WO2006033686A1 - Angiogenesis factor and biological material - Google Patents

Abstract

A method for promoting angiogenesis in a patient comprising providing crosslinkable biological solution to the target tissue, wherein the crosslinkable biological solution is loaded with at least one angiogenic agent. In one embodiment, the at least one angiogenic agent is a non-protein factor selected from the group consisting of ginsenoside Rg1, ginsenoside Re, combinations thereof and the like. In another embodiment, the crosslinkable biological solution of the present invention is broadly defined in a form or phase of solution, paste, gel, suspension, colloid or plasma that may be solidified thereafter.

Description

AfSKItUlAENBSHi PAaEiDOR AND BIOLOGTCAL MATERIAL

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority of U.S. patent application Ser. No. 10/916,170, filed

August U, 2004, entitled DRUG-ELUTING BIODEGRADABLE STENT. This application is also a co-pending application of U.S. patent application Ser. No. 10/827,673 filed April 19, 2004, entitled CROSSLINKABLE BIOLOGICAL MATERIAL AND MEDICAL USES, the entire contents of both applications are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention generally relates to chemical modification of biomedical materials, such as collagen and/or chitosan matrix with a naturally occurring crosslinking reagent, genipin. More particularly, the present invention relates to crosslinked collagen, chitosan, and/or fibrin glue as medical material prepared with a bioactive angiogenesis agent that is configured suitably for sustained drug release effective for therapeutic purposes.

BACKGROUND OF THE INVENTION

[0003] Crosslinking of biological molecules is often desired for optimum effectiveness in biomedical applications. For example, collagen, which constitutes the structural framework of biological tissue, has been extensively used for manufacturing bioprostheses and other implanted structures, such as vascular grafts, wherein it provides a good medium for cell infiltration and proliferation. However, biomaterials derived from collagenous tissue must be chemically modified and subsequently sterilized before they can be implanted in humans. The fixation, or crosslinking, of collagenous tissue increases strength and reduces antigenicity and immunogenicity.

[0004] Clinically, biological tissue has been used in manufacturing heart valve prostheses, small-diameter vascular grafts, and biological patches, among others. However, the biological tissue has to be fixed with a crosslinking or chemically modifying agent and subsequently sterilized before they can be implanted in humans. The fixation of biological tissue is to reduce antigenicity and immunogenicity and prevent enzymatic degradation. Various crosslinking agents have been used in fixing biological tissue. These crosslinking agents are mostly synthetic chemicals such as formaldehyde, glutaraldehyde, dialdehyde starch, glyceraldehydes, cyanamide, diimides, diisocyanates, and epoxy compound. However, most of these chemicals are highly cytotoxic which may impair the biocompatibility of biological tissue. Of these, glutaraldehyde is known to have allergenic properties, causing occupational dermatitis and is cytotoxic at concentrations greater than 10-25 ppm and as low as 3 ppm in tissue culture. It is therefore desirable to provide a crosslinking agent suitable for use in biomedical applications that is within acceptable cytotoxicity- and that forms stable and biocompatible crosslinked products.

[0005] To achieve this goal, a naturally occurring crosslinking agent (genipin) has been used to fix biological itissme.'Or.ffii;αsslϊiϊkab^'Ie. bf©ϊogi«al solution. The cytotoxicity of genipin was previously studied in vitro using 3T3 fibroblasts, indicating that genipin is substantially less cytotoxic than glutaraldehyde (Sung HW et al., J Biomater Sci Polymer Edn 1999;10:63-78). Additionally, the genotoxiciτy of genipin was tested in vitro using Chinese hamster ovary (CHO-Kl) cells, suggesting that genipin does not cause clastogenic response in CHO-Kl cells (Tsai CC et al., J Biomed Mater Res 2000;52:58-65). A biological material treated with genipin resulting in acceptable cytotoxicity is essential to biomedical applications.

[0006] In accordance with the present invention, there is provided genipin treated tissue grafts for orthopedic and other surgical applications, such as vascular grafts and heart valve bioprostheses, which have shown to exhibit many of the desired characteristics important for optimal graft function. In particular, the tissue regeneration capability in the genipin-fixed acellular tissue is suitable as a graft material for bone, tendon, ligament, cartilage, muscle, ophthalmology and cardiovascular applications.

[0007] It is generally agreed that an ideal implant or stent for atherosclerosis or vulnerable plaque treatment should be biodegradable after serving its purpose and its breakdown products must be biocompatible. It should possess physical properties sufficient to perform its mechanical function and have sufficient longitudinal flexibility to facilitate insertion. It should be able to deliver drugs locally in a sustained manner to prevent restenosis or treat lesions.

[0008] It is therefore desirable to provide a method for promoting autogenous ingrowth of a biological tissue material, comprising providing a natural tissue, optionally removing cellular material from the natural tissue with increased porosity of the natural tissue by at least 5%, loading an angiogenesis agent or autologous cells onto the natural tissue, and crosslinking the natural tissue. Furthermore, it is desirable to provide crosslinkable biological solution or material configured and adapted for promoting angiogenesis, wherein the crosslinkable biological material is incorporated with an organic non-protein angiogenic agent such as ginsenoside Rg_b ginsenoside Re or the like. The biological material may comprise collagen, gelatin, elastin or tropoelastin, chitosan, N, O, carboxylmethyl chitosan (NOCC), and the like (such as fibrin glue, biological sealant, fϊbronectin derivatives, and combination thereof) that has at least one amino functional group for reaction with genipin.

SUMMARY OF THE INVENTION

[0009] In general, it is an object of the present invention to provide a medical device with a capability of enhanced angiogenesis for a patient comprising a therapeutically effective amount of at least one non-protein angiogenesis factor. In one embodiment, the non-protein angiogenesis factor is an organic angiogenesis factor. In another embodiment, the non-protein angiogenesis factor is ginsenoside Rgi or ginsenoside Re. In a further embodiment, the medical device is an acellular tissue material or a wound dressing material, wherein the acellular tissue material has an increased porosity by at least 5%.

[0010] In one embodiment, the medical device of the present invention is crosslinked with a crosslinking agent or crosslinked via irradiation. In one embodiment, the medical device is an artificial organ selected from the group consisting of a biological patch, a vascular graft, a heart valve, a venous valve, a tendon, a ligament a. bqμe, s

a tartipage,. a ωreter, a urinary bladder, a dermal graft, a cardiac tissue, an anti-adhesion membrane, and a myocardial tissue. In another embodiment, the medical device is a cardiovascular stent, non-stent implant, a biodegradable/bioabsorbable stent or implant, or selected from the group consisting of a biological implant, non-biological implant, armuloplasty ring, heart valve prosthesis, venous valve bioprosthesis, orthopedic implant, dental implant, ophthalmology implant, and cerebral implants.

[0011] It is another object of the present invention to provide a biological substance configured and adapted for drug slow release and/or enhanced angiogenesis. In one aspect of the present invention, the biological substance may be a cardiovascular stent or implant (biodegradable or non-biodegradable). The "biological substance" is herein intended to mean a substance made of drug-loaded (particularly angiogenesis factor-loaded) biological material that is, in one preferred embodiment, solidifiable and biocompatible. The biological substance is crosslinkable with a crosslinker, such as genipin, its derivatives, analog (for example, aglycon geniposidic acid), stereoisomers and mixtures thereof. In one embodiment, the crosslinker may further comprise epoxy compounds, dialdehyde starch, glutaraldehyde, formaldehyde, dimethyl suberimidate, carbodiimides, succinimidyls, diisocyanates, acyl azide, reuterin, ultraviolet irradiation, dehydrothermal treatment, tris(hydroxymethyl)phosphine, ascorbate-copper, glucose-lysine and photo-oxidizers, or the like. The "biological material" is intended herein to mean collagen, gelatin, elastin, chitosan, NOCC (N, O, carboxylmethyl chitosan), fibrin glue, biological sealant, and the like that could be crosslinked with a crosslinker (also known as a crosslinking agent).

[0012] Some aspects of the invention relate to a biodegradable implant, wherein the biodegradable implant is made of a material selected from the group consisting of polylactic acid (PLA), polyglycolic acid (PGA), poly (D_jL-lactide-co-glycolide), polycaprolactone, and co-polymers thereof, polyhydroxy acids, polyalkanoates, polyanhydrides, polyphosphazenes, polyetheresters, polyesteramides, polyesters, and polyorthoesters.

[0013] Some aspects of the invention relate to a medical non-solid material for promoting angiogenesis at a tissue site of a patient, comprising at least one angiogenesis factor, wherein the non-solid material may be a crosslinkable biological solution. In one embodiment, the crosslinkable biological solution is crosslinked with a crosslinking agent or with ultraviolet irradiation. In one embodiment, the crosslinkable biological solution is selected from the group consisting of collagen extract, soluble collagen, elastin or tropoelastin, gelatin, chitosan, N, O, carboxylmethyl chitosan (NOCC), chitosan-containing solution, and collagen-containing solution.

[0014] In one embodiment, the medical solution or non-solid material of the present invention comprises at least one protein type angiogenesis factor selected from the group consisting of VEGF, VEGF 2, bFGF, VEGF121, VEGF165, VEGF189, VEGF206, PDGF, PDAF, TGF-β, TGF-α, PDEGF, PDWHF, epidermal growth factor, insulin-like growth factor, aFGF, human growth factor, and combinations thereof. In another embodiment, the medical solution of the present invention comprises at least one non-protein angiogenesis factor selected from the group consisting of ginsenoside Rgi, ginsenoside Re, and combinations thereof. The medical solution of the present invention is in a form of liquid, paste, gel, suspension, colloid, or plasma, whereas lie med'tødϊ setotioπ- ϊs^ι ouitfrgured and formulated for administering to the tissue site by an administration route selected from the group consisting of an oral administration, topical administration, percutaneous injection, intravenous injection, intramuscular injection, oral administration, and implantation.

[0015] Some aspects of the invention relate to a crosslinkable biological solution kit comprising a first readily mixable crosslinkable biological solution component and a second crosslinker component, wherein the first component and the second component are mixed at a point of need.

[0016] In one embodiment of the present invention, the first component of the kit further comprises at least one non-protein angiogenesis factor of ginsenoside Rgi, ginsenoside Re, combinations thereof, or the like. In another embodiment, the first component further comprises at least one protein angiogenesis factor selected from the group consisting of VEGF, VEGF 2, bFGF, VEGF121, VEGF165, VEGF189, VEGF206, PDGF, PDAF, TGF-β, TGF-α, PDEGF, PDWHF, epidermal growth factor, insulin-like growth factor, aFGF, human growth factor, and combinations thereof. In a further embodiment, the first component further comprises at least one bioactive agent selected from the group consisting of analgesics/antipyretics, antiasthamatics, antibiotics, antidepressants, antidiabetics, antifungal agents, antihypertensive agents, anti-inflammatories, antineoplastics, antianxiety agents, immunosuppressive agents, antimigraine agents, sedatives/hypnotics, antipsychotic agents, antimanic agents, antiarrhythmics, antiarthritic agents, antigout agents, anticoagulants, thrombolytic agents, antifϊbrinolytic agents, antiplatelet agents and antibacterial agents, antiviral agents, antimicrobials, and anti-infectives. In still another embodiment, the first component further comprises at least one bioactive agent selected from the group consisting of actinomycin D, paclitaxel, vincristin, methotrexate, and angiopeptin, batimastat, halofuginone, sirolimus, tacrolimus, everolimus, tranilast, ABT-578, dexamethasone, mycophenolic acid, lovastatin, thromboxane A₂ synthetase inhibitors, eicosapentanoic acid, ciprostene, trapidil, angiotensin convening enzyme inhibitors, heparin, and biological cells. In one embodiment, the crosslinkable biological solution kit is packaged in a form suitable for application selected from the group consisting of a topical administration, percutaneous injection, intravenous injection, intramuscular injection, oral administration, and loading on an implant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Additional objects and features of the present invention will become more apparent and the invention itself will be best understood from the following Detailed Description of Exemplary Embodiments, when read with reference to the accompanying drawings.

[0018] FIG. 1 shows chemical structures of glutaraldehyde (GA) and genipin (GP) that are used in the chemical treatment examples of the current disclosure.

[0019] FIG, 2 shows one embodiment of a biodegradable stent in a meshed tubular shape.

[0020] FIG. 3 shows one embodiment of a spiral (helical) biodegradable stent.

[0021] FIG. 4 shows one embodiment of an open-ring biodegradable stent.

[0022] FIG. 5 shows another embodiment of an open-ring biodegradable stent.

[0023] FIG. 6 shows one embodiment of an interlocking open-ring biodegradable stent. [00,2.4} PK&i ? sirøvre a αhemie&l formula of ginssnoside Rgi.

[OUAO] πvj. o snows a cnemical formula for Ginsenoside Re.

[0026] FIG. 9 shows cells infiltration extents of genipin-crosslinked acellular bovine pericardia tissue with angiogenesis factors for (a) specimen-AGP, without Rgi; (b) light microscopy of specimen a; (c) specimen- AGP, with Rgi; and (d) light microscopy of specimen c; all explants retrieved at 1-week postoperatively.

[0027] FIG. 10 shows a preparation method of loading an acellular tissue with growth factors Rg₁,

Re, or bFGF.

[0028] FIG. 11 shows 1-week postoperative results on animal angiogenesis study, photomicrographs of H&E (hematoxylin and eosin) stained tissue.

[0029] FIG. 12 shows effect of ginsenoside Rg₁ on human umbilical vein endothelial cell

(HUVEC) proliferation, migration, and tube formation, using bFGF as a control: effect of bFGF or Rg₁ on HUVEC proliferation.

[0030] FIG. 13 shows effect of ginsenoside Rg₁ on human umbilical vein endothelial cell

(HUVEC) proliferation, migration, and tube formation, using bFGF as a control: effect of bFGF or Rg₁ on EIUVEC migration obtained in a Transwell-plate assay.

[0031] FIG. 14 shows histological evaluation of the tissue responses to test ECMs implanted subcutaneously in a rat model retrieved at 1-week postoperatively: (A) photomicrograph of the ECM dip-coated in a gelatin hydrogel incorporated with Rg₁ at 70 μg stained with H&E before implantation; photomicrographs of (B) the ECM without loading any drug (ECM/control), (C) the ECM loaded with 0.7 μg bFGF (ECM/bFGF), (D) the ECM loaded with 0.7 μg Rgi (ECM/Rgi-0.7), and (E) the ECM loaded with 70 μg Rg₁ (ECM/Rg_r70) retrieved at 1-week postoperatively stained with H&E (20Ox magnification); (F) photomicrograph of the

ECMTRg₁^O retrieved at 1-week postoperatively stained with factor VIII (80Ox magnification).

[0032] FIG. 15 shows quantitative analyses of the cell density and the density and depth of blood vessels infiltrated and the tissue hemoglobin content observed in each test ECM retrieved at 1-week and 1 -month postoperatively, the density (in percentage of the depth of the whole test sample) of blood vessels infiltrated into each test ECM.

[0033] FIG. 16 shows quantitative analyses of the cell density and the density and depth of blood vessels infiltrated and the tissue hemoglobin content observed in each test ECM retrieved at 1-week and 1-month postoperatively: the tissue hemoglobin content observed in each test ECM.

[0034] FIG. 17 shows histological evaluation of the tissue responses to test ECMs implanted subcutaneously in a rat model retrieved at 1 -month postoperatively: photomicrographs of (A) ECM/control, (B) ECM/bFGF, (C) ECM/Rgi-0.7, and (D) ECM/Rg_r70 retrieved at 1 -month postoperatively stained with H&E (20Ox magnification).

[0035] FIG. 18 shows the neo-connective tissues observed in the pores of each test ECM loaded with bFGF or Rgi were identified by the immunohistochemical stains to contain neo-collagen type I and III fibrils regenerated from the host rat: photomicrographs of (A) ECM/bFGF, (B) ECM/Rg_r0.7, and (C) ECMZRg_I-¹ZO- sretrjeved at; ihflKJnfl- jxksatβ_jperatfsrely obtained by the immunohistocnemical stains to identify neo-collagen type III (40Ox magnification).

[0036] FIG. 19 shows a crosslinkable biological solution kit comprising a first crosslinkable biological solution component and a second crosslinker component.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0037] The following detailed description is of the best presently contemplated modes of carrying out the invention. This description is not to be taken in a limiting sense, but is made merely for the purpose of illustrating general principles of embodiments of the invention,

[0038] "Genipin" in this invention is meant to refer to the naturally occurring compound as shown in FIG. 1 and its derivatives, analog, stereoisomers and mixtures thereof.

[0039] "Tissue engineering" or "tissue regeneration" in meant to refer to cell seeding, cell ingrowth and cell proliferation into the acellular scaffold or collagen matrix in vivo or in vitro, optionally enhanced with an angiogenesis factor.

[0040] A "biological tissue material" refers to a biomedical material or device of biological tissue origin which is inserted into, or grafted onto, bodily tissue to remain for a period of time, such as an extended-release drug delivery device, tissue valve, tissue valve leaflet, vascular or dermal graft, ureter, urinary bladder, or orthopedic prosthesis, such as bone, ligament, tendon, cartilage, and muscle.

[0041] "Biological solution" is herein meant to refer to collagen extract, soluble. collagen, elastin or tropoelastin, gelatin, chitosan, N, O, carboxylmethyl chitosan (NOCC), chitosan-containing and other collagen-containing biological solution. For a preferred aspect of the present invention, the biological solution may also include a crosslinkable biological substrate that may comprise at least a genipin-crosslinkable functional group, such as amino group or the like, or crosslinkable with UV irradiation. The biological solution or crosslinkable biological solution of the present invention is broadly defined in a form or phase of solution, liquid, paste, gel, suspension, colloid or plasma that may be solidified thereafter.

[0042] An "implant" refers to a medical device (of biological and non-biological origin) which is inserted into, or grafted onto, bodily tissue to remain for a period of time.

[0043] A "scaffold" in this invention is meant to refer to a tissue matrix substantially or completely devoid of cellular materials. A scaffold may further comprise added structure porosity for cell ingrowth or proliferation.

[0044] "Drug" in this invention is meant to broadly refer to a chemical molecule(s), biological molecule(s) or bioactive agent providing a therapeutic, diagnostic, or prophylactic effect in vivo. "Drug" and "bioactive agent" (interchangeable in meaning) may comprise, but not limited to, synthetic chemicals, biotechnology-derived molecules, herbs, cells, genes, growth factors, health food and/or alternate medicines. In the present invention, the terms "drug" and "bioactive agent" are used interchangeably.

[0045] It is one object of the present invention to provide an acellular biological scaffold chemically treated with a naturally occurring crosslinking agent, genipin that is configured and adapted for tissue regetsiiftrøϊvaπdføpmssϊiie engineering iiift biomedical applications.

[0046] Genipin, shown in FIG. 1, is an iridoid glycoside present in fruits (Gardenia jasmindides

Ellis). It may be obtained from the parent compound geniposide, which may be isolated from natural sources as described in elsewhere. Genipin, the aglycone of geniposide, may be prepared from the latter by oxidation followed by reduction and hydrolysis or by enzymatic hydrolysis. Although FIG. 1 shows the natural configuration of genipin, any stereoisomer or mixture of stereoisomers of genipin may be used as a crosslinking reagent, in accordance with the present invention.

[0047] Genipin has a low acute toxicity, with LD50 i-v. 382 mg/kg in mice. It is therefore much less toxic than glutaraldehyde and many other commonly used synthetic crosslinking reagents. As described below, genipin is shown to be an effective crosslinking agent for treatment of biological materials intended for in vivo biomedical applications, such as prostheses and other implants, wound dressings, and substitutes.

[0048] Kyogoku et al. in U.S. Pat. No. 5,037,664, U.S. Pat. No. 5,270,446, and EP 0366998, entire contents of all three being incorporated herein by reference, teach the crosslinking of amino group containing compounds with genipin and the crosslinking of genipin with chitosan. They also teach the crosslinking of iridoid compounds with proteins which can be vegetable, animal or microbial origin. However, they do not teach loading drug onto a biological material or solution crosslinked with genipin as biocompatible drug carriers for drug slow-release.

[0049] Noishiki et al. in U.S. Pat. 4,806,595 discloses a tissue treatment method by a crosslinking agent, polyepoxy compounds. Collagens used in that patent include an insoluble collagen, a soluble collagen, an atelocollagen prepared by removing telopeptides on the collagen molecule terminus using protease other than collagenase, a chemically modified collagen obtained by succinylation or esterification of above-described collagens, a collagen derivative such as gelatin, a polypeptide obtained by hydrolysis of collagen, and a natural collagen present in natural tissue (ureter, blood vessel, pericardium, heart valve, etc.) The Noishiki et al. patent is incorporated herein by reference. "Collagen matrix" in the present invention is collectively used referring to the above-mentioned collagens, collagen species, collagen in natural tissue, and collagen in a biological implant preform.

[0050] In one embodiment, the crosslinker may comprise genipin, epoxy compounds, dialdehyde starch, glutaraldehyde, formaldehyde, dimethyl suberimidate, carbodiimides, succinimidyls, diisocyanates, acyl azide, ultraviolet irradiation, dehydrothermal treatment, tris(hydroxymethyl)phosphine, ascorbate-copper, glucose-lysine and photo-oxidizers, and the like.

[0051] FIGS. 2-6 show some embodiments of a stent or implant, either biodegradable or non-biodegradable for treating atherosclerosis or vulnerable plaque in cardiovascular applications and other medical applications, such as in ophthalmology, orthopedic, urology, and wound healing, etc. In one embodiment, the stent comprises a biological material loaded with at least one bioactive agent for therapeutic purposes. In a preferred embodiment, the stent or implant is loaded with non-protein angiogenesis factor for treating blood vessels in need of angiogenesis (for example, for diabetic patients).

[0052] In one embodiment of the present invention, bovine pericardia procured from a slaughterhouses are usetf as rø£iιmater.raik Thenprocured pericardia are transported to the laboratory in a cold normal saline. In the laboratory, the pericardia are first gently rinsed with fresh saline to remove excess blood on tissue. Adherent fat is then carefully trimmed from the pericardial surface. The cleaned/trimmed pericardium before acellular process is herein coded specimen-A. The procedure used to remove the cellular components from bovine pericardia is adapted from a method developed by Courtman et al (J Biomed Mater Res 1994;28:655-66), which is also referred to herein as "an acellularization process". A portion of the trimmed pericardia is then immersed in a hypotonic tris buffer (pH 8.0) containing a protease inhibitor (phenylmethyl-sulfonyl fluoride, 0.35 mg/L) for 24 hours at 4°C under constant stirring. Subsequently, they are immersed in a 1% solution of Triton X-IOO (octylphenoxypolyethoxyethanol; Sigma Chemical, St. Louis, Missouri, USA) in tris-buffered salt solution with protease inhibition for 24 hours at 4⁰C under constant stirring. Samples then are thoroughly rinsed in Hanks' physiological solution and digested with DNase and RNase at 37°C for 1 hour. This is followed by a further 24-hour extraction with Triton X-100 in tris buffer. Finally, all samples are washed for 48 hours in Hanks' solution and the acellular sample is coded specimen-B. Light microscopic examination of histological sections from extracted tissue revealed an intact connective tissue matrix with no evidence of cells.

[0053] A portion of the acellular tissue of bovine pericardia (specimen-B) is further treated with

1 % acetic acid at room temperature for one hour. The acidic component is thereafter removed from the tissue by lyophilization at about -5O⁰C for 24 hours, followed by thorough rinse with filtered water to obtain the acellular pericardia having enlarged pore or added porosity. The tissue is stored in phosphate buffered saline (PBS, 0.01M, pH 7.4, Sigma Chemical), which tissue is coded specimen-C. The procedure of acetic acid treatment to add porosity is referred herein as "acid treatment". Similar results could be achieved by following the acid treatment with other diluted acid solution, such as nitric acid or the like, at the comparable acidity or pH vales.

[0054] The mechanism of increasing the tissue porosity treated by a mild acidic solution lies in the effect of [H⁺] or [OH^"] values on the collagen fibers matrix of the acellular tissue. It is also disclosed that acellular tissue treated with a base solution (i.e., a solution pH value greater than 7.0) could have the same effect upon enlarged pores or added porosity.

[0055] A portion of the bovine pericardia tissue post-acid treatment (i.e., specimen-C) is further treated with enzymatic collagenase as follows. Add 0.01 gram of collagenase to a beaker of 40 ml TES buffer and incubate the specimen-C pericardia tissue at 37⁰C for 3 hours. The sample is further treated with 10 mM EDTA solution, followed by thorough rinse. The tissue is stored in phosphate buffered saline (PBS, 0.01M, pH 7.4, Sigma Chemical), which tissue is coded specimen-D. The procedure of collagenase treatment to add porosity is referred herein as "enzyme treatment" (referred to U.S. Pat. No. 6,645,042).

[0056] The cellular tissue (specimen-A) and acellular tissue (specimen-B) of bovine pericardia are fixed in 0.625% aqueous glutaraldehyde (Merck KGaA, Darmstadt, Germany) and are coded as specimen-A/GA and specimen-B/GA, respectively. Furthermore, the cellular tissue (specimen-A) and acellular tissue (specimen-B, specimen-C, and specimen-D) of bovine pericardia are fixed in genipin (Challenge Bioproducts, Taiwan) solution at 37⁰C for 3 days and are coded as specimen-A/GP, specimen-B/GP, SPeCImSn-QZ¹UiP!, &nd spu≥efaΦa D/GP_S respectively. The aqueous glutaraldehyde and genipin solutions used are buffered with PBS. The amount of solution used in each fixation was approximately 200 mL for a 10 x 10 cm bovine pericardium. After fixation, the thickness of each studied group is determined using a micrometer (Digimatic Micrometer MDC-25P, Mirutoyo, Tokyo, Japan). Subsequently, the fixed cellular and acellular tissues are sterilized in a graded series of ethanol solutions with a gradual increase in concentration from 20 to 75% over a period of 4 hours. Finally, the test tissue is thoroughly rinsed in sterilized PBS for approximately 1 day, with solution change several times, and prepared for tissue characterization as well as a subcutaneous study. The chemical structures of the crosslinking agents (genipin and glutaraldehyde as control) used in the study are shown in FIG. 1.

[0057] In the present invention, the terms "crosslinking", "fixation", "chemical modification", and/or "chemical treatment" for tissue, biological material or biological solution are used interchangeably.

[0058] It is hereby disclosed that a method of preparing a biological scaffold configured and adapted for tissue regeneration or tissue engineering comprises steps of removing cellular material from a natural tissue or collagen matrix; and chemically modifying the acellular tissue or collagen matrix with genipin, with an optional step of adding angiogenesis factors.

[0059] It is further disclosed that a biological scaffold for cells seeding, cell growth or enhanced cell proliferation with an angiogenesis factor may comprise a natural tissue devoid of cellular material and crosslinked by a crosslinker or with UV irradiation.

[0060] U.S. Pat. No. 6,506,398 issued to Tu et al. (a co-inventor of the present invention), the entire contents of which are incorporated herein by reference, discloses a vascular graft comprising Vascular Endothelial Growth Factor (VEGF) and/or Platelet Derived Growth Factor (PDGF) for enhanced site-specific angiogenesis and methods thereof. At least one VEGF, PDGF or angiogenesis factor is incorporated into the vascular graft to facilitate enhanced angiogenesis so as the cells are stimulated to migrate to environments having higher concentration of growth factors and start mitosis. With added porosity, it is provided a biological tissue material with loaded growth factors adapted for promoting tissue regeneration, wherein the growth factor is selected from the group consisting of VEGF, VEGF 2, bFGF, VEGF121, VEGF165, VEGF189, VEGF206, PDGF, PDAF, TGF-β, PDEGF, PDWHF₁ and combination thereof.

[0061] Vascular endothelial growth factor (VEGF) is mitogenic for vascular endothelial cells and consequently is useful in promoting neovascularization (angiogenesis) and reendothelialization. Angiogenesis means the growth of new capillary blood vessels. Angiogenesis is a multi-step process involving capillary endothelial cell proliferation, migration and tissue penetration. VEGF is a growth factor having a cell-specific mitogenic activity. It would be desirable to employ a wound healing substrate incorporating a mitogenic factor having mitogenic activity that is highly specific for vascular endothelial cells following vascular graft surgery, balloon angioplasty or to promote collateral circulation. U.S. Pat. No. 5,194,596 discloses a method for producing VEGF while U.S. Pat. No. 6,040,157 discloses a specific VEGF-2 polypeptide. Both patents are incorporated herein by reference.

[0062] Gordinier et al. in U.S. Pat. No. 5,599,558 discloses a method of making a platelet releasate pir«iuct and Methods of treating tissues with the platelet releasate. Platelet derived growth factor (PDGF) is a well-characterized dimeric glycoprotein with mitogenic and chemoattractant activity for fibroblasts, smooth muscle cells and glial cells. In the presence of PDGF, fibroblasts move into the area of tissue needing repair and are stimulated to divide in the lesion space itself. It has been reported that the cells exposed to lower PDGF concentrations are stimulated to move to environments having higher concentrations of PDGF and divide. The patent is incorporated hereby by reference.

[0063] In some aspects, there is provided a method for promoting autogenous ingrowth of a biological tissue material comprising at least one or more steps of providing a natural tissue, removing cellular material from the natural tissue, increasing a porosity of the natural tissue by at least 5%, loading an angiogenesis agent or autologous cells into the porosity, and crosslinking the natural tissue with a crosslinking agent. In one preferred embodiment, the angiogenesis agent is an organic angiogenesis factor (for example, ginsenoside Rgi, ginsenoside Re) or a protein angiogenesis factor selected from the group consisting of VEGF (vascular endothelial growth factor), VEGF 2, bFGF (basic fibroblast growth factor), VEGF121, VEGF165, VEGFl 89, VEGF206, PDGF (platelet-derived endothelial cell growth factor), PDAF, TGF-β (transforming growth factor-beta), TGF-α (transforming growth factor-alpha),PDEGF, PDWHF, and combination thereof. In another embodiment, the protein angiogenesis factor may further comprise, but not limited to, a fibroblast growth factor, an epidermal growth factor, an endothelial cell growth factor, an insulin-like growth factor, a periodontal ligament cell, aFGF (acidic fibroblast growth factor), and human growth factor (HGF).

[0064] In an alternate embodiment, there is provided a method for inhibiting autogenous ingrowth of a biological tissue material comprising at least one or more steps of providing a natural tissue, removing cellular material from the natural tissue, increasing porosity of the natural tissue by at least 5%, loading an anti-angiogenesis agent into the porosity, and crosslinking the natural tissue with a crosslinking agent. The anti-angiogenesis factor or angiogenesis inhibitors may include, for example, collagenase inhibitors; minocycline; medroxyprogesterone; chitin chemically modified with 6-O-sulfate and 6-O-carboxymethyl groups; angiostatic steroids, such as tetrahydrocortisol; and heparin, including fragments of heparin, such as, for example, fragments having a molecular weight of about 6,000, admixed with steroids, such as, for example, cortisone or hydrocortisone; angiogenesis inhibitors, including angioinhibin (AGM-1470 - an angiostatic antibiotic); platelet factor 4; protamine; sulfated polysaccharide peptidoglycan complexes derived from the bacterial wall of an Arthobacter species; fungal-derived angiogenesis inhibitors, such as fumagillin derived from Aspergillus fumigatus; D-penicillamine; gold thiomalate; thrombospondin; vitamin D3 analogues, including, for example, l-α-25-dihydroxyvitamin D3 and a synthetic analog, 22-oxa-l-α, 25-dihydroxyvitamin D3; α-interferon; cytokines, such as the interleukins, including, for example, interleukin-I (IL-I), interleukin-2 (IL-2), and interleukin-8 (IL-8) granulocyte macrophage colony stimulating factor (GMCSF); heparin, including low molecular weight fragments of heparin or analogues of heparin; simple sulfated polysaccharides, such as cyclodextrins, including α-, β- and γ-cyclodextrin; tetradecasulfate; transferrin; ferritin; platelet factor 4; protamine; Gly-His-Lys complexed to copper, ceruloplasmin; (12R)-hydroxyeicosatrienoic acid; okadaic acid; lectins; antibodies; CdI la/CD 18; and Very Late Acting Integrin-4 (VLA-4). [0OiErSj It is: known that protein type growth factors have relatively short shelf life. For medical device use, it is one object of the invention to provide an organic compound, non-protein type growth factors, such as ginsenoside Rg₁ (as shown in FIG. 7) and/or ginsenoside Re (as shown in FIG. 8).

[0066] Ginseng is one of the most widely used herbal drugs and is reported to have a wide range of therapeutic and pharmacological activities. The two major species of commerce are Panax ginseng CA. Meyer (Asian ginseng), and Panax quinquefolius L. (North American ginseng). Both species contain active ginsenoside saponins, but there are significant differences in their identity and distribution. It has been observed that over thirty ginsenosides have been identified from Panax spp., however six of these, Rg₁, Re, Rbi, Rc, Rb₂, and Rd constitute the major ginsenosides accounting for over 90% of the saponin content of ginseng root. Standard ginsenosides Rgi, Re, Rb₁, Rc, Rb₂ and Rd can be isolated and characterized by NMR. In contrary to general angiogenesis effects of ginsenoside Rgi and Re, ginsenoside Rg3 can block angiogenesis and inhibit tumor growth and metastasis by downregulating the expression of VEGF mRNA and protein and reducing microvascular density. Some aspects of the invention relate to a method of reducing angiogenesis for treating tissue comprising: providing crosslinkable biological solution to the target tissue, wherein the crosslinkable biological solution is loaded with at least one anti-angiogenic agent (also known as angiogenic antagonist or inhibitor) such as ginsenoside Rg₃ and the like.

[0067] FIG. 7 shows a chemical formula of ginsenoside Rgi, one of the principal active components of ginseng saponins which is isolated from the roots of Panax ginseng. In one embodiment as shown in FIG. 7, in which R_1A = OH or O-Glc, R_2A ⁼H or O-Glc, R3A ⁼ O-Glc, wherein GIc designates a β-D glucopyranosyl group. Rg₁ is believed to stimulate vascular endothelial cells proliferation, and tube formation in a patient. Ginseng's therapeutic uses were recorded in the oldest Chinese pharmacopeia, Shen Nong Ben Cao Jing, written about two thousand years ago. Ginseng action is non-local and non-specific. In Asian medicine, ginseng is used as a tonic to revitalize the function of organism as a whole and replenish vital energy ("chi")- It is traditionally used as the best supplemental and restorative nature agent during convalescence and as a prophylactic to build resistance, reduces susceptibility to illness, and promotes health and longevity.

[0068] Other functions of ginseng are to stimulate mental and physical activity, strengthen and protect human organism, increase physical and mental efficiency and to prevent fatigue. Ginseng has good effect on the stomach, the brain, and the nervous system. Ginseng is effective for reflex nervous disease. Ginseng has also been found to have an anti-cancer effect. There are more than 30 kinds of ginsenosides, and each one function differently. For example, ginsenoside Rh₂ has anti-tumor activity. Ginsenoside Rg₁ can enhance DNA and RNA formation, which may speed up the angiogenesis. In some aspect of the present invention, there is provided a method for promoting autogenous ingrowth of a biological tissue material comprising the steps of providing a natural tissue, removing cellular material from the natural tissue, increasing porosity of the natural tissue by at least 5%, loading an angiogenesis agent or autologous cells into the porosity, and crosslinking the natural tissue with a crosslinking agent. In one preferred embodiment, the angiogenesis agent is ginsenoside Rg₁. In still another aspect of the invention, there is provided a method for treating cancer or tumor by implanting a biological tissue material comprising the steps of providing a natural tissue, removing cellular material f-Qiϊi fe naϊβal ti&sap,¹ Snertasing porosity of the natural tissue by at least 5%, loading a cancer/tumor antagonist agent into the porosity, and crosslinking the natural tissue with a crosslinking agent. In one preferred embodiment, the cancer/tumor antagonist is ginsenoside Rh₂.

[0069] Some aspects of the invention relate to a method for promoting angiogenesis for treating tissue comprising: providing crosslinkable biological solution to the target tissue, wherein the crosslinkable biological solution is loaded with at least one angiogenic agent (also known as angiogenic growth factor) such as ginsenoside Rgi or Re. Some aspects of the invention relate to a method for treating cancer or tumor of a patient comprising: providing crosslinkable biological solution to the target tissue, wherein the crosslinkable biological solution is loaded with at least one cancer/tumor antagonist agent such as ginsenoside RI1₂.

[0070] Example 1 - In vitro Angiogeαesis study with Ginsenoside

[0071] In the in vitro study, effects of Rgi on HUVEC (human umbilical vein endothelial cell) proliferation, migration, and tube formation were investigated, using bFGF (basic fibroblast growth factor) as a reference control. During angiogenesis, activated existing endothelial cells proliferate and their mobile activity increases. The mobile endothelial cells migrate toward the attractants and connect each other to form tube-like structures in vitro or neo-vessels in vivo. The in vitro assays applied in the present study have been widely used and are appropriate models to examine various aspects of angiogenic behaviors of Rg₁. HUVECs (Cascade Biologies, Portland, OR) were cultured at 37⁰C in a humidified atmosphere of 5% CO₂ and 95% air in Medium 200 supplemented with low serum growth supplement (LSGS, Cascade Biologies). All experiments were carried out with the same batch of HUVECs.

[0072] Proliferation assay on in vitro specimens: HUVECs (6000 cells/0.1 ml) were added to a collagen-coated 96-well plate (Pierce) and incubated with 100 μl of Medium 200 supplemented with LSGS for 24 hours. The cells were then treated with the testing sample by replacing the media with 0.1 ml Medium 200 containing 2% fetal bovine serum (FBS, Cascade Biologies). The growth medium was supplemented with Rg₁ (Wako, Osaka, Japan) at 10 ng/ml or 50 μg/ml. A positive control, in which 10 ng/ml bFGF (PeproTech, Rockhill, NJ) was added, and a negative control, in which no supplemented Rgi or bFGF was added, were performed. After 48 hours, 100 μl of 20% CellTiter 96^® AQ_ueOus One Solution Reagent (Promega) was added to the wells and the plate was returned to the incubator for 3 hours. The optical density for each well was measured.

[0073] The number of viable cells was estimated by the [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, MTS] method using CellTiter 96 AQ_ueOus One Solution Cell Proliferation Assay. The quantity of formazan product as measured by the amount of 490 nm absorbance was directly proportional to the number of living cells in cultures (FIG. 12). For bFGF (10 ng/ml), HUVEC proliferation was increased 39% over untreated cells (p<0.05). At 10 ng/ml Rg₁, cell proliferation was increased 18% over untreated cells, which was found to be statistically significant (p<0.05). The proliferation rate of cells exposed to a higher concentration of Rgi (50 μg/ml) was found to have increased significantly 25% over untreated cells (p<0.05). hi FIGS. 12 and 13, the term "n.s." indicates no statistical difference; the term "*" indicates statistical significance at a level of p<0.05. [O0P4J Migration assay¹ BW asn Vttfo specimens: The ability of Rg₁ to stimulate HUVEC migration was assessed in Transwell plates (6.5 mm, 8 μm, COSTAR, Corning, NY). The upper chambers of these plates were coated with 50 μl of 5% Matrigel™ (BD Biosciences) diluted in Medium 200 containing 2% FBS. The plates were then incubated at 37⁰C for 2 hours. HUVECs were seeded at 1 x 10⁵ cells in 200 μl to each Transwell. The bottom chambers contained Medium 200 plus 2% FBS and Rgi at 10 ng/ml or 50 μg/ml. The positive control chambers contained 10 ng/ml bFGF. Medium 200 plus 2% FBS was used as the negative control. The plates were incubated at 37⁰C, 5% CO₂ for 12 hours. Cells were fixed with 1000 μl of 4% formaldehyde for 30 minutes. Cells on the upper surface of the membrane were removed by gentle wiping with a cotton-tipped swab. The membranes were counter-stained with hematoxylin, and then the number of migrated cells was counted. The assay was quantified by counting the number of cells per microscopic Field that migrated through the pores to the lower surface of each membrane with Nikon-E-800 at 20Ox magnification. Each data point was based on pentaplicate chambers and six microscopic fields per membrane.

[0075] FIG. 13 shows the effect of bFGF or Rgi on HUVEC migration was tested in Transwell plates using Matrigel™-coated membranes. Cells were added to the upper chamber; after 12 hours the number of cells migrated through the membrane in response to bFGF or Rgi in the lower chamber was quantified. HUVECs treated with bFGF showed more than three times the migratory activity over that of untreated cells, and cells treated with Rgi migrated at more than twice the rate of untreated cells (p<0.05, FIG. 13).

[0076] Tube formation assay on in vitro specimens: Tube formation assays were performed using

96-well plates coated with 50 μl of Matrigel™ per well. HUVECs were plated at a density of 10,000 cells/well in 150 μl of Medium 200 containing 2% FBS. Rgi was added to the wells at 10 ng/ml or 50 μg/ml, and the plates were incubated for 12 hours at 37⁰C. After 12 hours, cells were fixed in 100 μl of 4% formaldehyde for 30 min and the images were taken at 10Ox magnification. The images were converted into gray scale and the area of the formed tube networks was determined using Image-Pro^® Plus (Media Cybernetics, Silver Spring, MD). Each value was in pentaplicate.

[0077] The ability of HUVECs to form a network of tubular structures across the surface of a

Matrigel™ substratum is a complex phenomenon that combines elements of attachment, migration, organization, and differentiation. The complex organizational behavior of HUVECs on Matrigel™ models the type of coordinated activities required for atigiogenesis by endothelial cells. Although the in vitro Matrigel™ model does not represent true angiogenesis, it suggested that bFGF, Rgi, or the like is important for many of the activities that contribute to vessel formation. Thus, the aforementioned results indicated that both bFGF (a protein type angiogenesis factor) and Rg₁ (a non-protein type angiogenesis factor) enhanced several in vitro HUVEC activities that are relevant to angiogenesis, including proliferation, migration, and tube formation.

[0078] Rgi has a steroid backbone and contains two molecules of glucose at its 6th and 18th positions (FIG. 7). The steroid backbone of Rgi makes it a suitable candidate to interact and activate steroid receptors, such as glucocorticoid and estrogen receptors, It was reported that estrogen induces endothelial proliferation and migration mediated by the classic estrogen receptor, which is expressed by endothelial cells. Additionally, ginsenoside and its purified form Rgi were shown to induce nitric oxide in endothelial cells and caused vasodilatation (A¹IB J Ghlri Med 1995,T3.279-287). Nitric oxide has been reported to be a downstream mediator in the angiogenic response to a variety of growth factors, but the mechanisms by which nitric oxide promotes neo-vessel formation is not clear.

[0079] FIG. 9 show cells infiltration extents of genipin-crosslinked acellular bovine pericardia tissue with angiogenesis factors for (a) specimen- AGP, without Rg₁; (b) light microscopy of specimen a (specimen-AGP, without Rg₁); (c) specimen-AGP, with Rg₁; and (d) light microscopy of specimen c (specimen-AGP, with RgO; wherein all implants are retrieved at 1-week postoperatively. The micro-vessel numbers per field (on a reference basis) are measured under a microscope using an imaging processing software. The micro-vessel density for the Rgi loaded explant (specimen (b) in FIG. 9) is 778 vessels/mm² that is statistically significantly higher than the micro-vessel density for the control explant (specimen (d) in FIG. 9) of 341 vessels/mm².

[0080] In some aspects of the present invention, the acellular tissue structure with a porosity increase of more than 5% is also suitable for use in anti-adhesion patches for abdominal surgery, anti-adhesion patches for cardiovascular surgery, acellular matrix for regeneration of myocardiocytes, and vascular grafts. Rgi has shown properties of stimulating HUVEC proliferation, tube formation and chemoinvasion in in vitro studies (T.P.Fan at 3^rd Asian International Symposium on Biomaterials and Drug Delivery Systems, April 16, 2002). Some aspects of the invention relate to a method for promoting angiogenesis in a subject in need thereof, comprising administering to the subject a substrate loaded with therapeutically effective amount of a non-protein angiogenesis factor (for example, ginsenoside Rg₁ and/or ginsenoside Re), wherein the substrate may comprise acellular tissue, artificial, organs, wound dressing device in wound care, and prostheses/implants. The therapeutically effective amount may range from 1 ppm to 10%, preferably in a range of 10 ppm to 1%, of the substrate.

[0081] Example 2 - In vivo Angiogenesis study

[0082] Preparation of test ECMs: The procedures used to remove the cellular components from bovine pericardia were based on a method previously reported. To increase pore sizes and porosities within test samples, the acellular tissues were treated additionally with acetic acid and collagenase. Subsequently, acellular tissues were fixed in a 0.05% genipin (Challenge Bioproducts, Taiwan) aqueous solution (pH 7.4) at 37⁰C for 3 days, The chemical structure of genipin can be found in the literature. The denaturation temperature and porosity of the fixed ECMs were measured in a differential scanning calorimeter and by helium pycnometery, respectively (n=5). The pore size of the fixed ECMs stained with hematoxylin and eosin (H&E) was determined under a microscope (n=5). The fixed ECMs were sterilized in a graded series of ethanol solutions. Finally, the sterilized ECMs were rinsed in sterilized phosphate buffered saline (PBS).

[0083] FIG. 10 shows a preparation method of loading an acellular tissue with ginsenoside Rgi or ginsenoside Re (both are organic compound growth factors, belonging to non-protein angiogenesis factor category), or bFGF (a protein type growth factor which has a short shelf life). As shown in FIG. 10, extracellular membranes of 1-cm by 1-cm specimens are used to load model growth factors onto the specimens by air-sucking, dip coating and liquid nitrogen cooling steps. The animal implant study includes a rat intramuscular model, wheρ*|i the tesft

are- loaded' wiA « 7μg Rgi, 0.7μg bFGF, 70μg Rgi or 70μg Re growth factors.

[0084] To incorporate bFGF (0.7 μg) or Rgi (0.7 or 70 μg) in the ECMs, bFGF or Rg₁ were dissolved in a sterilized gelatin (300 rag/ml PBS, from porcine skin, 225 Bloom, Sigma) aqueous solution. Following vigorous mixing of the solution, the prepared ECMs were dip-coated in the drug-containing gelatin solution under a reduced pressure environment and subsequently gelled in liquid nitrogen. The ECM without loading any drug was used as a blank control.

[0085] An extracellular matrix (ECM) is prepared by removing the cellular components of bovine pericardia. Additionally, to increase the pore size and porosity within the ECM, the acellular tissue was further treated with acetic acid and subsequently with collagenase. It is generally accepted that a tissue-engineering extracellular matrix must be highly porous for a sufficient cell density to be seeded in vitro, for blood invasion to occur in vivo, and for oxygen and nutrients to be supplied to cells. It was found by transmission electron microscopy in our previous study that the cellular extraction process used in the study produced a complete extraction and left no cell membrane or nuclear structures within the tissue. The denaturation temperature of the genipin fixed ECM was 74.6±0.6°C and its pore size and porosity were approximately 130.3±14.6 μm and 94.9-bl.7%, respectively.

[0086] Genipin can be obtained from its parent compound, geniposide, which may be isolated from the fruits of Gardenia jasminoides ELLIS. Genipin and its related iridoid glucosides have been widely used as an antiphlogistic and cholagogue in herbal medicine. It was found in our previous study that genipin can react with free amino groups such as lysine, hydroxylysine, or arginine residues in biological tissues. The cytotoxicity of genipin was previously studied by our group in vitro using 3T3 fibroblasts. Glutaraldehyde was used as a control. The results indicated that genipin is significantly less cytotoxic than glutaraldehyde. Additionally, the genotoxicity of genipin was tested in vitro using Chinese hamster ovary (CHO-Kl) cells. The results suggested that glutaraldehyde may produce a weakly clastogenic response in CHO-Kl cells. In contrast, genipin does not cause clastogenic response in CHO-Kl cells.

[0087] Example 3 - In vivo Angiogenesis study with Ginsenoside

[0088] In vivo rat subcutaneous study: In total, 40 rats (4-week-old male Wistar) divided into four groups (ECM/control, ECM/bFGF, ECM/Rg_r0.7, and ECM/Rg_r70) were used in the study. Two test samples (10x10 mm) of the same type were separately implanted subcutaneously in each rat under aseptic conditions. The implanted samples were retrieved at 1-week and 1 -month postoperatively (n=5 rats at each time point). At retrieval, the appearance of each retrieved sample was grossly examined and photographed. Subsequently, half of each retrieved sample was fixed and embedded in paraffin for the histological examination, and the remainder of the sample was used to quantify the amount of tissue hemoglobin.

[0089] FIG. 11 shows 1-week postoperative results on animal angiogenesis study: photomicrographs of H&E (hematoxylin and eosin) stained tissue explant. Both organic compound growth factor and protein growth factor promote angiogenesis as evidenced by enhanced neo-capillaries and tissue hemoglobin measurements as compared to control. However, the protein growth factors tend to have a shorter shelf life than the organic growth factors. [0^Q9QI M tM ftfafol'oilcal c-xsmsraation, the fixed samples were stained with H&E. The stained sections of each test sample were examined using light microscopy for tissue inflammatory reaction and tissue regeneration. The number of inflammatory cells observed in each studied case was quantified with a computer-based image analysis system (Image-Pro^® Plus) at 20Ox magnification. Also, the density and depth (in percentage of the depth of the whole test sample) of blood vessels infiltrated into each studied sample were quantified with the same image analysis system. A minimum of five fields was counted for each retrieved sample.

[0091] Immunohistochemical staining was performed on the paraffin sections with a labeled streptavidin-biotin immunoenzymatic antigen detection system (DAKO LSAB^®2 System, Dako Co., Carpinteria, CA). The paraffin sections were digested enzymatically with pepsin (1 mg/ml in 0.0 IN of HCl) for 30 minutes at 37⁰C. Collagen types I and III mouse monoclonal antibodies were obtained from ICΝ Biomedicals Inc. (Aurora, OH). The subcutaneous tissue of a healthy rat and the ECM used in the study were used as the positive and negative controls, respectively. Additional sections were stained for factor VIII with immunohistological technique with a monoclonal anti-factor VIII antibody (Dako Co., Carpinteria, CA).

[0092] The extent of vascularization in each retrieved sample was determined by measuring the amount of tissue hemoglobin. Test samples were fragmented with a scalpel and immersed in YJmM Tris-HCl buffer solution (pH 7.6) containing 0.75% ammonium chloride for 24 hours at 4⁰C to extract hemoglobin in test samples. The extracted hemoglobin was quantitated using a hemoglobin assay kit (Wako, Osaka, Japan).

[0093] Angiogenesis and tissue regeneration in the genipin-fixed ECMs loaded with Rg₁ at 0.7 μg

(ECM/Rgi-0.7) or 70 μg (ECM/Rgi-70) were investigated subcutaneously in a rat model. FIG. 14(A) shows photomicrograph of the ECM dip-coated in a gelatin hydrogel incorporated with Rgi at 70 μg stained with H&E before implantation. The ECMs without drug (ECM/control) or loaded with 0.7 μg bFGF (ECM/bFGF) were used as blank and positive controls, respectively. At 1-week postoperatively, a thin layer of transparent tissue enriched with blood capillaries surrounded the ECM/bFGF, ECMZRg₁-OJ and ECM/Rgi-70. In contrast, there was no macroscopic evidence of any angiogenesis for the ECM/control.

[0094] FIGS. 14(B) to 14(E) present photomicrographs of each studied group retrieved at 1-week postoperatively stained with H&E. The solid line in each photograph represents the interface between the host tissue (rat) and the implanted test sample. As shown, host cells (inflammatory cells, endothelial cells, and red blood cells) were able to infiltrate into the open spaces of all test ECMs. The amount of inflammatory cells infiltrated into the ECM/control was the most remarkable among all studied groups. The density of neo-capillaries infiltrated into the ECMs loaded with bFGF or Rgi were significantly greater than the control ECM without drug loading (FIG. 15, p<0.05). FIG. 14(F) presents a photomicrograph of the ECM/Rgi-70 retrieved at 1-week postoperatively stained with factor VIII.

[0095] As shown, in-growing capillaries were coated with an inner endothelial layer. These results indicates that angiogenesis in the ECMs was significantly enhanced by loading with bFGF (a protein angiogenesis factor) or Rgi (a non-protein angiogenesis factor). It is known that site-specific delivery of angiogenic molecules may provide an efficient means of stimulating localized vessel formation. The ECMs dip-coated witti a gei&ln Ifydϊϋsgel foioΘiforatted with bFGF or Rgi prepared in the study may allow one to optimize this process. It was noted that there were more neo-capillaries and tissue hemoglobin measured in the ECM/bFGF and ECM/Rg!-70 than in the ECM/Rgi-0.7 (p<0.05, FIGS. 15 and 16); however, there were no significant differences between the former two test samples (p>0.05). In FIGS. 15 and 16, the term "n.s." indicates no statistical difference; the term "*" indicates statistical significance at a level of p<0.05.

[0096] At 1-month postoperatively, inflammatory cells in the outer layers of the ECM/Rgi-0.7 and

ECM/Rgi-70 had almost disappeared, while there were still some inflammatory cells observed in the ECM/bFGF (FIGS. 17(B)-17(D)). Instead, fibroblasts (migration from the host tissue), neo-capillaries, and neo-connective-tissue fibrils were found to fill the pores in this area, indicating that the tissue was being regenerated. The neo-connective tissues were identified by the immunohistochemical stains to contain neo-collagen type I and type III fibrils regenerated from the host rat (FIGS. 18(A)-18(C)).

[0097] In contrast, there were still a large number of inflammatory cells with minimal neo-capillaries and neo-connective-tissue fibrils seen in the ECM/control (FIG. 17(A)). These results indicated that Rgj-associated induction of angiogenesis enhanced tissue regeneration, supporting the concept of therapeutic angiogenesis in tissue-engineering strategies.

[0098] The densities of neo-capillaries and tissue hemoglobin infiltrated into the ECM/Rgi-0.7 and

ECM/Rgi-70 were significantly greater than their counterparts observed at 1-week postoperatively (p<0.05), while those seen in the ECM/bFGF stayed approximately the same (p>0.05, FIGS. 15 and 16). These results suggested that the delivered Rgi continued to be effective in enhancing angiogenesis. In contrast, although bFGF can enhance angiogenesis at 1-week postoperatively, it is difficult to achieve long-term delivery of functional properties because of the limitations of protein stability. It was reported that at physiological pH and temperature, the in vitro half-lifetime of bFGF activity (a protein angiogenesis factor) is approximately 12 hours. In contrast, it was shown that degradation of Rg₁ (a non-protein angiogenesis factor) under neutral intestinal pH conditions is negligible throughout the experimental period (about 40 hours). In conclusions, the aforementioned results indicated that Rgi is an effective agent for angiogenesis and may be load in an extracellular matrix for accelerating tissue regeneration.

[0099] Some aspects of the invention relate to a method for promoting angiogenesis in a subject in need thereof, comprising administering to the subject a substrate loaded with therapeutically effective amount of a non-protein angiogenesis factor, wherein the non-protein angiogenesis factor may be an organic angiogenesis factor. In one embodiment, the non-protein angiogenesis factor is ginsenoside Rgi, ginsenoside Re Oi the like extracted from a plant. In another embodiment, the substrate is configured and formulated for administering to the subject by a route selected from the group consisting of oral administration, topical administration, percutaneous injection, intravenous injection, intramuscular injection, oral administration, and implantation.

[0100] In one embodiment, the substrate is an acellular tissue or a wound dressing, wherein the acellular tissue may have an increased porosity over the substrate by at least 5%. In another embodiment, the method for administering to a subject a substrate loaded with a therapeutically effective amount of a non-protein angiogenesfs factor uuraprtses a step of crosslinking the substrate with a crosslinking agent. In a further embodiment, the substrate is an artificial organ sele< ted from the group consisting of biological patch, vascular graft, heart valve, venous valve, tendon, ligament, bone, muscle, cartilage, ureter, urinary bladder, dermal graft, cardiac tissue, anti-adhesion membrane, and myocardial tissue.

[0101] Biological Solution Kits

[0102] FIG. 19 shows a crosslinkable biological solution kit 90 comprising a first crosslinkable biological solution component 93B and a second crosslinker component 93A. The kit has a double-barrel cylinder 91 with a divider 99 that separates the crosslinkable biological solution component 93B from the crosslinker component 93A before use, wherein each barrel is appropriately sized and configured to provide a desired amount and ratio of each component for later mixing and application. The kit further comprises an end portion 92A with (optionally) appropriate mixing means 92B for mixing the liquid/solution from each of the double-barrel. A control valve 96 is provided to maintain the components 93A, 93B in their own barrels before use or is activated to start the mixing process. The plunger means 94 for pressurizing the components 93A, 93B toward the end portion 92A has a first plunger 95A and a second plunger 95B. In an alternate embodiment, the plunger means 94 can be either mechanical or equipped with a gas or liquid compressor. In one preferred embodiment, the mixed solution can be sprayed onto an implant or a stent. In another embodiment, the mixed solution is used directly onto a target tissue. In a further embodiment, the cylinder comprises a liquid input port 93C, wherein the bioactive agent(s) 98 can be injected via the injecting applicator 97 into and mixed with the crosslinkable biological solution component 93B.

[0103] Example 4 - Biological Solution as Medical Material

[0104] The first step for preparing a biological solution as medical material is to load the double-barrel cylinder with 4 mg/ml collagen solution at a pH4 as crosslinkable biological solution component 93B. The second step is to load 0.5% genipin solution as the crosslinker component 93A. Each of the double-barrel is appropriately sized and configured to provide a desired ratio and amount of each component 93A, 93B for later mixing in the end portion 92A. One example is to provide 0.6 ml of component 93A with respect to 4 ml of component 93 B. Upon receiving the cylinder in sterile conditions, an operator as end-users prepares a paclitaxel solution (Solution A) by mixing 20mg paclitaxel in one ml absolute alcohol, wherein Solution A is readily mixed into the component 93B by the operator. Paclitaxel is used as a bioactive agent in this example. When use, two barrels are pushed to mix the component 93A and component 93B that contains the desired bioactive agent. In one embodiment, the mixed crosslinkable biological solution is loaded onto a stent at about 3O⁰C temperature and subsequently leave the coated stent at 37⁰C to solidify collagen, evaporate acetic acid, and crosslink collagen on the stent. The loading process may comprise spray coating, dip coating, plasma coating, painting or other known techniques. In another embodiment, the crosslinkable biological solution is administered or delivered to the target tissue accompanied with means for adjusting the biological solution to pH7, either by removing excess acetic acid or by neutralizing with a base solution.

[0105] It is one object of the present invention to provide a drug-collagen-genipin and/or drug-chitosan-genipin compound that is loadable onto an implant/stent or deliverable to a target tissue enabling drug slow-ife%as*s to tire target" tissue, ϊn one "'preferred embodiment, the compound is loaded onto the outer periphery of the stent enabling drug slow-release to the surrounding tissue.

[0106] The drugs used in the current drug eluting cardiovascular stents include two major mechanisms: cytotoxic and cytostatic. Some aspects of the invention relating to the drugs used in biological compound from the category of cytotoxic mechanism comprise actinomycin D, paclitaxel, vincristin, methotrexate, and angiopeptin. Some aspects of the invention relating to the drugs used in biological compound from the category of cytostatic mechanism comprise batimastat, halofuginone, sirolimus, tacrolimus, everolimus, tranilast, ABT-578 (a sirolimus analog manufactured by Abbott Labs) dexamethasone, and mycophenolic acid (MPA). Some aspects of the present invention provide a bioactive agent in a bioactive agent-eluting device, wherein the bioactive agent is selected from the group consisting of actinomycin D, paclitaxel, vincristin, methotrexate, and angiopeptin, batimastat, halofuginone, sirolimus, tacrolimus, everolimus, tranilast, dexamethasone, and mycophenolic acid.

[0107] Everolimus with molecular weight of 958 (a chemical formula of C₅₃H_S3NO₁₄) is poorly soluble in water and is a novel proliferation inhibitor. There is no clear upper therapeutic limit of everolimus. However, thrombocytopenia occurs at a rate of 17% at everolimus trough serum concentrations above 7.8 ng/ml in renal transplant recipients (Expert Opin Investig Drugs 2002;l 1(12): 1845-1857). In a patient, everolimus binds to cytosolic immunophyllin FKBP 12 to inhibit growth factor-driven cell proliferation. Everolimus has shown promising results in animal studies, demonstrating a 50% reduction of neointimal proliferation compared with a control bare metal stent.

[0108] Preferred drugs useful in the present invention may include albuterol, adapalene, doxazosin mesylate, mometasone furoate, ursodiol, amphotericin, enalapril maleate, felodipine, nefazodone hydrochloride, valrubicin, albendazole, conjugated estrogens, medroxyprogesterone acetate, nicardipine hydrochloride, Zolpidem tartrate, amlodipine besylate, ethinyl estradiol, omeprazole, rubitecan, amlodipine besylate/ benazepril hydrochloride, etodolac, paroxetine hydrochloride, paclitaxel, atovaquone, felodipine, podofϊlox, paricalcitol, betamethasone dipropionate, fentanyl, pramipexole dihydrochloride, Vitamin D₃ and related analogues, finasteride, quetiapine fumarate, alprostadil, candesartan, cilexetil, fluconazole, ritonavir, busulfan, carbamazepine, flumazenil, risperidone, carbemazepine, carbidopa, levodopa, ganciclovir, saquinavir, amprenavir, carboplatin, glyburide, sertraline hydrochloride, rofecoxib carvedilol, clobustasol, diflucortolone, halobetasolproprionate, sildenafil citrate, celecoxib, chlorthalidone, imiquimod, simvastatin, citalopram, ciprofloxacin, irinotecan hydrochloride, sparfloxacin, efavirenz, cisapride monohydrate, lansoprazole, tamsulosin hydrochloride, mofafinil, clarithromycin, letrozole, terbinafine hydrochloride, rosiglitazone maleate, diclofenac sodium, lomefloxacin hydrochloride, tirofiban hydrochloride, telmisartan, diazapam, loratadine, toremifene citrate, thalidomide, dinoprostone, mefloquine hydrochloride, trandolapril, docetaxel, mitoxantrone hydrochloride, tretinoin, etodolac, triamcinolone acetate, estradiol, ursodiol, nelfinavir mesylate, indinavir, beclomethasone dipropionate, oxaprozin, flutamide, famotidine, nifedipine, prednisone, cefuroxime, lorazepam, digoxin, lovastatin, griseofulvin, naproxen, ibuprofen, isotretinoin, tamoxifen citrate, nimodipine, amiodarone, and alprazolam.

©samples of some drugs that fall under the above categories include paclitaxel, docetaxel and derivatives, epothilones, nitric oxide release agents, heparin, aspirin, Coumadin, PPACK, hirudin, polypeptide from angiostatin and endostatin, methotrexate, 5-fluorouracil, estradiol, P-selectin Glycoprotein ligand-1 chimera, abciximab, exochelin, eleutherobin and sarcodictyin, fludarabine, sirolimus, tranilast, VEGF, transforming growth factor (TGF)-beta, Insulin-like growth factor (IGF), platelet derived growth factor (PDGF), fibroblast growth factor (FGF), RGD peptide, beta or gamma ray emitter (radioactive) agents, and dexamethasone, tacrolimus, actinomycin-D, batimastat etc.

[0110] Sirolimus is a naturally occurring macrolide antibiotic produced by the fungus

Streptomyces found in Easter Island. It was discovered by Wyeth-Ayerst in 1974 while screening fermentation products. Sirolimus with molecular weight of 916 (a chemical formula of C₅₁H₇₉NO₁₃) is non-water soluble and is a potential inhibitor of cytokine and growth factor mediated cell proliferation. FDA approved its use as oral immunosuppressive agents with a formulation of 2 to 5 mg/dose. The suggested drug-eluting efficacy is about 140 micrograms/cm², 95% drug release at 90 days and 30% drug-to-polymer ratio. Some aspects of the invention provide a method for administering to a subject a substrate loaded with therapeutically effective amount of at least one bioactive agent formulated for oral administration.

[0111] In some aspect of the present invention, the drug (also referred as a bioactive agent) may broadly comprise, but not limited to, synthetic chemicals, biotechnology-derived molecules, herbs, health food, extracts, and/or alternate medicines; for example, including allicin and its corresponding garlic extract, ginsenosides and the corresponding ginseng extract, flavone/terpene lactone and the corresponding ginkgo biloba extract, glycyrrhetinic acid and the corresponding licorice extract, and polyphenol/proanthocyanides and the corresponding grape seed extract.

[0112] While the preventive and treatment properties of the foregoing therapeutic substances, agents, drugs, or bioactive agents are well known to those having ordinary skill in the art, the substances or agents are provided by way of example and are not meant to be limiting. Other therapeutic substances are equally applicable for use with the disclosed methods, devices, and compositions.

[0113] It is another object of the present invention to provide a crosslinkable biological solution kit comprising a first readily mixable crosslinkable biological solution component and a second crosslinker component, wherein an operator can add appropriate drug or bioactive agent to the kit and obtain a drug-collagen-genipin and/or drug-chitosan-genipin compound that is loadable onto an implant/stent or deliverable to a target tissue enabling drug slow-release to the target tissue. In a further embodiment, the crosslinkable biological solution kit is packaged in a form for topical administration, for percutaneous injection, for intravenous injection, for intramuscular injection, for loading on an implant or biological tissue material, and/or for oral administration.

[0114] Some aspects of the invention provide a method for promoting angiogenesis for treating tissue, comprising: providing crosslinkable biological solution to the target tissue, wherein the crosslinkable biological solution is loaded with at least one angiogenesis factor. In one embodiment, the crosslinkable biological solution to treat the target tissue is a kit comprising a first readily mixable crosslinkable biological solution cQittJJonpnt and a second ctsosslinker component, wherein the first component and the second component are mixed at point of need. The point of need may comprise the operating suite, a hospital room, a physician clinic, the local tissue site of a patient needed for treatment, or the device to have enhanced angiogenesis, and the like. In one embodiment, the at least one angiogenesis factor is a protein factor selected from the group consisting of VEGF, VEGF 2, bFGF, VEGF121, VEGF165, VEGF189, VEGF206, PDGF, PDAF, TGF-β, TGF-α, PDEGF, PDWHF, epidermal growth factor, insulin-like growth factor, aFGF, human growth factor, and combination thereof. In another embodiment, the at least one angiogenesis factor is a non-protein factor selected from the group consisting of ginsenoside Rgi, ginsenoside Re, and combination thereof. In s a further embodiment, the crosslinkable biological solution is in a form of solution, paste, gel, suspension, colloid, or plasma, wherein the crosslinkable biological solution is crosslinkable with a crosslinking agent or with ultraviolet irradiation.

[0115] Some aspects of the invention relate to a crosslinkable biological solution kit comprising at least one bioactive agent selected from the group consisting of analgesics/antipyretics, antiasthamatics, antibiotics, antidepressants, antidiabetics, antifungal agents, antihypertensive agents, anti-inflammatories, antineoplastics, antianxiety agents, immunosuppressive agents, antimigraine agents, sedatives/hypnotics, antipsychotic agents, antimanic agents, antiarrhythmics, antiarthritic agents, antigout agents, anticoagulants, thrombolytic agents, antifϊbrinolytic agents, antiplatelet agents and antibacterial agents, antiviral agents, antimicrobials, and anti-infectives. In a further embodiment, the crosslinkable biological solution kit may comprise at least one bioactive agent selected from the group consisting of actinomycin D, paclitaxel, vincristin, methotrexate, and angiopeptin, batimastat, halofuginone, sirolimus, tacrolimus, everolimus, tranilast, ABT-578, dexamethasone, mycophenolic acid, lovastatin, thromboxane A₂ synthetase inhibitors, eicosapentanoic acid, ciprostene, trapidil, angiotensin convening enzyme inhibitors, heparin, and biological cells.

[0116] Some aspects of the invention provide the crosslinkable biological solution kit that is configured and packaged in a form suitable for application selected from the group consisting of topical administration, percutaneous injection, intravenous injection, intramuscular injection, oral administration, and loading on an implant before implantation or after implantation.

[0117] Some aspects of the invention relate to a method for promoting angiogenesis comprising administering ginsenoside Rgi and/or ginsenoside Re onto tissue after radiation therapy to promote neovascularization. Some further aspects of the invention relate to a method for promoting angiogenesis comprising administering ginsenoside Rgi and/or ginsenoside Re onto tissue of ulcer or diabetes to promote neovascularization.

[0118] From the foregoing description, it should now be appreciated that a novel and unobvious process for promoting angiogenesis has been disclosed for tissue engineering applications. While the invention has been described with reference to a specific embodiment, the description is illustrative of the invention and is not to be construed as limiting the invention. Various modifications and applications may occur to those who are skilled in the art, without departing from the true spirit and scope of the invention.

Claims

What is elated' Js:

1. A medical device with a capability of enhanced angiogenesis for a patient comprising a therapeutically effective amount of at least.one non-protein angiogenesis factor.

2. The medical device of claim 1, wherein the non-protein angiogenesis factor is an organic angiogenesis factor.

3. The medical device of claim 1, wherein the non-protein angiogenesis factor is ginsenoside Rg₁ or ginsenoside Re.

4. The medical device of claim 1, wherein the medical device is an acellular tissue material or a wound dressing material.

5. The medical device of claim 4, wherein the acellular tissue material has an increased porosity by at least 5%.

6. The medical device of claim 1, wherein the medical device is crosslinked with a crosslinking agent.

7. The medical device of claim 1, wherein the medical device is crosslinked via irradiation.

8. The medical device of claim 1, wherein the medical device is an artificial organ selected from a group consisting of a biological patch, a vascular graft, a heart valve, a venous valve, a tendon, a ligament, a bone, a muscle, a cartilage, a ureter, a urinary bladder, a dermal graft, a cardiac tissue, an anti-adhesion membrane, and a myocardial tissue.

9. The medical device of claim 1, wherein the medical device is a cardiovascular stent or non-stent implant.

10. The medical device of claim 1, wherein the medical device is a biodegradable stent or implant.

11. The medical device of claim 1, wherein the medical device is selected from a group consisting of a biological implant, non-biological implant, annuloplasty ring, heart valve prosthesis, venous valve bioprosthesis, orthopedic implant, dental implant, ophthalmology implant, and cerebral implants.

12. A medical solution for promoting angiogenesis at a tissue site of a patient, comprising at least one angiogenesis factor.

13. The medical solution of claim 12, wherein the solution is a crosslinkable biological solution, the crosslinkable biological solution being crosslinked with a crosslinking agent or with ultraviolet irradiation.

14. The medical solution of claim 12, wherein the crosslinkable biological solution is selected from a group consisting of collagen extract, soluble collagen, elastin or tropoelastin, gelatin, chitosan, N, O, carboxylmethyl chitosan (NOCC), chitosan-containing solution, and collagen-containing solution.

15. The medical solution of claim 12, wherein the at least one angiogenesis factor is a protein factor selected from a group consisting of VEGF, VEGF 2, bFGF, VEGF121, VEGF165, VEGF189, VEGF206, PDGF, PDAF, TGF-β, TGF-α, PDEGF, PDWHF, epidermal growth factor, insulin-like growth factor, aFGF, human growth factor, and combinations thereof.

16. The medical solution of claim 12, wherein the at least one angiogenesis factor is a non-protein factor selected from a group consisting of ginsenoside Rgi, ginsenoside Re, and combinations thereof.

17. The medical solution of claim 12, wherein the medical solution is in a form of liquid, paste, gel, suspension, caαlkid, or .plasma,.

18. The medical solution of claim 17, wherein the medical solution is configured and formulated for administering to said tissue site by an administration route selected from a group consisting of an oral administration, topical administration, percutaneous injection, intravenous injection, intramuscular injection, oral administration, and implantation.

19. A crosslinkable biological solution kit comprising a first readily mixable crosslinkable biological solution component and a second crosslinker component, wherein the first component and the second component are mixed at a point of need.

20. The kit of claim 19, wherein said first component further comprises at least one non-protein angiogenesis factor.

21. The kit of claim 20, wherein said non-protein angiogenesis factor comprises ginsenoside Rg₁, ginsenoside Re, or combinations thereof.

22. The kit of claim 19, wherein said first component further comprises at least one protein angiogenesis factor selected from a group consisting of VEGF, VEGF 2, bFGF, VEGF121, VEGF165, VEGFl 89, VEGF206, PDGF, PDAF, TGF-β, TGF-α, PDEGF, PDWHF, epidermal growth factor, insulin-like growth factor, aFGF, human growth factor, and combinations thereof.

23. The kit of claim 19, wherein said first component further comprises at least one bioactive agent selected from a group consisting of analgesics/antipyretics, antiasthamatics, antibiotics, antidepressants, antidiabetics, antifungal agents, antihypertensive agents, anti-inflammatories, antineoplastics, antianxiety agents, immunosuppressive agents, antimigraine agents, sedatives/hypnotics, antipsychotic agents, antimanic agents, antiarrhythmics, antiarthritic agents, antigout agents, anticoagulants, thrombolytic agents, antifibrinolytic agents, antiplatelet agents and antibacterial agents, antiviral agents, antimicrobials, and anti-infectives.

24. The kit of claim 19, wherein said first component further comprises at least one bioactive agent selected from a group consisting of actinomycin D, paclitaxel, vincristin, methotrexate, and angiopeptin, batimastat, halofuginone, sirolimus, tacrolimus, everolimus, tranilast, ABT-578, dexamethasone, mycophenolic acid, lovastatin, thromboxane A₂ synthetase inhibitors, eicosapentanoic acid, ciprostene, trapidil, angiotensin convening enzyme inhibitors, heparin, and biological cells.

25. The kit of claim 19, wherein the crosslinkable biological solution kit is packaged in a form suitable for application selected from a group consisting of a topical administration, percutaneous injection, intravenous injection, intramuscular injection, oral administration, and loading on an implant.