RU2808448C1 - Device based on collagen-hydroxyapatite for non-surgical treatment of paradontium - Google Patents

Abstract

FIELD: medicine; dentistry.

SUBSTANCE: invention concerns a non-surgical method of periodontitis treating. The following is proposed: a non-surgical method of periodontitis treating which involves inserting a compressed article into a periodontal pocket in which the article is substantially composed of cross-linked collagen and hydroxyapatite; the compression is applied with a force of 0.005 to 10 ton-force.

EFFECT: use of the method can contribute to the reconstruction and growth of bone tissue in the area of the defect.

14 cl, 7 dwg, 2 ex

Description

Field of technology to which the invention relates

[001] The present invention provides a non-surgical method for treating periodontitis, comprising inserting an article into a periodontal pocket, wherein the article contains cross-linked collagen and hydroxyapatite. The present invention also provides articles for use in these methods.

Information about the prior art

[002] Periodontitis is a disease in which periodontal tissue is chronically infected, continually recedes and exposes the root surface to the oral environment, often forming a pocket that can become infected. This can lead to loosening and eventual loss of the tooth and its supporting bone. Periodontal treatment often focuses on debridement of the root surface and an attempt to reduce pocket depth by reattaching the root surface or reducing loose gingival tissue.

[003] Periodontal treatment usually begins with non-surgical debridement (scaling and root planing) followed by careful care (both professional and the patient's own oral hygiene measures). When this is not enough and further loss of attachment is detected, resection or regenerative surgical procedures are used in an attempt to stop the progression of the disease and even achieve regeneration of lost periodontal tissue.

[004] Accordingly, there is an unmet need for alternative, minimally invasive, safer, less painful and easier to perform methods of treating periodontitis.

[005] The present invention provides a non-surgical method for treating periodontitis, comprising inserting an article into a periodontal pocket, wherein the article contains cross-linked collagen and hydroxyapatite.

Brief description of the invention

[006] In one embodiment, the present invention provides a non-surgical method for treating periodontitis, comprising inserting an article into a periodontal pocket, wherein the article comprises cross-linked collagen and hydroxyapatite. In another embodiment, the method results in a reduction in the depth of the periodontal pocket.

Brief description of drawings

[007] The subject matter of the invention is specifically stated and clearly stated at the end of the specification. However, the invention, both with respect to organization and method of operation, together with its objects, features and advantages, can best be understood with reference to the following detailed description, which should be read in conjunction with the accompanying drawings, in which:

[008] in FIG. 1 shows product forms used in this invention;

[009] in FIG. 2A-2B show micrographs of histological analysis of a 5 mm defect in Animal 1, which was filled with a cross-linked collagen-hydroxyapatite product and covered with OSSIX® PLUS, where: “I” means inflammation, “NB” means new bone, “OB” means original bone, “OP” means Ossix® Plus, and “OPB” means cross-linked collagen-hydroxyapatite product. Fig. 2A - double magnification; and figs. 2B - five-fold magnification and focusing on the interface between the product based on cross-linked collagen-hydroxyapatite and the newly formed bone;

[0010] in FIG. 3A-3B show radiographic analysis of animals 1 and 2 as part of a bone grafting study. Fig. 3A: animal 1; and figs. 3B: animal 2;

[0011] in FIG. 4A-4B show micrographs of histological analysis of a 10 mm defect in animal 2, which was filled with a cross-linked collagen-hydroxyapatite product and covered with OSSIX® PLUS, where: “F” means fibroblasts, “NB” means new bone, “OB” means original bone, “OP” means Ossix® Plus, and “OPB” means cross-linked collagen-hydroxyapatite product. Fig. 4A: double magnification; and figs. 4B: 5x magnification and focusing on the area containing the newly formed bone; residual defect; and a cross-linked collagen-hydroxyapatite product.

[0012] It should be understood that for simplicity and clarity of illustration, elements shown in the figures are not necessarily drawn to scale. For example, for the sake of clarity, the size of some elements may be exaggerated compared to other elements. Additionally, where appropriate, reference numerals may be repeated among the figures to designate corresponding or similar elements.

Detailed Description of the Present Invention

[0013] The following detailed description sets forth numerous specific details in order to provide a thorough understanding of the invention. However, those skilled in the art will appreciate that the present invention can be practiced without these specific details. In other cases, well-known methods, procedures and components are not described in detail so as not to obscure the present invention.

Non-surgical methods for treating periodontitis

[0014] In one aspect, the present invention provides a non-surgical method for treating periodontitis, comprising inserting an article into a periodontal pocket, wherein the article comprises cross-linked collagen and hydroxyapatite. In one embodiment, the periodontal pocket is infected or not infected. In another embodiment, the periodontal pocket is infected. In another embodiment, the infection is a bacterial or microbial infection. In another embodiment, the periodontal pocket is not infected. Each possibility represents a different embodiment of the invention.

[0015] In one embodiment, the method of the present invention results in a reduction in periodontal pocket depth. Without being limited by any mechanism or theory, it is believed that the collagen-hydroxyapatite product stimulates the growth of bone and soft tissue in the pocket area, resulting in “closure” of the pocket and a decrease in its depth. In addition, it is believed that the collagen (in the product) serves as a substrate for the collagenase enzyme in the pocket and thus may saturate the active site of the enzyme, inhibiting the degradation of gingival tissue; Once applied to the periodontal pocket, the product is absorbed into the periodontal fluid and converted into a hydrated soft framework that slows collagenase-mediated destruction of gingival tissue by absorbing the enzyme and saturating its active site. In addition, it is believed that the hydroxyapatite (in the article) serves as a solid resin to absorb and immobilize the collagenase enzyme.

[0016] In one embodiment, the method of this invention does not include the administration of an additional pharmaceutically active agent.

[0017] In one further embodiment, the method of this invention further comprises administering at least one pharmaceutically active agent selected from the group consisting of: antibacterial agents, antifungal agents, antiseptic agents, anti-inflammatory agents, antibiotic agents, vitamins and vitamers, and any combination of them. In another embodiment, the pharmaceutically active agent is an antibacterial agent. In another embodiment, the pharmaceutically active agent is an antifungal agent. In another embodiment, the pharmaceutically active agent is an antiseptic agent. In another embodiment, the pharmaceutically active agent is an anti-inflammatory agent. In another embodiment, the pharmaceutically active agent is an antibiotic agent. In another embodiment, the pharmaceutically active agent is a vitamin and/or vitamer. Each possibility represents a separate embodiment of the invention.

[0018] In another embodiment, non-limiting examples of antibacterial agents include: amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, arbekacin, plazomycin, streptomycin, apramycin, geldanamycin, herbimycin, loracarbef, faropenem, ertapenem, doripenem, imipenem , meropenem , cefazolin, cephasetril, cefadroxil, cephalexin, cephaloglycine, cephalonium, cephaloridine, cephalothin, cephapirin, cefatrizin, cefazedon, cefazaflur, cefradine, cefroxadine, ceftezol, cefaclor, cefamandole, cefminox, cefonicide, ceforanide, cefotiam, cefprozil, cefbuperazone, cefuroxime, cefuzonam , cephamycin, cefoxitin, cefotetan, cefmetazole, carbacefem, cefixime, ceftazidime, ceftriaxone, cefcapen, cefdaloxime, cefdinir, cefditoren, cefetameth, cefmenoxime, cefodizime, cefoperazone, cefotaxime, cefpimizole, cefpira mid, cefpodoxime, cefsulodin, cefteram, ceftibuten, cefthiolene, ceftizoxime , oxacephem, cefepime, cefozopran, cefpirome, cefquin, ceftiofur, cefquin, cefovecin, CXA-101, ceftaroline, ceftobiprole, clindamycin, lincomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, Trolean omycin, telithromycin, spectinomycin, solithromycin, aztreonam, furazolidone , nitrofurantoin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, temocillin, ticarcillin, iprofloxacin , enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidix acid, levonadifloxacin, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, delafloxacin, mafenide, sulfonamidochryzoidin, sulfacetamide, sulfadiazine, sulfamethizole, sulfamethoxazole, sulfasalazine, sulfisoxazole, trimethoprim, emec locycline, doxycycline, minocycline, oxytetracycline, tetracycline, tigecycline, tedizolid, linezolid, ranbezolid, torezolid, radezolid, any combination thereof and any pharmaceutically acceptable salts thereof. Each possibility represents a different embodiment of the invention.

[0019] In another embodiment, non-limiting examples of antifungal agents include: terbinafine, naftifine, amphotericin B, butenafine, chloroxylenol, ciclopirox, flucytosine, caspofungin, griseofulvin, clotrimazole, fluconazole, itraconazole, ketoconazole, miconazole, oxyconazole, nystatin, undecylene oic acid, any a combination thereof and any pharmaceutically acceptable salt thereof. Each possibility represents a different embodiment of the invention.

[0020] In another embodiment, non-limiting examples of antiseptic agents include: naftifine, tolnaftate, mediocidin, candicidin, trichomycin, gamycin, aurefungin, ascozine, aifattine, azacolutin, trichomycin, levorin, heptamicin, candymycin, griseofulvin, pradimycins, benanomycin; ambisome; nikkomycin Z; flucytosine, perimycin, any combination thereof and any pharmaceutically acceptable salt thereof. Each possibility represents a different embodiment of the invention.

[0021] In another embodiment, non-limiting examples of anti-inflammatory agents include: aspirin, ibuprofen, naproxen, celecoxib, diclofenac, ketoprofen, ketorolac, oxaprozin, salsalate, sulindac, any combination thereof, and any pharmaceutically acceptable salt thereof. Each possibility represents a different embodiment of the invention.

[0022] In another embodiment, non-limiting examples of antibiotics include penicillin, cephalosporin, ciprofloxacin, erythromycin, any combination thereof, and any pharmaceutically acceptable salt thereof. Each possibility represents a different embodiment of the invention.

[0023] In another embodiment, non-limiting examples of vitamins and vitamers include: vitamin A, retinol, retinal, carotenoid, vitamin B1, thiamine, vitamin B2, riboflavin, vitamin B3, niacin, niacinamide, nicotinamide, riboside, vitamin B5, pantothenic acid, vitamin B6, pyridoxine, pyridoxamine, pyridoxal, vitamin B7, biotin, vitamin B9, folate, vitamin B12, cyanocobalamin, hydroxocobalamin, methylcobalamin, adenosylcobalamin, vitamin C, ascorbic acid, vitamin D, cholecalciferol (D3), ergocalciferol (D2), vitamin E, tocopherols, tocotrienols, vitamin K, phylloquinone, menaquinones, any combination thereof and any pharmaceutically acceptable salt thereof. Each possibility represents a different embodiment of the invention.

[0024] In one embodiment, introducing the article into a periodontal pocket includes inserting or pushing the article into the pocket. In another embodiment, insertion or pushing is performed to allow at least partial contact between the article and the pocket. In another embodiment, insertion or pushing is performed to allow full contact between the product and the pocket.

[0025] In one embodiment, the method of this invention further includes scaling and root planing, wherein the scaling and root planing is performed prior to insertion of the product. In another embodiment, scaling and root planing are performed by any method known in the art.

[0026] In one embodiment, the method of the present invention further includes covering the article of the present invention with a cross-linked collagen membrane (e.g., Ossix® Plus) such that the article is in contact with (e.g., inserted into) a periodontal pocket and the collagen membrane is contained over products. In another embodiment, fixation sutures are placed on the collagen membrane.

Articles used in the methods of this invention

[0027] In another aspect, the present invention provides an article containing cross-linked collagen and hydroxyapatite for use in the methods described above herein. In one embodiment, the product is biodegradable and biocompatible.

[0028] In some embodiments, the weight ratio of collagen to hydroxyapatite in the article used in this invention is from 95:5 to 20:80. In another embodiment, the ratio is from 90:10 to 25:75. In another embodiment, the ratio is from 85:15 to 30:70. In another embodiment, the ratio is from 80:20 to 35:65. In another embodiment, the ratio is from 75:25 to 40:60. In another embodiment, the ratio is from 70:30 to 45:55. In another embodiment, the ratio is 70:30. Each possibility represents a different embodiment of the invention.

[0029] In some embodiments, the concentration of cross-linked collagen in the article used in this invention is from 20 to 95 wt./wt. In another embodiment, the concentration is from 20 to 30 wt./mass. In another embodiment, the concentration is from 30 to 40 wt./mass. In another embodiment, the concentration is from 40 to 50 wt./mass. In another embodiment, the concentration is from 50 to 60 wt./mass. In another embodiment, the concentration is from 60 to 70 wt./mass. In another embodiment, the concentration is from 70 to 80 wt./mass. In another embodiment, the concentration is from 80 to 90 wt./mass. In another embodiment, the concentration is from 90 to 95 wt./mass. Each possibility represents a different embodiment of the invention.

[0030] In some embodiments, the concentration of hydroxyapatite in the article used in this invention is from 5 to 80 wt./wt. In another embodiment, the concentration is from 5 to 10 w/w. In another embodiment, the concentration is from 10 to 20 w/w. In another embodiment, the concentration is from 20 to 30 wt./mass. In another embodiment, the concentration is from 30 to 40 wt./mass. In another embodiment, the concentration is from 40 to 50 wt./mass. In another embodiment, the concentration is from 50 to 60 wt./mass. In another embodiment, the concentration is from 60 to 70 wt./mass. In another embodiment, the concentration is from 70 to 80 wt./mass. Each possibility represents a different embodiment of the invention.

[0031] In some embodiments, the article used in this invention further contains one pharmaceutically active agent, as described above herein.

[0032] In some embodiments, a pharmaceutically active agent, as described above herein, can be administered additionally and/or in an administration article that contains cross-linked collagen and hydroxyapatite. In one embodiment, when the method of this invention further includes administering a pharmaceutically active agent, the administered article may or may not contain a pharmaceutically active agent as described above, in addition to cross-linked collagen and hydroxyapatite. In one embodiment, when the method of this invention does not include administration of a pharmaceutically active agent, the administered article may or may not contain a pharmaceutically active agent as described above, in addition to cross-linked collagen and hydroxyapatite. Each possibility represents a different embodiment of the invention.

[0033] In some embodiments, the term "article" as used herein refers to a matrix that is in some shape or form, that is, compressed, designed, engineered, manufactured, or produced by methods known in the art to provide some two- or three-dimensional structure of such a product. In other embodiments, non-limiting examples of two- or three-dimensional structures are depicted in FIGS. 1. In other embodiments, any conceivable design, construction, manufacturing, or production method may be used to obtain the product. In another embodiment, the matrix is designed, compressed and/or shaped to provide a product, as described above, and is placed in the periodontal pocket. In another embodiment, the product is pressed with a force from 0.005 to 10 tf. In another embodiment, the product is pressed with a force from 0.1 to 6 tf. In another embodiment, the product is pressed with a force of 0.0055 to 0.2 tf. In another embodiment, the product is pressed with a force of 0.005 to 0.02 tf. In another embodiment, the product is pressed with a force of 0.02 to 0.05 tf. In another embodiment, the product is pressed with a force of 0.05 to 0.1 tf. In another embodiment, the product is pressed with a force of 0.1 to 0.2 tf. In another embodiment, the product is pressed with a force of 0.2 to 1 tf. In another embodiment, the product is pressed with a force of 1 to 2 tf. In another embodiment, the product is pressed with a force of 2 to 5 tf. In another embodiment, the product is pressed with a force of 5 to 10 tf. Each possibility represents a different embodiment of the invention.

[0034] In some embodiments, the term “matrix” as used herein refers to a physical volume of solid matter (eg, cross-linked collagen and hydroxyapatite). In other embodiments, the solid is dry and does not contain any solvent or liquid. In other embodiments, the solid contains at least one single component. In another embodiment, non-limiting examples of the component include: a chemical compound, a small molecule, a metal, an alloy, a composite material, a biomaterial, a polymer, and an organometallic complex. In another embodiment, the solid contains more than one component selected from the above list. In another embodiment, the matrix contains a biocompatible material or composition. In another embodiment, the matrix is a biopolymer or protein. In other embodiments, the physical properties of the matrix allow it to be designed, molded, cut, or constructed, as is known in the art, into a desired shape. In another embodiment, non-limiting examples of matrix properties include the following characteristics: dense, porous or non-porous, viscous, rigid, soft or moldable. In other embodiments, cutting is performed by any method known in the art. In another embodiment, the cutting method in accordance with the method of the invention is performed on a computer numerical control (CNC) machine, laser cutting machine, water jet cutting machine, drilling machine or grinding machine. Each possibility represents a different embodiment of the invention. In another embodiment, the matrix has a porosity of 10-90%. In another embodiment, the matrix has a porosity of 10-20%. In another embodiment, the matrix has a porosity of 20-30%. In another embodiment, the matrix has a porosity of 30-40%. In another embodiment, the matrix has a porosity of 40-50%. In another embodiment, the matrix has a porosity of 50-60%. In another embodiment, the matrix has a porosity of 60-70%. In another embodiment, the matrix has a porosity of 70-80%. In another embodiment, the matrix has a porosity of 80-90%. In another embodiment, the matrix has a porosity of 10-30%. In another embodiment, the matrix has a porosity of 30-50%. In another embodiment, the matrix has a porosity of 50-70%. In another embodiment, the matrix has a porosity of 70-90%. Each possibility represents a different embodiment of the invention.

[0035] In some embodiments, the term “collagen” refers to a biopolymer organized in fibrillar networks or other non-fibrillar superstructures, and is a major component of connective tissue in humans or many animals. Numerous types of collagen are known and occur in nature. Non-limiting examples include types I-V. In some embodiments, the collagen has a fibrillar (eg, type I) or non-fibrillar structure. Each possibility represents a different embodiment of the invention.

[0036] In one embodiment, the collagen used in this invention refers to native collagen, fibrillar collagen, fibrillar atelopeptide collagen, lyophilized collagen, collagen derived from animal sources, human collagen, recombinant collagen, pepsinized collagen, reconstituted collagen, and any of these combinations. In another embodiment, the collagen includes fibrillar collagen reconstituted from monomolecular atelopeptide collagen. In another embodiment, the collagen is atelopeptide fibrillar collagen obtained by reconstituting monomolecular atelopeptide collagen obtained by proteolytic cleavage of native collagen. Each possibility represents a different embodiment of the invention.

[0037] In some embodiments, the term “cross-linked collagen” refers to a covalent network containing biopolymer chains of collagen covalently and intermolecularly linked by cross-linking agents.

[0038] In some embodiments, the term “cross-linking agents” refers to small molecules or polymers containing at least two ends that can covalently connect polymer/oligomeric chains and thereby cross-link those chains.

[0039] In some embodiments, the collagen is cross-linked with sugar. In another embodiment, the sugar is selected from the group consisting of glycerose (glyceraldehyde), threose, erythrose, lyxose, xylose, arabinose, ribose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, and any combination thereof. In another embodiment, the sugar is glycerose (glyceraldehyde). Each possibility represents a different embodiment of the invention.

[0040] In another embodiment, the sugar is a disaccharide. Each possibility represents a different embodiment of the invention.

[0041] In another embodiment, the disaccharide is selected from the group consisting of maltose, lactose, sucrose, cellobiose, gentiobiose, melibiose, turanose, trehalose, and any combination thereof. Each possibility represents a different embodiment of the invention.

[0042] In another embodiment, the crosslinking agent can be any crosslinking agent known in the art. In another embodiment, the crosslinking agent is a sugar. In another embodiment, the sugar is a compound represented by at least one of the following formulas I or II:

(I)

(II),

Where:

R ¹ represents H or alkyl or alkenyl, an amino acid fragment, a peptide fragment, a saccharide fragment, a purine or pyrimidine fragment, a phosphorylated purine or pyrimidine fragment;

n is an integer from 2 to 9, and

each of p and q is independently an integer from 0 to 8, and the sum of p and q is at least 2 and at most 8.

[0043] In another embodiment, the term “alkyl” group refers to a saturated aliphatic hydrocarbon, including straight or branched chain. In one embodiment, the alkyl group is straight or branched. In another embodiment, the alkyl is optionally substituted linear or branched. In one embodiment, the alkyl group has from 1 to 20 carbon atoms. In one embodiment, the alkyl group has from 1 to 10 carbon atoms. In one embodiment, the alkyl group has from 2 to 10 carbon atoms. In one embodiment, the alkyl group has from 1 to 6 carbon atoms. In one embodiment, the alkyl group has from 2 to 8 carbon atoms. In another embodiment, non-limiting examples of alkyl groups include methyl, ethyl, propyl, isopropyl, isobutyl, butyl, pentyl, 3-pentyl, hexylheptyl, octyl and hexadecyl. In another embodiment, the alkyl group is optionally substituted with one or more halogens, hydroxides, alkoxides, carboxylic acids, phosphates, phosphonates, sulfates, sulfonates, amidates, cyanates, and a nitro group. Each possibility represents a different embodiment of the invention.

[0044] In another embodiment, the term “alkenyl” group refers to alkyl groups, as described herein, having at least one carbon-carbon double bond, including straight and branched chain groups. In one embodiment, the alkene has one double bond. In another embodiment, the alkene has more than one double bond. In another embodiment, the alkene has from 2 to 6 double bonds, with each possibility representing a different embodiment of the present invention. In one embodiment, the alkene has 2-20 carbon atoms. Non-limiting examples include ethylenyl, propylenyl, 2-methylpropyl-1-enyl and butenyl, each possibility representing a different embodiment of the present invention.

[0045] In another embodiment, the term “amino acid” refers to an organic compound containing amine (-NH ₂ ) and carboxyl (-COOH) functional groups along with a side chain specific to each amino acid. In another embodiment, any amino acid known in the art may be used. In another embodiment, the amino acid is alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0046] In another embodiment, the term “peptide” refers to short chains of amino acids covalently linked by amide (peptide; -C(O)-N(H)-) bonds. In another embodiment, the peptide contains from 2 to 20 amino acids. In another embodiment, the peptide is a dipeptide. In another embodiment, the peptide is a tripeptide. In another embodiment, the peptide is a tetrapeptide. In another embodiment, the peptide is a pentapeptide. In another embodiment, the peptide is a hexapeptide.

[0047] In another embodiment, the term “saccharide” refers to the group including sugars, cellulose and starch as described herein.

[0048] In another embodiment, the term “purine” refers to a heterocyclic aromatic organic compound that consists of a pyrimidine ring fused to an imidazole ring. Non-limiting examples of purines include: purine, adenine, guanine, hypoxanthine, xanthine, theobromine, caffeine, uric acid and isoguanine.

[0049] In another embodiment, the term “pyrimidine” refers to a heterocyclic aromatic organic compound similar to pyridine but having an additional nitrogen on the aromatic ring such that the nitrogen atoms are in the 1, 3 positions of the ring. Non-limiting examples of purines include: cytosine, thymine and uracil.

[0050] In another embodiment, the term "phosphorylated purine or pyrimidine" refers to a purine or pyrimidine, as described herein, where the purine or pyrimidine is connected to a phosphoryl group (the chemical compound PO ₃ ^x- ; "x" denotes any possible protic state ).

[0051] In another embodiment, the sugar is a naturally occurring reducing sugar.

[0052] In another embodiment, the sugar is diose, triose, tetrose, pentose, hexose, septose, octose, nanose, or decose. Each probability represents a different embodiment of the invention.

[0053] In another embodiment, the sugar is selected from the group consisting of glycerose (glyceraldehyde), threose, erythrose, lyxose, xylose, arabinose, ribose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, and any combination thereof . Each probability represents a different embodiment of the invention.

[0054] In another embodiment, the sugar is a disaccharide. Each possibility represents a different embodiment of the invention.

[0055] In another embodiment, the disaccharide is selected from the group consisting of maltose, lactose, sucrose, cellobiose, gentiobiose, melibiose, turanose, trehalose, and any combination thereof. Each possibility represents a different embodiment of the invention.

[0056] In some embodiments, the term “hydroxyapatite” refers to the naturally occurring mineral form of calcium with the formula Ca ₅ (PO ₄ ) ₃ (OH) or Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ . In other embodiments, hydroxyapatite can be manufactured or obtained by any method known in the art. In another embodiment, bulk hydroxyapatite or hydroxyapatite in the form of nanoparticles is used and/or manufactured. Each possibility represents a different embodiment of the invention.

[0057] In some embodiments, the term “nanoparticle substance” (eg, hydroxyapatite) refers to a substance that has at least one physical nanoscale size. In another embodiment, the nanoparticle material is in the form of nanoparticles, nanospheres, nanocubes, nanoplates, nanoribbons, nanowires, nanorods, or any other nanosized form known in the art.

Method for manufacturing products according to this invention

[0058] In another aspect, the present invention provides a method of making an article for use in a non-surgical method of treating periodontitis, wherein the article contains cross-linked collagen and hydroxyapatite, and wherein the method includes:

A. providing a matrix containing cross-linked collagen and hydroxyapatite; And

b. giving the matrix shape, thereby obtaining a product containing cross-linked collagen and hydroxyapatite.

[0059] In one embodiment, the present invention provides a method for making a matrix containing cross-linked collagen and hydroxyapatite, the method comprising:

(i) mixing collagen and hydroxyapatite in solution to fibrillate collagen;

(ii) cross-linking the collagen with a cross-linking agent, thereby obtaining a cross-linked suspension;

(iii) concentrating the cross-linked suspension, thereby obtaining a concentrated suspension;

(iv) lyophilizing the concentrated suspension, thereby obtaining a dry precipitate; And

(v) grinding the dry sediment, thereby obtaining a matrix containing cross-linked collagen and hydroxyapatite.

[0060] In one embodiment, the present invention provides a method for making a matrix containing cross-linked collagen and hydroxyapatite, the method comprising:

(i) providing an acidic solution of collagen followed by neutralizing the solution, wherein the neutralizing solution contains hydroxyapatite;

(ii) concentrating the solution obtained in step (i);

(iii) lyophilizing the concentrated mixture obtained in step (ii), thereby obtaining a dried collagen composition;

(iv) incubating the composition with a cross-linking agent and a first solvent;

(v) washing the incubated composition obtained in step (iv) with a second solvent; And

(vi) lyophilizing the washed composition obtained in step (v), thereby obtaining a matrix containing cross-linked collagen and hydroxyapatite.

[0061] In one embodiment, the present invention provides a method for making a matrix containing cross-linked collagen, hydroxyapatite and a pharmaceutically active agent, the method comprising:

(i) providing an acidic solution of collagen followed by neutralizing the solution, wherein the neutralizing solution contains hydroxyapatite;

(ii) concentrating the solution obtained in step (i);

(iii) lyophilizing the concentrated mixture obtained in step (ii), thereby obtaining a dried collagen composition;

(iv) incubating the composition with a cross-linking agent, a first solvent and adding a pharmaceutically active agent;

(v) washing the incubated composition obtained in step (iv) with a second solvent; And

(vi) lyophilizing the washed composition obtained in step (v), thereby obtaining a matrix containing cross-linked collagen, hydroxyapatite and a pharmaceutically active agent.

[0062] In some embodiments, compression steps (application of mechanical pressure using special equipment) are used in addition to or instead of lyophilization steps.

Method for manufacturing an article of the present invention using a mold

[0063] In one further aspect, the present invention provides a method for making an article containing cross-linked collagen, hydroxyapatite, and the method includes:

(i) providing an acidic solution of collagen followed by neutralizing the solution, wherein the neutralizing solution contains hydroxyapatite;

(ii) concentrating the solution obtained in step (i); and pouring it into a mold with a pre-designed matrix shape;

(iii) lyophilizing the concentrated mixture obtained from step (ii), thereby obtaining a dried composition containing collagen and hydroxyapatite;

(iv) incubating the composition with a cross-linking agent and a first solvent;

(v) washing the incubated composition obtained in step (iv) with a second solvent; And

(vi) lyophilizing the washed composition obtained in step (v), thereby obtaining an article containing cross-linked collagen and hydroxyapatite.

[0064] In one embodiment, the present invention provides a method of making an article containing cross-linked collagen, hydroxyapatite and a pharmaceutically active agent, and the method includes:

(i) providing an acidic solution of collagen and a cross-linking agent, followed by neutralizing the solution, wherein the neutralizing solution contains hydroxyapatite;

(ii) concentrating the solution obtained in step (i); and pouring it into a mold with a pre-designed matrix shape;

(iii) lyophilizing the concentrated mixture obtained from step (ii), thereby obtaining a dried composition containing collagen and hydroxyapatite;

(iv) incubating the composition with a cross-linking agent, a first solvent and a pharmaceutically active agent;

(v) washing the incubated composition obtained in step (iv) with a second solvent; And

(vi) lyophilizing the washed composition obtained in step (v), thereby obtaining an article containing cross-linked collagen, hydroxyapatite and a pharmaceutically active agent.

[0065] In some embodiments, compression steps (application of mechanical pressure using special equipment) are used in addition to or instead of lyophilization steps.

[0066] In some embodiments, the mold is cooled to a freezing temperature of -10°C to -190°C before the lyophilization step (iii). In another embodiment, the mold is cooled to a freezing temperature of -10 to -80°C over a period of 0.5 to 24 hours, followed by a lyophilization step (iii). In another embodiment, cooling is carried out for 0.5-24 hours. In another embodiment, cooling is carried out for 0.5-1 hour. In another embodiment, cooling is carried out for 1-2 hours. In another embodiment, cooling is carried out for 2-5 hours. In another embodiment, cooling is carried out for 5-10 hours. In another embodiment, cooling is carried out for 10-24 hours. Each probability represents a different embodiment of the invention.

[0067] In another embodiment, a mold is manufactured to obtain the desired die shape using a method including 3D printing, injection molding, or any combination thereof. Each possibility represents a different embodiment of the invention.

Method for manufacturing an article according to the present invention using granulate

[0068] In another aspect, the present invention provides a method for making an article containing cross-linked collagen, hydroxyapatite, and the method includes:

(i) providing an acidic solution of collagen followed by neutralizing the solution, wherein the neutralizing solution contains hydroxyapatite;

(ii) concentrating the solution obtained in step (i);

(iii) incubating the composition with a cross-linking agent and a first solvent;

(iv) washing the incubated composition obtained in step (iv) with a second solvent;

(v) homogenizing, casting and grinding the composition to obtain a cross-linked collagen granulate;

(vi) wetting the granulate obtained in step (v) with the first or second solvent and cutting to obtain an article containing cross-linked collagen and hydroxyapatite.

[0069] In one embodiment, the present invention provides a method of making an article containing cross-linked collagen, hydroxyapatite and a pharmaceutically active agent, and the method includes:

(i) providing an acidic solution of collagen followed by neutralizing the solution, wherein the neutralizing solution contains hydroxyapatite;

(ii) concentrating the solution obtained in step (i);

(iii) incubating the composition with a cross-linking agent, a first solvent and adding a pharmaceutically active agent;

(iv) washing the incubated composition obtained in step (iv) with a second solvent;

(v) homogenizing, casting and grinding the composition to obtain a cross-linked collagen granulate;

(vi) wetting the granulates obtained in step (v) with a first or second solvent and cutting to obtain an article containing cross-linked collagen, hydroxyapatite and a pharmaceutically active agent.

[0070] In some embodiments, the matrix produced by the methods of the present invention is ground to form a granulate and then wetted with a first or second solvent and cut to form an article of the present invention. In other embodiments, the granule size ranges from 1 to 2000 microns.

[0071] In some embodiments, an article manufactured in accordance with the methods described above is sterilized by any method known in the art (eg, ethylene oxide (EtO) gas).

[0072] In some embodiments, the collagen used in the methods of the present invention in solution in step "(i)" is selected from the following non-limiting examples, including: native collagen, fibrillar collagen, fibrillar atelopeptide collagen, lyophilized collagen, collagen derived from animal sources, human collagen, recombinant collagen, pepsinized collagen, reconstituted collagen, and any combination thereof. In another embodiment, the collagen includes fibrillar collagen reconstituted from monomolecular atelopeptide collagen. In another embodiment, the collagen is atelopeptide fibrillar collagen obtained by reconstituting monomolecular atelopeptide collagen obtained by proteolytic cleavage of native collagen. Each possibility represents a different embodiment of the invention.

[0073] In some embodiments, the neutralization solution used in the methods of the present invention contains a base or a buffer. In another embodiment, the buffer is selected from phosphate buffered saline, NaHCO ₃ /Na ₂ CO ₃ buffer, Tris buffer or Tricine buffer, or any other buffer that maintains a neutral pH. In another embodiment, the acidic solution contains HCl, acetic acid, nitric acid, citric acid, sulfuric acid, phosphoric acid, or any other acid known in the art. In another embodiment, the stock solution contains NaOH, KOH, NaHCO ₃ , Na ₂ CO _{3 ,} Na ₂ HPO ₄ or any other base known in the art.

[0074] In some embodiments, the term “neutral pH” refers to a pH range that resembles the physiological pH in a biological organism and/or system; and is defined as 6.5-7.5. In some other embodiments, the neutral pH is 6.5-6.7. In some other embodiments, the neutral pH is 6.7-6.9. In some other embodiments, the neutral pH is between 6.9 and 7.1. In some other embodiments, the neutral pH is 7.1-7.3. In some other embodiments, the neutral pH is 7.3-7.5. In some other embodiments, the neutral pH is 7.1-7.2. In some other embodiments, the neutral pH is 7.2-7.3. In some other embodiments, the neutral pH is 7.3-7.4. In some other embodiments, the neutral pH is 7.4-7.5.

[0075] In some embodiments, the concentration step used in the methods of the present invention is performed by centrifugation. In another embodiment, centrifugation is carried out at a speed of 50-20,000 rpm (cycles per minute). In another embodiment, centrifugation is carried out at a speed of 50-100 rpm. In another embodiment, centrifugation is carried out at a speed of 100-1000 rpm. In another embodiment, centrifugation is carried out at a speed of from 1000 to 5000 rpm. In another embodiment, centrifugation is carried out at a speed of from 5000 to 10000 rpm. In another embodiment, centrifugation is carried out at a speed of 10,000-20,000 rpm. In another embodiment, centrifugation is carried out for 1-120 minutes. In another embodiment, centrifugation is carried out for 1-5 minutes. In another embodiment, centrifugation is carried out for 5-10 minutes. In another embodiment, centrifugation is carried out for 10-20 minutes. In another embodiment, centrifugation is carried out for 20-50 minutes. In another embodiment, centrifugation is carried out for 50-100 minutes. In another embodiment, centrifugation is carried out for 100-120 minutes. Each probability represents a different embodiment of the invention.

[0076] In another embodiment, the lyophilization in step (iii) is carried out for 1-48 hours. In another embodiment, the lyophilization in step (iii) is carried out for 1-2 hours. In another embodiment, the lyophilization in step (iii) is carried out for 2-5 hours. In another embodiment, the lyophilization in step (iii) is carried out for 5-10 hours. In another embodiment, the lyophilization in step (iii) is carried out for 10-24 hours. In another embodiment, the lyophilization in step (iii) is carried out for 24-48 hours. Each probability represents a different embodiment of the invention.

[0077] After lyophilization, the dried collagen-hydroxyapatite composition of the present invention is obtained. The dried composition is incubated with a crosslinking agent, a first solvent and optionally a pharmaceutically active agent. The incubated composition is further washed with a second solvent and lyophilized to obtain a matrix containing cross-linked collagen, hydroxyapatite and optionally a pharmaceutically active agent.

[0078] In another embodiment, the first and second solvents are the same or different and are selected from any solvent known in the art. In another embodiment, the solvent is selected from the group consisting of: water, ethanol, saline, methanol, phosphate-buffered saline, or any combination thereof.

[0079] In another embodiment, the lyophilization in step (vi) is carried out for 24 to 72 hours. In another embodiment, the lyophilization in step (vi) is carried out for 24 to 36 hours. In another embodiment, the lyophilization in step (vi) is carried out for 36 to 48 hours. In another embodiment, the lyophilization in step (vi) is carried out for 48 to 72 hours. Each probability represents a different embodiment of the invention.

[0080] In some other embodiments, the designed shape and cut are planned, developed and/or designed using computer-aided design (CAD) and/or computer-aided manufacturing (CAM) techniques and software known in the art. Each possibility represents a different embodiment of the invention.

[0081] In another embodiment, the cutting used in the method of the invention is performed by any method known in the art. In another embodiment, cutting in accordance with the method of the invention is performed on a computer numerical control (CNC) machine, laser cutting machine, water jet cutting machine, drilling machine or grinding machine. Each probability represents a different embodiment of the invention.

[0082] The following examples are presented to more fully illustrate preferred embodiments of the invention. However, they should in no way be construed as limiting the broad scope of the invention.

Examples

Example 1

Making a product from collagen-hydroxyapatite

[0083] The components of 30 wt% hydroxyapatite (CAS® number 12167-74-7, Plasma Biotal Limited (UK)) and 70 wt% collagen (purified from porcine tendon) were mixed together during collagen fibrillation. Subsequently, this suspension was cross-linked with sugar (glyceraldehyde). The cross-linked suspension was concentrated by centrifugation and lyophilized. The resulting intermediate dry sediment was crushed and pressed into round, rectangular or trapezoidal pieces. The compressive force applied to compact the ground intermediate product varied from 0.005 to 0.2 tf. Sterilization of the condensed substance was carried out using EtO (ethylene oxide gas).

Example 2

Study of bone grafting of a cross-linked collagen-hydroxyapatite product in a rat model

[0084] Target

[0085] The purpose of this study was to preliminarily evaluate the effectiveness and safety of a cross-linked collagen-hydroxyapatite product (prepared as described in Example 1) as a bone graft in terms of: 1) bone regeneration and/or augmentation in the model calvarial bone defect, and 2) foreign body and inflammatory reactions assessed by histological analysis.

[0086] Surgical procedure

[0087] After anesthesia, an incision was made behind the line between the ears. The full-thickness flap was peeled away to expose the calvarial bone. The soft tissue and periosteum were separated from the bone to the area in front of the eyes and a defect was created in the calvarial bone without perforating the dura using a 5 mm trephine. Two animals were used in the study. In the first, a defect with a diameter of 5 mm was created and in the second, 2 defects of 5 mm each were created, forming a defect of approximately 10 x 5 mm. The defects were filled with a cross-linked collagen-hydroxyapatite product and covered with a cross-linked collagen membrane (OSSIX® Plus). Anchorage sutures using 5-0 absorbable Vicryl suture were placed over OSSIX® Plus and secured bilaterally in the temporalis muscles.

[0088] Histological evaluation

[0089] Animals were sacrificed 9 weeks after implantation. The calvaria was removed with the overlying skin and placed in a 10% buffered formalin solution. After decalcification, samples were cut and stained with hematoxylin and eosin (H&E). Evaluation was carried out under a light microscope.

[0090] Results

[0091] Clinical observations

[0092] The animals appeared clinically normal throughout the two studies, based on their weight and food intake.

[0093] Microscopic examination

[0094] FIG. 2A-B are micrographs of a 5 mm defect in Animal 1 that was filled with a cross-linked collagen hydroxyapatite product and coated with OSSIX® PLUS. Sections were taken from an off-center location where the length of the defect was only about 2 mm. Micrographs show a completely enclosed bone structure underneath the implanted material. The newly formed bone structures are located next to and above the original bone tissue. It should be noted that this animal also experienced extensive intraoperative bleeding and postoperative suture dehiscence not related to the device. As a result, signs of inflammation appeared.

[0095] The opacity of the x-ray image in FIG. 3A indicates closure of the defect, but with a layer of bone tissue that is thinner than the original bone tissue.

[0096] FIG. 4A-B are overview micrographs of a 10 mm defect in Animal 2 that was filled with a cross-linked collagen hydroxyapatite product and coated with OSSIX® PLUS. Sections were taken from an off-center location where the length of the defect was approximately 6.5 mm. The cross-linked collagen-hydroxyapatite product showed signs of soft tissue remodeling or ossification. Micrographs show 90% closure of the bone tissue defect with newly formed bone tissue. New bone structures are found adjacent to and above the original bone tissue within the cross-linked collagen and hydroxyapatite product. Remodeling of connective tissue is confirmed by the infiltration of fibroblasts over the newly formed bone tissue.

[0097] The opacity of the defect on x-ray (Fig. 3B) 9 weeks after implantation indicates almost complete coverage of the defect by a layer of bone tissue with a thickness similar to that of the original bone tissue.

[0098] Conclusions

[0099] A bone grafting material that is a cross-linked collagen-hydroxyapatite product (combined with a dental membrane as a barrier membrane (OSSIX® PLUS)) can promote the reconstruction and expansion of bone tissue beyond the size of the original bone tissue.

[00100] Although only certain features of the invention have been illustrated and described herein, many modifications, substitutions, alterations and equivalents will be apparent to those skilled in the art. Thus, it is to be understood that the appended claims cover all such modifications and changes that fall within the spirit of the invention.

Claims (14)

1. A non-surgical method of treating periodontitis, comprising inserting a compressed article into a periodontal pocket, in which the article is substantially composed of cross-linked collagen and hydroxyapatite and the compression is applied with a force of from 0.005 to 10 ton-force. 2. The method according to claim 1, in which the compression is carried out with a force of 0.005 to 0.2 ton-force. 3. The method according to claim 1, in which the compression is carried out with a force of 2 to 5 ton-force. 4. The method according to any of the preceding paragraphs, in which the product has a porosity in the range of 40-50%. 5. The method according to any one of the preceding claims, in which the concentration of cross-linked collagen is from 20 to 95 wt.%. 6. The method according to any of the preceding paragraphs, in which the mass ratio of collagen and hydroxyapatite is from 95:5 to 20:80. 7. The method according to claim 6, in which the mass ratio is 70:30. 8. The method according to any of the preceding claims, wherein the periodontal pocket is infected. 9. The method according to any of the preceding claims, wherein the periodontal pocket is not infected. 10. The method according to any of the preceding claims, which does not include the administration of an additional pharmaceutically active agent. 11. Method according to any one of paragraphs. 1-9, which further includes the administration of at least one pharmaceutically active agent selected from the group consisting of: antibacterial agents, antifungal agents, antiseptic agents, anti-inflammatory agents, antibiotic agents, vitamins and vitamers, and any combination thereof. 12. The method of claim 11, wherein the pharmaceutically active agent is an antibiotic agent or an antiseptic agent. 13. A method as claimed in any one of the preceding claims, which includes inserting or pushing the article into a pocket. 14. The method according to any of the preceding claims, which further includes scaling and root smoothing, wherein the scaling and root smoothing is performed before insertion of the product.