least partially denatured collagen, at least partially oxidized collagen, at least partially crosslinked collagen, dehyrothermal treated collagen, freeze-dried collagen, methacrylate collagen, or any combination thereof.

3. The composition according to claim 1 or claim 2, wherein the water-soluble biopolymer comprises a biopolymer selected from the group consisting of collagen, alginate, chitosan, and hyaluronic acid.

4. The composition according to any one of claims 1 through 3, wherein the water- soluble biopolymer in the form of an aqueous solution, a viscous fluid, a hydrogel, a film, a powder, a sponge, or any combination thereof.

5. The composition according to any one of claims 1 through 4, wherein the calcium- binding composition comprises 0.1 wt-% to 70 wt-% of the water-soluble biopolymer based on the weight of the calcium-binding composition.

6. The composition according to any one of claims 1 through 5, wherein the calcium- binding composition comprises 1 wt-% to 40 wt-% of the calcium-binder based on the weight of the calcium-binding composition.

7. The composition according to any one of claims 1 through 6, wherein the calcium- binder comprises EDTA, citric acid, ascorbic acid, acetic acid, N-acetylneuraminic acid, or any combination thereof.

8. A method for treating a vascular calcification, the method comprising: delivering to a treatment site a calcium-binding composition comprising:Medtronic Ref. No. A0011118WOO 1 a water-soluble biopolymer; and a calcium-binder; wherein the calcium-binding composition removes calcium from the vascular calcification.

9. The method according to claim 8, wherein the water-soluble biopolymer comprises collagen, porcine collagen, biosynthetic collagen, at least partially denatured collagen, at least partially oxidized collagen, at least partially crosslinked collagen, dehyrothermal treated collagen, freeze-dried collagen, methacrylate collagen, or any combination thereof.

10. The method according to claim 8 or claim 9, wherein the water-soluble biopolymer comprises a biopolymer selected from the group consisting of collagen, alginate, chitosan, and hyaluronic acid.

11. The method according to any one of claims 8 through 10, wherein the water-soluble biopolymer in the form of an aqueous solution, a viscous fluid, a hydrogel, a film, a powder, a sponge, or any combination thereof.

12. The method according to any one of claims 8 through 11, wherein the calcium- binding composition comprises 0.1 wt-% to 70 wt-% of the water-soluble biopolymer based on the weight of the calcium-binding composition.

13. The method according to any one of claims 8 through 12, wherein the calcium- binding composition comprises 1 wt-% to 40 wt-% of the calcium-binder based on the weight of the calcium-binding composition.

14. The method according to any one of claims 8 through 13, wherein the calcium-binder comprises EDTA, citric acid, ascorbic acid, acetic acid, N-acetylneuraminic acid, or any combination thereof.

15. The method according to any one of claims 8 through 14, wherein delivering to the treatment site the calcium-binding composition comprises positioning the calcium-binding composition against the treatment site using a balloon, a stent, a plug, a filter, or any combination thereof.

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