artificial periosteum

Abstract

To provide an artificial periosteum which has better integrated characteristics, makes it necessary to collect an osteochondral implant, and has biochemical and mechanical characteristics of own osteochondral implant, and an improved bone restoration method, especially, an artificial periosteum which is usable to locally transmit a bone activator such as BMP-2 over a long period so as to promote bone growth and restore bone defects.SOLUTION: An artificial periosteum contains a functionalized collagen-containing film and a medicine-carrier mixture, wherein the medicine-carrier mixture contains at least one therapeutic agent and a calcium-containing carrier mixture.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an artificial periosteum, system and method for bone repair, and the use of the artificial periosteum for local delivery of therapeutic agents such as bone activators. [Background technology]

[0002] Today, many medical procedures rely on the regeneration of bone that has deteriorated due to disease or aging, or that has been damaged (e.g., fracture). While various surgical procedures are available, advances in modern medicine have enhanced certain techniques, sometimes even making it possible to replace these surgical procedures.

[0003] The periosteum is connective tissue surrounding bone that has the ability to regenerate both cartilage and bone. This tissue consists of two distinct layers: the inner cambium, which is thought to contain undifferentiated mesenchymal stem cells responsible for fracture repair, and the outer fibrous layer. The periosteum has been successfully used in biological surface reconstruction for the repair of damaged articular cartilage. For deep osteochondral defects, bone grafts can be used to replace damaged subchondral bone. However, problems associated with the use of bone grafts include obtaining grafts of appropriate size and shape, the condition of the transplant site, and integration with surrounding tissues.

[0004] The development of an artificial periosteum with better integration properties, eliminating the need to harvest osteochondral grafts, and possessing the biochemical and mechanical characteristics of autologous osteochondral grafts would be highly appealing.

[0005] A further advantage of artificial periosteum is that therapeutic agents, such as osteoactivators like BMP-2, can be used for delivery to promote cortical bone regeneration.

[0006] The existing material used for delivering recombinant human BMP-2 (rhBMP-2) is the FDA-approved porous collagen sponge. Other rhBMP-2 carriers have also been documented in the literature (Morales et al., (2017), J Drug Delivery Sciences and Technology, Vol 42). Current problems associated with approved biomaterials are their rapid degradation, leading to the abrupt release of proteins, and their secondary osteoclast-promoting effects, which reduce overall net bone formation. While porous biomaterials are used for general bone regeneration by delivering rhBMP-2, Horstmann and collaborators have reported that these materials tend to protrude into cortical bone and delay cortical bone healing (Horstmann et al. (2018), Tissue Eng. Part A, Vol. 23). Therefore, from a clinical standpoint, there is a need for a membrane in the form of a thin biomaterial that can prevent trabecular space fillers from protruding into cortical bone by providing a cell template and local release of growth factors, while simultaneously promoting the natural process of cortical bone healing. The process of cancellous bone healing differs from that of cortical bone healing, and this invention primarily aims at cortical bone regeneration. While cancellous bone can be treated with any bone substitute, cortical bone requires specific biomaterial properties.

[0007] Therefore, there remains a need for improved bone repair methods, particularly the development of artificial periosteum that can also be used to locally deliver bone activators such as BMP-2 over long periods to promote bone growth and repair bone defects. [Overview of the Initiative]

[0008] The present invention provides an artificial periosteum, as well as a method for bone repair and delivery of therapeutic agents to bone. The therapeutic agent may be a bone activator for repairing bone defects or promoting bone growth, a bone activator for treating bone-related pain, an anti-inflammatory agent for treating inflammation-related conditions (e.g., arthritis), an anticancer drug for treating bone cancer, an antimicrobial agent for treating or preventing infection at the treatment site, or a combination thereof.

[0009] One aspect of the present invention provides an artificial periosteum comprising a functionalized collagen-containing membrane and a drug-carrier mixture, wherein the drug-carrier mixture comprises at least one therapeutic agent and a calcium-containing carrier mixture.

[0010] Another aspect of the present invention provides a method for repairing bone, comprising the step of implanting an artificial periosteum comprising a functionalized collagen-containing membrane and a drug-carrier mixture, wherein the drug-carrier mixture comprises at least one therapeutic agent and a calcium-containing carrier mixture.

[0011] Also disclosed is an artificial periosteum for use in a method of bone repair, comprising a functionalized collagen-containing membrane and a drug-carrier mixture, wherein the drug-carrier mixture comprises at least one therapeutic agent and a calcium-containing carrier mixture.

[0012] In a particular embodiment of the present invention, the functionalized collagen-containing membrane is a hydroxyapatite-functionalized collagen-containing membrane, while the calcium-containing carrier mixture is collagen-based.

[0013] Therefore, in one aspect, the present invention provides an artificial periosteum comprising a hydroxyapatite-functionalized collagen-containing membrane and a drug-carrier mixture, wherein the drug-carrier mixture comprises BMP-2 and zoledronic acid (ZA).

[0014] In one particular embodiment of the present invention, the therapeutic agent is a bone activator comprising compounds that activate osteoblasts. In another embodiment, the therapeutic agent is a bone activator that inhibits osteoclasts. In yet another embodiment, the therapeutic agent is a bone activator comprising one or more of the following: PGE1; PGE2; EP2 receptor agonists; EP4 receptor agonists; EP2 receptor / EP4 receptor dual agonists; organobisphosphonates; cathepsin K inhibitors; estrogens or estrogen receptor modulators; calcitonin; osteoclast proton ATPase inhibitors; HMG-CoA reductase inhibitors; integrin receptor antagonists; RANKL inhibitors; anabolic agents; osteogenic agents; vitamin D or synthetic vitamin D analogs; androgens or androgen receptor modulators; SOST inhibitors; platelet-derived growth factors; pharmaceutically acceptable salts thereof; and mixtures thereof.

[0015] One aspect of the present invention provides a method for repairing bone, comprising the step of implanting an artificial periosteum containing a functionalized collagen-containing membrane and a drug-carrier mixture, wherein the drug-carrier mixture contains BMP-2 and zoledronic acid.

[0016] In one embodiment, the functionalized collagen-containing membrane is a hydroxyapatite-functionalized collagen-containing membrane.

[0017] Also disclosed is an artificial periosteum comprising a functionalized collagen-containing membrane and a drug-carrier mixture for use in a method of bone repair in a patient, wherein the drug-carrier mixture comprises BMP-2 and zoledronic acid.

[0018] In one aspect of the present invention, a method for repairing bone defects in a patient: (i) The step of implanting bone graft material into the bone defect; and (ii) A step of coating the graft with a hydroxyapatite-functionalized collagen-containing membrane; and This provides a method that includes [something].

[0019] Also disclosed is a bone graft material coated with a hydroxyapatite-functionalized collagen-containing membrane for use in a method of repairing bone defects in a patient.

Brief Description of the Drawings

[0020] [Figure 1] Figure 1 shows an overview of the structure of the material and the alignment of collagen fibers. [Figure 2] Figure 2 shows the surgical procedure of the tibial defect model. [Figure 3] Figure 3 shows the quantification by micro-CT of the tibial defect experiment 8 weeks after the surgical procedure. [Figure 4] Figure 4 shows the evaluation of cortical bone healing using micro-CT. [Figure 5] Figure 5 shows the histological analysis of tibial defect healing. [Figure 6] Figure 6 shows the radiograph of a sample taken from the rectus abdominis pouch 4 weeks after the surgical procedure. [Figure 7] Figure 7 shows the role of the collagen membrane as a storage device for ceramic or polymer biomaterials placed within the bone gap. [Figure 8] Figure 8 shows a comparative experiment between an absorbent collagen sponge (ACS) manufactured by Medtronic and sold as an INFUSE® bone graft used together with a rhBMP-2-containing solution, and the collagen-containing membrane of the present invention containing rhBMP-2 and ZA. The data are CT data, representing the mean ± standard deviation of n = 8 / group for ACS and n = 5 / group for the collagen membrane (shown at the top).

Modes for Carrying Out the Invention

[0021] In repairing bone defects caused by trauma, infection, or tumors, it is common practice to replace lost / removed material with allogeneic, autologous, or synthetic graft material. If the bone defect involves cortical bone loss, even if healing has occurred in cancellous bone, it takes considerable time for new cortical bone to be constructed. Depending on the size of the cortical bone defect, especially if it is segmental, healing may not occur. The same is true for poorly ununiting fractures, which account for up to 5% of all fractures caused by strong impact.

[0022] Because periosteal cells play a powerful role in the healing of cortical bone, the periosteum is considered to be involved in the successful healing of bone defects. A special method for treating cortical bone defects involves temporarily inserting a spacer to create a shell of soft tissue similar to the periosteum (with high metabolic activity) around the spacer, and then removing the spacer and grafting bone after several months. The temporary periosteum thus formed is re-sutured, allowing the graft to become normal bone.

[0023] The artificial periosteum of the present invention is an ideal material for repairing bone defects because the collagen-containing membrane acts as a covering for the bone defect and, in some aspects, also provides an alternative material. The artificial periosteum of the present invention reduces bulging and leakage and, when functionalized with biomolecules, forms new cortical bone bridges. It may be attached to or around the bone by adhesion, suture, or periphery loops. It may be inserted and applied under the cortical bone using onlay or inlay techniques. When functionalized with bone activators, it will accelerate cortical bone regeneration.

[0024] In a broad range of aspects, the present invention relates to functionalized collagen-containing membranes.

[0025] The term “collagen” as used herein refers to all collagen, including processed or otherwise modified collagen. Preferred collagen is processed to remove immunogenic telopeptide regions (“atelopeptide collagen”) and reconstituted into a soluble, fibrous form.

[0026] The term "collagen-containing membrane" refers to a piece or segment of collagen-containing tissue manufactured by methods known in the art, for example, as disclosed in U.S. Patent No. 7,096,688. Collagen-containing membranes may have a geometric shape, but are usually substantially planar and may conform to the shape of the underlying or overlying tissue at a given location.

[0027] Collagen-containing membranes preferably have the following properties: a) Pores that are interconnected in a manner that promotes integration with tissue and angiogenesis; b) Biodegradability and / or bioabsorption such that the collagen-containing membrane is eventually replaced by normal tissue; c) Surface chemical properties that promote cell adhesion, proliferation, and differentiation; d) Strength and flexibility, and e) Low immunogenicity.

[0028] Collagen-containing membranes are typically prepared or manufactured from “collagen-containing tissues” that contain high-density connective tissue found in mammals. The term “collagen-containing tissue” refers to skin, muscle, etc., that can be isolated from collagen-containing mammals. The term “collagen-containing tissue” also includes “synthetically” produced tissues, where collagen or collagen-containing materials are assembled or manufactured outside the body.

[0029] In some embodiments, collagen-containing tissue is isolated from mammals including, but not limited to, sheep, cattle, pigs, or humans. In other embodiments, collagen-containing tissue is isolated from humans.

[0030] In some embodiments, the collagen-containing tissue is isolated from the "self," i.e., from the body of a patient requiring treatment.

[0031] In some embodiments, the collagen-containing membrane contains more than 80% type I collagen. In other embodiments, the collagen-containing membrane contains at least 85% type I collagen. In yet another embodiment, the collagen-containing membrane contains more than 90% type I collagen.

[0032] The collagen-containing membrane is prepared by a method known in the art, however one preferred method includes the following steps (i) to (iv): (i) Separate the collagen-containing tissue and incubate the tissue in an ethanol solution; (ii) Incubate the collagen-containing tissue from step (i) in a first solution containing an inorganic salt and an anionic surfactant to denature the non-collagenous proteins present; (iii) Incubate the collagen-containing tissue obtained in step (ii) in a second solution containing an inorganic acid until the collagen in the material is denatured; and (iv) Incubate the collagen-containing tissue obtained in step (iii) in a third solution containing an inorganic acid for a time sufficient to allow the collagen bundles within the tissue to align, with simultaneous mechanical stimulation, wherein the mechanical stimulation includes periodically applying tension to the collagen-containing tissue. It may be manufactured by [another method].

[0033] It is understood that an inorganic salt capable of forming a complex with a Lewis acid can be used as the first solution. In some embodiments, the inorganic salt is selected from the group consisting of trimethylammonium chloride, tetramethylammonium chloride, sodium chloride, lithium chloride, perchlorates, and trifluoromethanesulfonates. In other embodiments, the inorganic salt is lithium chloride (LiCl).

[0034] Any anionic surfactant can be used as the first solution, but in some embodiments, the anionic surfactant is selected from the group consisting of alkyl sulfates, alkyl ether sulfates, alkyl sulfonates, and alkylaryl sulfonates. Particularly useful anionic surfactants include alkyl sulfates such as sodium dodecyl sulfate (SDS).

[0035] In some embodiments, the first solution contains about 1% (v / v) SDS and about 0.2% (v / v) LiCl.

[0036] In some embodiments, the inorganic acid in the second solution contains approximately 0.5% (v / v) HCl, and the inorganic acid in the third solution contains approximately 1% (v / v) HCl.

[0037] It will be understood by those skilled in the art that the incubation time in each step varies depending on (i) the type of collagen-containing tissue; (ii) the type of inorganic salt / acid and / or anionic surfactant; (iii) the strength (concentration) of each inorganic salt / acid and / or anionic surfactant used; and (iv) the incubation temperature. In some embodiments, the incubation time for step (i) is at least 8 hours. In other embodiments, the incubation time for step (ii) is less than 60 minutes, while in other embodiments, the incubation time for step (iii) is at least 20 hours.

[0038] In some embodiments, the incubation in step (ii) is carried out at approximately 4°C. In other embodiments, the incubation in step (ii) is carried out for at least 12 hours.

[0039] In some embodiments, the second solution contains approximately 0.5% (v / v) HCl.

[0040] In some embodiments, the incubation of step (iii) is performed for approximately 30 minutes. In other embodiments, the incubation of step (iii) is performed with shaking.

[0041] In some embodiments, the third solution contains approximately 1% (v / v) HCl solution.

[0042] In some embodiments, the incubation in step (iv) is carried out for about 12 to 36 hours, preferably about 24 hours. In other embodiments, the incubation in step (iv) is carried out with shaking.

[0043] In some embodiments, the method further includes a neutralization step between steps (iii) and (iv), which includes incubation of the collagen-containing tissue with about 0.5% (v / v) NaOH.

[0044] In some embodiments, the method further includes step (v), which involves incubating the collagen-containing tissue of step (iv) with acetone, and then drying the collagen-containing tissue.

[0045] In some embodiments, the method further includes a step of contacting collagen-containing tissue with glycerol between steps (ii) and (iii), and / or between steps (iii) and (iv), in order to visualize and facilitate the removal of fat and / or blood vessels.

[0046] Glycerol may be allowed to come into contact with collagen-containing tissue for a time that facilitates the removal of fat and / or blood vessels. In some embodiments, the contact time is at least 10 minutes.

[0047] In some embodiments, the method further includes a washing step of the collagen-containing tissue between steps (ii) and (iii) and / or between steps (iii) and (iv). The purpose of the washing step performed between steps (ii) and (iii) is the removal of denatured proteins. Therefore, a washing solution capable of removing denatured proteins can be used. In some embodiments, the washing solution used between steps (ii) and (iii) is acetone.

[0048] After washing with acetone, the collagen-containing tissue is further washed with sterile water.

[0049] In some embodiments, the collagen-containing tissue is further washed with a NaOH:NaCl solution. When washing the collagen-containing tissue with NaOH:NaCl, it is preferable to further wash it with sterile water.

[0050] In some embodiments, after step (iv), the collagen-containing tissue is further washed with the first solution.

[0051] As used herein, the term “simultaneous mechanical stimulation” refers to the process of stretching the collagen-containing tissue during the chemical treatment of the collagen-containing tissue. The collagen-containing tissue may be subjected to static and / or periodic stretching. Therefore, in some embodiments, simultaneous mechanical stimulation is (i) Prolongation of a specified period of time for collagen-containing tissue; (ii) Relaxation of collagen-containing tissue for a specified period of time; and (iii) Repeat steps (i) and (ii) n times (where n is an integer greater than or equal to 1) It may include.

[0052] When stretching collagen-containing tissue with mechanical stimulation, the collagen-containing tissue is preferably stretched along its long axis.

[0053] In some embodiments, simultaneous mechanical stimulation includes periodically applying tension to collagen-containing tissue, the periodicity of which includes a stretch time of about 10 to about 20 seconds and a relaxation time of about 10 seconds, the resulting strain being about 10%, and the mechanical stimulation is continued until the collagen bundles in the collagen-containing tissue are aligned as described herein.

[0054] The resulting collagen-containing tissue comprises collagen fibers or bundles in a mesh-like structure. In this specification, the term “mesh-like structure” refers to a structure comprising first and second groups of fibers or bundles, where the first group of fibers or bundles extends primarily in a first direction, and the second group of fibers or bundles extends primarily in a second direction, the first and second directions being distinct from each other, and the fibers or bundles of the first group being alternately arranged or otherwise interwoven with those of the second group. The difference in direction may be approximately 90°.

[0055] Collagen-containing tissue prepared by a preferred method has a "maximum tensile load strength" of more than 20 N. In some embodiments, the collagen-containing tissue of the present invention has a maximum tensile load strength of more than 25 N, 40 N, 60 N, 80 N, 100 N, 120 N, or 140 N.

[0056] Furthermore, the mesh structure in the embodiment of collagen-containing tissue is thought to increase the elastic modulus of the collagen-containing patch while reducing elongation under maximum load.

[0057] In this specification, the term "elastic modulus" refers to Young's modulus, which is determined as the ratio of stress to strain. It is a measure of the stiffness of collagen-containing tissues and / or patches.

[0058] In some embodiments, the elastic modulus of the collagen-containing tissue exceeds 100 MPa. In other embodiments, the elastic modulus of the collagen-containing tissue exceeds 200 MPa, 300 MPa, 400 MPa, or 500 MPa.

[0059] In this specification, the term “elongation under maximum load” means the elongation of collagen-containing tissue at the maximum tensile load strength relative to the original length of the collagen-containing tissue under no load. This is in contrast to maximum expansion, which is the greater the tissue becomes.

[0060] In some embodiments, the elongation of collagen-containing tissue under maximum load is less than 85% of its original length.

[0061] The collagen-containing membrane to be used may be formed after the collagen-containing tissue has been generated. In some embodiments, the collagen-containing membrane is in the shape of a membrane, which improves in situ handling.

[0062] Preferably, the collagen-containing membrane of the present invention is thick enough to support the drug-carrier mixture, but not so thick that it impairs the in situ operability of the collagen-containing membrane. Therefore, in some embodiments, the thickness of the collagen-containing membrane is 25 μm to 200 μm. In some embodiments, the thickness of the collagen-containing membrane is 30 μm to 180 μm. In other embodiments, the thickness of the collagen-containing membrane is 35 μm to 170 μm. In yet another embodiment, the thickness of the collagen-containing membrane is 40 μm to 160 μm. In yet another embodiment, the thickness of the collagen-containing membrane is 45 μm to 150 μm. In yet another embodiment, the thickness of the collagen-containing membrane is 50 μm to 140 μm. In yet another embodiment, the thickness of the collagen-containing membrane is 50 μm to 100 μm. Also, in some embodiments, the thickness of the collagen-containing membrane is approximately 50 μm.

[0063] In one form of collagen-containing membrane, the membrane is perforated, allowing for the transport of natural bone-activating molecules or therapeutic activators in graft material through the membrane, and enabling the recruitment of circulating stem cells and pericytes from the overlying muscle.

[0064] The collagen-containing membrane preferably has two surfaces (one on each side): a smooth surface characterized by dense collagen bundles and a rough, porous surface with loose collagen fibers. The rough surface is particularly suitable for promoting cell bonding, and in fact, if muscle overlaps the membrane, it is important to orient the rough surface toward the muscle. However, if there is no muscle overlapping, for example in the repair of the distal tibia, it is not as important to designate one of the surfaces as the rough surface.

[0065] The collagen-containing membrane is functionalized on each surface of the membrane with bioactive molecules such as bone morphogenetic protein-2 (BMP-2) and zoledronic acid (ZA), and / or hydroxyapatite nanoparticles (nHAP).

[0066] In one embodiment, nHAP is synthesized using the wet method described in Teotia et al. (2017), ACS Appl. Mater. Interfaces, 9(8), pp 6816-6828. Briefly, an alkaline solution (pH 10.0) of calcium nitrate tetrahydrate (Ca(NO3)2·4H2O, 0.96M) is maintained at 90-100°C under constant stirring, and then mixed with an aqueous solution of diammonium hydrogen orthophosphate ((NH4)2HPO4, 0.6M). The pH of this system is constantly monitored and maintained at 10.0 by adding NH4OH solution. nHAP precipitates from the solution as white crystals. After the reaction is complete, the crystals are kept in the mother liquor under alkaline conditions at room temperature for 48 hours for maturation. After maturation, the crystals are filtered from the solution and thoroughly washed with Milli-Q Type I water (DI-H2O). Next, dry it at 120°C.

[0067] In some embodiments, the synthesized nHAP is heat-treated to enhance its crystallinity, density, and phase purity. The temperature range is from 500°C to a maximum of 1000°C, with a holding time of 1 to 4 hours.

[0068] The synthesized nHAP is then applied to the collagen-containing membrane by simply immersing the membrane in sterile saline containing nHAP.

[0069] The bioactive molecules may also be incorporated into the nHAP solution simultaneously or applied independently to the collagen-containing membrane. The bioactive molecules (ZA, BMP-2) are either mixed in sterile water or saline solution and then mixed with nHAP using 600 μL of water per gram of dried nHAP, or applied to the collagen-containing membrane by immersion.

[0070] The final result of the above method is the artificial periosteum of the present invention.

[0071] In one form of artificial periosteum, a drug-carrier mixture is applied to a functionalized collagen-containing membrane.

[0072] Therefore, one form of the artificial periosteum of the present invention comprises a drug-carrier mixture. The carrier component of the drug-carrier mixture may be a calcium-containing carrier mixture known in the art, comprising calcium phosphate cement (CPC). The carrier component may also comprise other additional carriers.

[0073] The therapeutic agent in the drug-carrier mixture of the artificial periosteum of the present invention includes a bone activator that can stimulate, promote, enhance, or induce bone formation, or inhibit bone resorption. The therapeutic agent may be a bone repair agent or other bone activator that relieves pain and / or inflammation at the treatment site, treats cancer, or treats or prevents microbial infections. The drug-carrier mixture provides release of the therapeutic agent at the treatment site. Preferably, the drug-carrier mixture releases the therapeutic agent over a long period of time.

[0074] As will be described in detail below, the drug-carrier mixture is prepared by mixing the therapeutic agent with a suitable carrier material such as calcium phosphate cement powder. Depending on the specific embodiment, the drug-carrier mixture may be pulverized into a fine powder. The drug-carrier mixture may be further combined with a suitable bone matrix material to form the artificial periosteum of the present invention, as will be described below. Alternatively, the drug-carrier mixture may be applied to a functionalized collagen-containing membrane to similarly form the artificial periosteum of the present invention. The artificial periosteum may then be applied to the treatment site, for example, by implantation.

[0075] Examples of calcium phosphate cement (CPC) that can be used in the carrier component of a drug-carrier mixture include tricalcium phosphate mixtures such as α-tricalcium phosphate (α-TCP) and β-tricalcium phosphate (β-TCP). Other CPCs that may be used include combinations of dicalcium phosphate and tetracalcium phosphate. Commercially available calcium phosphate cements, such as Hydroset (sold by Stryker Corp.), used in the examples, may also be used. Hydroset is a soft tricalcium phosphate cement characterized by a mixture of α-TCP and β-TCP (1:3). In some embodiments, hydroxyapatite may be added to the calcium phosphate cement and mixtures thereof (e.g., 2.5% wt / wt hydroxyapatite crystals). α-TCP and β-TCP may be used in various ratios. For example, in some embodiments, the CPC comprises a mixture of α-TCP and β-TCP (1:3) and optionally contains hydroxyapatite. In other embodiments, α-TCP and β-TCP may be used in a 1:1 or 1:0 ratio. In another embodiment, the CPC is α-TCP cement to which 2.5% hydroxyapatite is added, resulting in a harder cement when installed.

[0076] The drug-carrier mixture may contain at least partially demineralized bone matrix. The bone matrix may be demineralized bone matrix putty or partially or completely demineralized intact bone matrix. Intact bone matrix may be used in bone grafting and serve as a scaffold for delivering bone repair agents.

[0077] Human demineralized bone matrix putty may be used as a carrier component in drug-carrier mixtures. This is commercially available, for example, Puros Demineralized Bone Matrix Putty manufactured by RTI Biologics (Alachua, Fla.). Demineralized bone matrix putty can also be prepared by the method described by Urist & Dowell (Inductive Substratum for Osteogenesis in Pellets of Particulate Bone Matrix, Clin. Orthop. Relat. Res., 1968, 61, 61-78). This method involves demineralizing bone, degreasing the solid demineralized bone, cutting it into small pieces, and grinding these pieces under liquid nitrogen to a coarse powder. The ground demineralized bone matrix has the consistency of a putty after thawing.

[0078] Where necessary, curing solutions for tricalcium phosphate cement powder are well known in the art, including a 2.5% by weight / volume Na2HPO4 solution or a commercially available solution. See Dorozhkin, Materials 2009, 2, 221-291.

[0079] Another example of a carrier component is one prepared by combining gelatin with calcium sulfate (CaS), sometimes in the presence of hydroxyapatite (HA), using the cryogel formation technique described by Kumar et al. (Mater. Today, 13, (2010), 42-44). An example of using a gelatin-CaS-HA composite material is described in Raina et al. (2018), J Control Release, Vol. 272, 83-96, and similar composite materials of silk, chitosan, bioactive glass, and HA are described by Raina et al. (J Control Release, Vol. 235, 365-378. (2016)). Murphy et al. also describe porous collagen hydroxyapatite-based carriers for delivering rhBMP-2 and ZA, both of which result in cancellous bone regeneration (Murphy et al. (2014), Acta Biomaterialia, Vol 10, Issue 5).

[0080] A drug-carrier mixture can be prepared by dissolving the therapeutic agent in a suitable solvent such as ethanol and adding this solution to the carrier mixture. After removing the solvent, the therapeutic agent-carrier mixture is mixed to uniformly (i.e., homogeneously) disperse the therapeutic agent throughout the carrier mixture, and if necessary, the therapeutic agent-carrier mixture is moistened with a suitable curing solution to produce the drug-carrier mixture.

[0081] Treatment method The artificial periosteum of the present invention is useful in the treatment of fractures and bone loss resulting from periodontal disease, surgical procedures, cancer, or trauma. Further applications of the artificial periosteum of the present invention include its use in increasing bone density during bone preparation for dental or orthopedic implants, as an implant coating for enhanced osseointegration, and in all forms of spinal fusion.

[0082] The present invention relates to a treatment method comprising using the artificial periosteum of the present invention on a patient in need thereof, wherein the artificial periosteum may contain a therapeutically effective amount of a bone repair agent as described in the specification. The treatment method generally includes stimulating, promoting, enhancing or inducing bone formation, or inhibiting bone resorption. The treatment method also includes, for example, promoting bone remodeling, activating osteoblasts, promoting osteoblast differentiation, inhibiting osteoclasts, increasing the number and activity of osteoblasts, increasing mean wall thickness, increasing cancellous bone volume, improving bone structure, improving trabecular connectivity, increasing cortical bone thickness, inhibiting bone loss, maintaining / improving bone strength, and increasing total bone volume or osteoid volume. The treatment method also includes treating one or more of osteoporosis, fractures, low bone density, or periodontal disease.

[0083] In one embodiment of the treatment method, one or more bone repair agents are released from a drug-carrier mixture described in the specification. In another embodiment, the bone repair agents are released from a drug-carrier mixture in combination with another therapeutic agent administered systemically (e.g., orally). For example, the bone repair agents may be sustainably released from an artificial periosteum in combination with one or more additional therapeutic agents administered systemically to treat bone loss or osteoporosis.

[0084] The treatment method further includes the use of artificial periosteum on a localized area of ​​choice in humans, other mammals, and birds, such as a gap in the bone, an adjacent area of ​​the alveolar bone, or a bone defect caused by surgery, trauma, or disease.

[0085] The present invention also provides a method for treating bone-related pain, inflammation, infection and / or bone cancer, comprising applying an artificial periosteum containing a therapeutically effective amount of an analgesic, anti-inflammatory, anticancer and / or antimicrobial agent. The method for treating pain, inflammation, cancer and / or infection may be combined with any of the aforementioned methods for treating bone disorders.

[0086] Combination therapy includes the administration of a single formulation containing one or more of the compounds described in the specification and one or more additional pharmaceuticals, as well as the administration of the compounds and each additional pharmaceutical in separate formulations of themselves. For example, the compounds described in the specification and one or more additional pharmaceuticals may be administered together to the patient in an artificial periosteum having fixed ratios of their respective active ingredients, or each drug may be administered as a separate formulation. For example, the patient may be treated by locally delivering the active drug to the site of the bone defect via an artificial periosteum in combination with another drug administered systemically. When separate formulations are used, the compounds and one or more additional pharmaceuticals may be administered essentially simultaneously (e.g., in parallel) or staggered (e.g., sequentially).

[0087] In one aspect of the present invention, the interosseous space is filled, for example, with a bone graft, or an independent or composite substitute for a bone graft derived from a natural or synthetic source, and then coated with an artificial periosteum containing the functionalized collagen-containing membrane and drug-carrier mixture of the present invention, or coated with the hydroxyapatite and / or bioactive agent-functionalized collagen-containing membrane of the present invention. The collagen-containing membrane functions as a crosslinking agent and regenerates bone by inducing tissue regeneration.

[0088] One use of the artificial periosteum of the present invention is as an external covering for bone defects and / or gaps filled with bone graft material. The artificial periosteum may be kept in place using methods known in the art, including suturing, clamping, or fixation with medical adhesives. The artificial periosteum may also be simply inserted into the endothelium of the bone.

[0089] In some embodiments, the artificial periosteum of the present invention is fixed in situ using a medical adhesive. Medical adhesives have the advantage of being suitable for contact with bodily fluids. With respect to the artificial periosteum, medical adhesives can be used to facilitate the fixation of the artificial periosteum or to structurally hold together a portion of the artificial periosteum. For convenience, in this specification, adhesives generally refer to adhesives in application form and adhesive compositions after curing in a set form. Suitable medical adhesives must be biocompatible in that they are non-toxic, non-carcinogenic, and do not induce hemolysis or immunological responses. Suitable biocompatible adhesives include commercially available surgical adhesives and mixtures thereof such as cyanoacrylates (e.g., 2-octyl cyanoacrylate, DERMABOND® from Ethicon Products) and fibrin glues (e.g., TISSUCOL® from Baxter), but a wide range of suitable adhesives are available.

[0090] For the repair of long segment defects of bone, the bone cavity may be filled with any bone graft substitute (synthetic, natural, or progenitor), which may or may not include internal or external fixation. The periosteum of the present invention can then be wrapped around the cortical bone to avoid the two-step maskelier technique by holding the bone graft substitute in the appropriate position. The maskelier technique is used in long bone trauma applications when there is a large intermediate defect, such as when a portion of a long bone is lost. The maskelier technique typically involves two steps: a first step of placing a spacer and forming soft tissue around the spacer, and a second step of covering the bone graft with the formed soft tissue. Therefore, in some embodiments, the periosteum of the present invention may be used for the repair of trauma in long bone segment defects. For example, a relatively large covering may be provided with a material suitable for trauma repair provided inside, where the periosteum is used to preserve the space of the long bone (without soft tissue) and allow soft tissue to form around it. The second step of the Maskelet procedure can be avoided because the graft material is provided when the artificial periosteum is originally placed.

[0091] A more preferred embodiment includes cells that are seeded or placed on the artificial periosteum of the present invention. Any cells may be used, but cells that are generally associated with promoting the growth of bone and bone-related tissues are clearly preferred. Some preferred examples include, but are not limited to, differentiated cells, including stem cells, undifferentiated stem cells, and bone marrow stem cells. Other examples of cells that can be used in various embodiments include, but are not limited to, osteoblasts, fibroblasts, chondrocytes, and connective tissue cells.

[0092] It will be understood that the present invention also provides an artificial periosteum for use in such therapeutic methods.

[0093] It will be understood that the use of functionalized collagen-containing membranes and drug-carrier mixtures in the manufacture of artificial periosteum for use in such therapeutic methods is also provided.

[0094] Definition of Terms The term "therapeutic dose" refers to the amount of compound sufficient to treat a disorder with a reasonable benefit / risk ratio applicable to medical treatment. However, the total dose of the compound in the periosteum can be determined by the attending physician within the bounds of sound medical judgment. The specific therapeutic dose for a particular patient may depend on a variety of factors, including: the disorder being treated and its severity; the activity of the compound used; the periosteum used; the rate of drug release from the periosteum; the patient's age, weight, general condition, medical history, sex, and diet; the method of delivery; drugs used in combination with or concurrently with the compound; and similar factors well known in the field of medicine. The actual dose of the active ingredient in the periosteum may be varied to obtain an amount of the active compound that is effective in achieving the desired therapeutic response for a particular patient and a particular dosage form.

[0095] The terms “bone repair agent” or “bone activator” as used herein refer to agents that can stimulate, promote, enhance or induce bone formation, or inhibit bone resorption. Accordingly, a bone activator may be an anabolic agent or an anticatabolic agent. A bone activator may have one or more of the following effects: promotion of bone remodeling, activation of osteoblasts, promotion of osteoblast differentiation, inhibition of osteoclasts, increase in the number and activity of osteoblasts, increase in mean wall thickness, increase in cancellous bone volume, improvement of bone structure, improvement of trabecular connectivity, increase in cortical bone thickness, inhibition of bone loss, maintenance / improvement of bone strength, increase in total bone volume or osteoid volume. Osteoactivators include: prostaglandin E1 (PGE1); prostaglandin E2 (PGE2); EP2 receptor agonists; EP4 receptor agonists; EP2 / EP4 receptor dual agonists; organic bisphosphonates (e.g., alendronate or sodium alendronate); cathepsin K inhibitors; estrogen or estrogen receptor modulators; calcitonin; osteoclast proton ATPase inhibitors; HMG-CoA reductase inhibitors (i.e., statins); αvβ-integrin receptor antagonists; and RAs such as denosumab. Examples of bone activators include, but are not limited to, NKL inhibitors; anabolic agents such as parathyroid hormone; bone morphogenetic proteins (e.g., BMP-2, BMP-4, BMP-7); vitamin D or synthetic vitamin D analogs such as ED-70; androgens or androgen receptor modulators; activators of Wnt / β-catenin signaling (e.g., GSK-3 inhibitors, sclerostin antagonists, SOST inhibitors); bortezomib; strontium ranelate; platelet-derived growth factor; pharmaceutically acceptable salts thereof; and mixtures thereof. Bone activators are preferably not decomposed into an inactive form when exposed to a pH of about 4-5.

[0096] The term "calcium phosphate cement" herein refers to a bone repair composition comprising dicalcium phosphate, tricalcium phosphate (e.g., α-tricalcium phosphate and β-tricalcium phosphate), or tetracalcium phosphate, or a bone repair composition made from any of the aforementioned, or a mixture thereof after curing. Calcium phosphate cement may also include hydroxyapatite incorporated together with the calcium phosphate compound.

[0097] The term "drug-carrier mixture" as used herein refers to a mixture of therapeutic agents incorporated into a calcium-containing carrier component.

[0098] The term "agonist" as used herein refers to a compound whose biological action mimics the action of a natural agonist. Agonists may have full efficacy (i.e., equivalent to a natural agonist), partial efficacy (lower maximum efficacy compared to a natural agonist), or supermaximal efficacy (higher maximum efficacy compared to a natural agonist). Agonists with partial efficacy are referred to as "partial agonists," and agonists with supermaximal efficacy are referred to as "superagonists." In one embodiment, the natural agonist may be PGE2.

[0099] Pain relievers that may be released from the artificial periosteum include sodium channel blockers (e.g., Nav1.8 inhibitors, Nav1.9 inhibitors, ropivacaine, bupivacaine, etc.), TRPV1 antagonists, endothelin antagonists (e.g., atrasentan, dibotentan), bradykinin antagonists, ASIC inhibitors, TrkA inhibitors, and radionuclides. 89 Sr, 153 Sm-Lexidronam, 186 Re-ethidronate is one example.

[0100] Anti-inflammatory agents that may be released from the artificial periosteum include NSAIDs, corticosteroids, and cytokine inhibitors (e.g., inhibitors of TNFα, IL-1β, etc.).

[0101] Antimicrobial agents that may be released from the artificial periosteum include antibacterial and antifungal agents. Examples of antibacterial agents include well-known drugs such as cephalosporins, cephalosporins, quinolone antibiotics (e.g., ciprofloxacin, levofloxacin, etc.), and macrolides (e.g., azithromycin, clarithromycin, erythromycin, etc.). Examples of antifungal agents include fluconazole, clotrimazole, and itraconazole.

[0102] Anticancer agents that may be released from the artificial periosteum include vincristine, doxorubicin, etoposide, gemcitabine, methotrexate, and SRC kinase inhibitors (e.g., dasatinib, salakatinib, bosutinib) as described in Saad in Cancer Treat Rev. 2010, 36(2) 177-84.

[0103] The bone activator may be prostaglandin E1, prostaglandin E2, strontium ranelic acid, calcitonin, parathyroid hormone, vitamin D or synthetic vitamin D analog (e.g., ED-70), BMP-2, BMP-4, BMP-7, or platelet-derived growth factor.

[0104] The bone activator may also be an organic bisphosphonate. Examples of organic bisphosphonates include alendronate, sodium alendronate, ibandronate, risedronate, zoledronate, zoledronic acid, etidronate, pamidronate, tildronate, neridronate, and olpadronate.

[0105] The osteoactivator may also be a cathepsin K inhibitor, such as a compound disclosed and cited in Bromme in Expert Opin. Investig. Drugs 2009, 18(5) 585-600 (e.g., odanacatib).

[0106] The bone activator may be an estrogen or estrogen receptor modulator, including, for example, raloxifene, bazedoxifene, and rasofoxifene, which include compounds described at http: / / en.wikipedia.org / wiki / Selective_estrogen-receptor_modulator.

[0107] The bone activator may be, for example, an androgen or androgen receptor modulator, including testosterone.

[0108] The osteoactivator may be an inhibitor of osteoclast proton ATPase, such as a compound described in Nyman in Potential of the Osteoclast's Proton Pump as a Drug Target in Osteoporosis, Annales Universitatis Turkuensis 2011, including SB242784, bafilomycin (e.g., bafilomycin A1), concanamycin A, apiclarene, alkazolides (archazolides), benzolactone enamides (salicilharamid A, lobatamide A), FR167356, FR177995, and diphylline.

[0109] The bone activator may be, for example, an HMG-CoA reductase inhibitor (i.e., a statin), such as atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin, as described at http: / / en.wikipedia.org / wiki / Statin.

[0110] The bone activator may be, for example, a compound described in Millard et al. in Integrin Targeted Therapeutics, Theranostics 2011, 154-188, and may include silendide (EMD 121974), L000845704, SB2730005, etc., which are αvβ-integrin receptor antagonists.

[0111] The bone activator may also be a RANKL inhibitor such as denosumab.

[0112] The bone activator may also be an EP2 receptor agonist such as ONO-AE1-259-01 and CP-533536.

[0113] The bone activator may be an EP2 receptor / EP4 receptor dual agonist as described in, for example, Bioorganic & Medicinal Chemistry Letters, 2012, 22(1), 396-401, U.S. Patent No. 7,402,605, and U.S. Patent No. 7,608,637. A typical EP2 receptor / EP4 receptor dual agonist is 2-((2-((R)-2-((S,E)-3-hydroxy-4-(m-tolyl)buto-1-en-1-yl)-5-oxopyrrolidine-1-yl)ethyl)thio)thiazole-4-carboxylic acid (CAS#494223-86-8).

[0114] Osteoactivators are listed under U.S. Patent Nos. 6,043,275, 6,462,081, 6,737,437, 7,169,807, 7,276,531, 7,402,605, 7,419,999, and 7,608,637; International Publication No. 2002 / 024647; Bioorganic & Medicinal Chemistry Letters, 2001, 11(15), 2029-2031; Bioorganic & Medicinal Chemistry Letters, 2002, 10(4), 989-1008; Bioorganic & Medicinal Chemistry Letters, 2002, 10(6), 1743-759; Bioorganic & Medicinal Chemistry Letters, 2002, 10(7), 213-2110;Journal of Medicinal Chemistry, 2004, 47(25), 6124-6127;Bioorganic & Medicinal Chemistry Letters, 2005, 15(10), 2523-2526;Bioorganic & Medicinal Chemistry Letters, 2003, 13(6), 1129-1132;Medicinal Chemistry Letters, 2006, 16(7), 1799-1802;Bioorganic & Medicinal Chemistry Letters, 2004, 14(7), 1655-1659;Bioorganic & Medicinal Chemistry Letters, 2003, 13(6), 1129-1132;Journal of Medicinal Chemistry, 1977, 20(10), 1292-1299;Bioorganic & Medicinal Chemistry Letters, 2008, 18(2), 821-824;Bioorganic & Medicinal Chemistry Letters, 2007, 17(15), 4323-4327;The compounds disclosed in Bioorganic & Medicinal Chemistry Letters, 2006, 16(7), 1799-1802; Tetrahedron Letters, 2010, 51(11), 1451-1454; Osteoporosis International, 2007, 18(3), 351-362; Journal of Bone and Mineral Research, 2007, 22(6), 877-888; and Heterocycles, 2004, 64, 437-445 may include, but are not limited to, EP4 receptor agonists.

[0115] Specific EP4 receptor agonists include, but are not limited to, CP-734432, ONO-4819 (i.e., rivenprost), AE1-329, and L-902,688.

[0116] In some embodiments, the bone activator contained in the artificial periosteum is one or more of the following: alendronate, sodium alendronate, ibandronate, risedronate, zoledronate, zoledronic acid, etidronate, pamidronate, tildronate, neridronate, olpadronate, odanacatib, raloxifene, bazedoxifene, rasofoxifene, atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, strontium ranelate, calcitonin, parathyroid hormone, or bone morphogenetic protein-2.

[0117] In another embodiment, the osteoactivating agent contained in the artificial periosteum is one or more of the following: EP2 receptor agonists, EP2 receptor / EP4 receptor dual agonists, EP4 receptor agonists, organic bisphosphonates, estrogen receptor modulators, HMG-CoA reductase inhibitors, and strontium ranelic acid.

[0118] The present invention also provides an artificial periosteum containing the agents, drugs, or combinations of drugs described in the specification. For example, one or more agents / drugs that activate osteoblasts may be combined with one or more agents / drugs that inhibit osteoclasts.

[0119] Alternatively, a combination of multiple drugs / medicines that activate osteoblasts and inhibit osteoclasts may be used.

[0120] In some embodiments, the artificial periosteum may contain an EP4 receptor agonist along with one or more of the following: bisphosphonates; cathepsin K inhibitors; estrogen or estrogen receptor modulators; calcitonin; osteoclast proton ATPase inhibitors; HMG-CoA reductase inhibitors (i.e., statins); αvβ3-integrin receptor antagonists; RANKL inhibitors such as denosumab; anabolic agents such as parathyroid hormone; bone morphogenetic proteins (e.g., BMP-2, BMP-4, BMP-7); vitamin D or synthetic vitamin D analogs such as ED-70; androgens or androgen receptor modulators; activators of Wnt / β-catenin signaling (e.g., GSK-3 inhibitors, sclerostin antagonists, SOST inhibitors); bortezomib; strontium ranelic acid; or platelet-derived growth factor.

[0121] In some embodiments, for example, the EP4 receptor agonist is combined with one or more bisphosphonates selected from alendronate, sodium alendronate, ibandronate, risedronate, zoledronate, zoledronic acid, etidronate, pamidronate, tildronate, neridronate, and olpadronate.

[0122] In another embodiment, the EP4 receptor agonist is combined with one or more of raloxifene, bazedoxifene, and rasofoxifene.

[0123] In other embodiments, EP4 receptor agonists are combined with osteomorphogenetic proteins such as BMP-2, BMP-4, or BMP-7. For example, one combination includes CP-734432 with either BMP-2 or BMP-7. Another combination includes ONO-4819 (rivenprost) with either BMP-2 or BMP-7. Yet another combination includes AE1-329 with either BMP-2 or BMP-7. Yet another combination includes L-902,688 with either BMP-2 or BMP-7. A further combination includes 7-((R)-3,3-difluoro-5-((3S,4S,E)-3-hydroxy-4-methylnon-1-en-6-in-1-yl)-2-oxopyridine-1-yl)heptanoic acid with either BMP-2 or BMP-7. Another combination includes 7-((R)-2-((3S,4S,E)-3-hydroxy-4-methylnon-1-en-6-in-1-yl)-5-oxopyridine-1-yl)heptanoic acid together with BMP-2 or BMP-7.

[0124] In other embodiments, EP4 receptor agonists are combined with statins such as atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.

[0125] All patents, patent applications, provisional applications, and publications mentioned or cited in this specification, including drawings and tables, are incorporated by reference to the extent that they are not inconsistent with the express teachings herein.

[0126] When prior art publications are referenced in the specification, it is understood that such references do not constitute a recognition that they form part of the common general knowledge in the art in Australia or any other country.

[0127] The following are illustrative examples illustrating the procedures for carrying out the present invention. These examples are not to be construed as limitations. [Examples]

[0128] Example 1: In Vivo Experiment In the first experiment, we used the tibial defect model previously described by Horstmann et al. Briefly, a 4.5 mm defect was created by drilling a hole in and beneath the cortical bone of the metaphysis of the proximal tibia in male Sprague Dolly rats. Figure 2 shows an example of the surgical procedure of the tibial defect model. As an interosseous space filler, a gelatin-calcium sulfate-hydroxyapatite scaffold was filled into the cancellous osteocoryal cavity of the defect with or without ZA added, and with or without rhBMP-2. This was done to provide support for the collagen-containing membrane that was to be layered on top, without which layering would have been difficult. In some groups, the scaffold was also covered with a 6 mm piece of collagen membrane, and the remaining membrane was inserted into the endoosseous membrane. This prevented the membrane from leaking the scaffold outside the circular defect. A detailed description of the groups and the doses of the bioactive molecules is shown in Table 1.

[0129] [Table 1]

[0130] Eight weeks after surgical intervention, the animals were euthanized, and quantitative and histological examinations using micro-CT were performed to assess the healing of the lesions.

[0131] In the second experiment, collagen membranes (4 mm circular) alone, or membranes functionalized with 1 mg of hydroxyapatite nanoparticles on both sides, were analyzed using the abdominal muscle pouch model described by Raina et al. The following groups were used: 1. Collagen membrane (CM) alone, 2. CM+ hydroxyapatite nanoparticles (nHA), 3. CM+rhBMP-2 (10 μg) 4. CM+nHA+rhBMP-2 (10 μg) 5. CM+rhBMP-2(10μg)+ZA(10μg), 6. CM+nHA+rhBMP-2(10μg)+ZA(10μg).

[0132] Five animals from each group were euthanized four weeks after surgical intervention, and then quantitative analysis was performed using X-ray and micro-CT.

[0133] result Experiment 1: Micro-CT Target Region 1 (ROI 1): For analysis by micro-CT, the inventors defined three ROIs. In ROI 1, the calcification volume (MV) / tissue volume (TV)% within the defect void, excluding cortical bone, was measured. The diameter of ROI 1 was not uniform, ranging from 4.5 mm at the top to 1.5 mm at the bottom. The depth of ROI 1 was 2 mm. Micro-CT measurements showed that all scaffold and membrane treatment groups regenerated significantly higher volumes of calcified tissue or MV / TV% compared to the empty group, regardless of the type or presence of bioactive molecules (Figure 3, top).

[0134] Target Region 2 (ROI 2): The inventors used values ​​obtained from ROI 2 to evaluate cortical bone healing. ROI 2 was a 4.5 mm circular ROI extending upward from the base of the old cortical bone. The inventors measured regenerated bone in the area of ​​regenerated cortical bone that had previously been removed during surgery. The S+ZA+rhBMP-2+(CM) group (group 6) and the S+ZA+(CM+rhBMP-2) group (group 7) had significantly higher cortical bone mineralization volume (MV) compared to the empty group (group 1) or the scaffold-only group (group 2) (Figure 3, center).

[0135] Figure 3 shows the quantitative analysis of tibial defect 8 weeks after surgical intervention using micro-CT. * This shows a comparison with the empty group. The central figure * The value shown is the p-value compared to the S+ZA+(CM+rhBMP-2) group. δ shows the comparison with S+ZA+rhBMP-2+(CM+rhBMP-2). See the figure below. * δ shows a comparison with an empty group, and δ shows a comparison with a group consisting only of scaffolding. * Alternatively, δ indicates p<0.05, **Or δδ indicates p < 0.01, *** or δδδ indicates p < 0.001.

[0136] Target region 3 (ROI 3): Using ROI 3, complete defects, as well as proximal and distal bone with respect to the defect, were measured. This not only provides measurement of the bone regenerated in the defect area, but also provides insight into the volume of calcified tissue regenerated around the implanted scaffold and membrane. ROI 3 had a height of 6.5 mm and included a 4.5 mm defect, as well as a 1 mm proximal and 1 mm distal range from the defect. Groups 3 - 8 were shown to have significantly higher MV compared to Group 1. Furthermore, Groups 4, 5, 6, and 8 also had significantly higher MV compared to Group 2. No significant differences were seen between Group 1 and 2, or between any of Groups 3 - 8 (Figure 3, lower part).

[0137] First experiment: Cortical bone healing by micro-CT Figure 4 shows the evaluation of cortical bone healing using micro-CT. The white arrows indicate the defect position of the cortical bone and the degree of cortical bone regeneration (the image is only for display).

[0138] In the empty group, almost all animals had healed on the cortical bone side, and the cortical bone at the defect position was very thin. Little or no bone formation within the defect was confirmed. The groups treated with only the scaffold (2 - 4) had varying degrees of bone formation in the defect but did not achieve cortical bone regeneration. In Groups 5 and 6, an edge that did not transmit white radiation could be confirmed along the surface of the membrane, which also confirmed the placement of the membrane on the inner membrane side. In all membrane-treated groups (5 - 8), the scaffold was confined within the defect. Groups 7 (5 / 10) and Group 8 (7 / 10) significantly improved the formation of cortical bone bridges, and these were the only groups among those treated with the scaffold or membrane to form the maximum cortical bone bridges. For details, refer to Figure 4.

[0139] In Figure 5, the left image is the overall view of the defect healing at low magnification, and the right image is the figure of the cortical bone healing at high magnification. The framed area indicates the range of the cortical bone defect, and the approximate center of the cortical bone defect is indicated by the black arrow.

[0140] The results of the histological analysis fully supported the micro-CT imaging. The empty group showed thin but healed cortical bone with bone marrow infiltration in the metaphysical region. The scaffold in group 2 showed some bone formation at the periphery of the scaffold, but the cortical bone did not heal. Groups 3-6 showed a significant amount of new cancellous bone around the defect, and similarly some bone formation within the scaffold pores. However, cortical bone regeneration was only observed in some areas. Representative histological images show cortical bone bridging formation in groups 7 and 8. The interior of the defect was filled with cancellous bone at the periphery of the scaffold, similar to groups 3-6, but bone formation within the scaffold was limited.

[0141] Experiment 2: X-ray imaging The X-ray images in Figure 6 show that adding rhBMP-2 to collagen-containing membranes with or without hydroxyapatite increased the radiation-impermeable region in the sample compared to the collagen-containing membrane alone. Adding both ZA and rhBMP-2 to collagen-containing membranes with or without nHA significantly increased the radiation-impermeable region in the sample.

[0142] conclusion Experiment 2 demonstrated the true carrier properties of collagen-containing membranes by inducing bone formation in an abdominal muscle pouch model. Bone formation was induced to varying degrees by delivering both rhBMP-2 and rhBMP-2+ZA, regardless of the presence of nHA. Simultaneous delivery of rhBMP-2 and ZA induced higher bone formation than the rhBMP-2 group. Adding nHA to the collagen membrane further increased its osteogenic capacity when rhBMP-2 and ZA were delivered using the membrane. Such an effect was not observed when rhBMP-2 alone was added.

[0143] Experiment 1 elucidated the true capacity of membranes in cortical bone healing through the phenomenon of tissue regeneration induction. Apart from the empty group, groups 2-4 failed to demonstrate complete cortical bone regeneration. In groups 5-8, adding a membrane on top of the scaffold prevented the scaffold from being forcibly ejected from the defect and hindered cortical bone regeneration. This experiment also showed that delivering ultra-low doses of rhBMP-2 via a collagen-containing membrane significantly increased cortical bone regeneration, as was observed in both groups 7 and 8. Therefore, membranes functionalized with low doses of rhBMP-2 can be used to regenerate bone in demanding orthopedic settings.

[0144] Example 2 Functionalized collagen-containing membrane As described above, the artificial periosteum of the present invention, i.e., the hydroxyapatite-functionalized collagen-containing membrane described herein, can be used as a containment device to prevent biomaterials filled in interosseous spaces from leaking into cortical bone. The inventors believe that when biomaterials (ceramics or polymers) are placed in a bone defect, they tend to be pushed out of the bone due to hydrostatic pressure generated within the bone. This phenomenon is highly likely to cause damage to cortical bone healing. However, when the artificial periosteum of the present invention is used to cover the biomaterial placed in the interosseous space, particularly on the endothelial side, i.e., below the medial end of the cortical bone (endoosseous membrane), the artificial periosteum prevents the biomaterial from being pushed out into the cortical bone. Although not essential, the inventors believe it is important to place the artificial periosteum on the endothelial side because it provides a strong gripping force that covers the embedded material throughout the experiment.

[0145] Figure 7 illustrates the role of the artificial periosteum of the present invention as a storage device for ceramic or polymer biomaterials placed within the interosseous space. The dashed lines indicate the medial and lateral edges of the cortical bone. The arrows in the upper left and upper right indicate the ceramic and polymer biomaterials protruding from and nestled between the ends of the cortical bone, respectively, as indicated by the lower dashed line. The lower arrow in the lower left panel indicates leakage of the ceramic material into the cortical bone, and the upper arrow indicates calcification of the collagen membrane placed on the outer membrane side. The arrow in the lower right points to the artificial periosteum covering the polymer scaffold placed in the bone defect. Note that the membrane is calcified to some extent and ensures that the material remains beneath the cortical bone rather than between the cortical bone ends. All images are representative micro-CT slices taken 8 weeks after in vivo treatment.

[0146] Naturally, another role of the artificial periosteum is to function as a crosslinking agent, inducing cortical bone regeneration through tissue regeneration (see Figure 4). Experiments conducted showed that when the membrane was placed on the outer side, it tended to be pushed out from the cortex and calcified by the overlapping muscle because it did not properly cover the defect. However, when membranes with or without rhBMP-2 were placed on the inner side, both the containment of biomaterials in the interosseous space and cortical bone regeneration were demonstrated (this was more pronounced when small doses of rhBMP-2 were used in the membrane).

[0147] Example 3: Comparative Experiment The inventors compared a commercially available ACS collagen sponge (Medtronic) with the functionalized collagen-containing membrane of the present invention in the presence of BMP-2 and ZA, using a publicly available abdominal muscle pouch model (Raina et al. (2018), J. Control Release, Volume 272 Pages 83-96).

[0148] Micro-CT data was acquired from the ACS group and compared with data from the muscle pouch of the artificial periosteum described in this invention.

[0149] Both experiments were conducted using abdominal pouch models with the same doses of rhBMP-2 (10 μg / scaffold) and ZA (10 μg / scaffold). While a direct comparison of these data is not entirely possible because the experiments were conducted at two different time points and calibration with a micro-CT phantom was not available, the inventors noticed that the same micro-CT settings were used and the voxel size was the same (10 μm). Furthermore, the X-ray images were acquired with the same settings and also show differences.

[0150] The inventors found that the artificial periosteum of the present invention was superior to the ACS group in terms of bone formation (Figure 8).

[0151] summary Based on the above embodiments, the present invention has several advantages over the prior art, for example: • Ease of use, regardless of the availability of bone grafts of appropriate size and shape, the condition of the graft site, and integration with surrounding tissues; and • Providing alternative bone repair methods, particularly the development of artificial periosteum that can be used to locally deliver bone activators such as BMP-2 over extended periods to promote bone growth and repair bone defects.

[0152] In this specification and in the claims (where applicable), the word “comprising” and its derivatives such as “comprises” and “comprise” include, but do not exclude, the inclusion of one or more further integers.

[0153] Throughout this specification, any reference to “one aspect” or “a certain aspect” means that the specific characteristics, structures, or features described in conjunction with the aspect are included in at least one aspect of the present invention. Therefore, the phrases “in one aspect” or “a certain aspect” in various parts of this specification do not necessarily all refer to the same aspect. Furthermore, specific characteristics, structures, or features may be combined in one or more combinations in a preferred manner.

[0154] Since the means described in the specification include preferred forms for carrying out the present invention, it should be understood that the present invention is not limited to the specific characteristics shown or described. Accordingly, the present invention can be claimed in any form or modification thereof. The following are the embodiments of the claims originally filed for this application. [1] An artificial periosteum comprising a functionalized collagen-containing membrane and a drug-carrier mixture, wherein the drug-carrier mixture comprises at least one therapeutic agent and a calcium-containing carrier mixture. [2] The artificial periosteum according to [1], wherein the functionalized collagen-containing membrane is a hydroxyapatite-functionalized collagen-containing membrane. [3] The artificial periosteum according to [1] or [2], wherein the therapeutic agent is a bone activator. [4] The artificial periosteum according to [3], wherein the bone activator activates osteoblasts. [5] The artificial periosteum according to [3], wherein the bone activator inhibits osteoclasts. [6] The artificial periosteum according to any one of [3] to [5], wherein the osteoactivating agent comprises one or more of the following: PGE1; PGE2; EP2 receptor agonists; EP4 receptor agonists; EP2 receptor / EP4 receptor dual agonists; organic bisphosphonates; cathepsin K inhibitors; estrogen or estrogen receptor modulators; calcitonin; osteoclast proton ATPase inhibitors; HMG-CoA reductase inhibitors; integrin receptor antagonists; RANKL inhibitors; osteoanabolic agents; osteogenic agents; vitamin D or synthetic vitamin D analogs; androgens or androgen receptor modulators; SOST inhibitors; platelet-derived growth factors; pharmaceutically acceptable salts thereof; and mixtures thereof. [7] The artificial periosteum according to [6], wherein the organic bisphosphonate is selected from the group consisting of alendronate, sodium alendronate, ibandronate, risedronate, zoledronate, zoledronic acid, etidronate, pamidronate, tildronate, neridronate, and olpadronate. [8] The artificial periosteum according to [6], wherein the bone morphogenetic protein is selected from the group consisting of BMP-2, BMP-4, and BMP-7. [9] The artificial periosteum according to any one of [1] to [8], wherein the drug-carrier mixture comprises a sodium channel blocker, a TRPV1 antagonist, an endothelin antagonist, a bradykinin antagonist, an ASIC inhibitor, a TrkA inhibitor, or a radionuclide.

[10] The artificial periosteum according to any one of [1] to [9], wherein the drug-carrier mixture comprises an anti-inflammatory agent selected from the group consisting of NSAIDs, corticosteroids and cytokine inhibitors.

[11] The artificial periosteum according to any one of [1] to

[10] , wherein the drug-carrier mixture comprises an antibacterial agent and / or an antifungal agent.

[12] The artificial periosteum according to

[11] , wherein the antibacterial agent is a cephalosporin, a quinolone antibiotic and / or a macrolide.

[13] The artificial periosteum according to

[11] , wherein the antifungal agent is fluconazole, clotrimazole and / or itraconazole.

[14] The artificial periosteum according to any one of [1] to

[13] , wherein the drug-carrier mixture comprises an anticancer agent.

[15] The artificial periosteum according to

[14] , wherein the anticancer agent is vincristine, doxorubicin, etoposide, gemcitabine and / or methotrexate.

[16] An artificial periosteum comprising a hydroxyapatite-functionalized collagen-containing membrane and a drug-carrier mixture, wherein the drug-carrier mixture comprises BMP-2 and zoledronic acid.

[17] A method for repairing bone, comprising the step of implanting an artificial periosteum comprising a functionalized collagen-containing membrane and a drug-carrier mixture, wherein the drug-carrier mixture comprises at least one therapeutic agent and a calcium-containing carrier mixture.

[18] The method according to

[17] , wherein the functionalized collagen-containing membrane is a hydroxyapatite functionalized collagen-containing membrane.

[19] The method according to

[17] or

[18] , wherein the drug-carrier mixture comprises a bone activator.

[20] The method according to

[19] , wherein the bone activator activates osteoblasts.

[21] The method according to

[19] wherein the bone activator inhibits osteoclasts.

[22] The method according to any one of

[19] to

[21] , wherein the osteoactivator comprises one or more of the following: PGE1; PGE2; EP2 receptor agonists; EP4 receptor agonists; EP2 receptor / EP4 receptor dual agonists; organobisphosphonates; cathepsin K inhibitors; estrogens or estrogen receptor modulators; calcitonin; osteoclast proton ATPase inhibitors; HMG-CoA reductase inhibitors; integrin receptor antagonists; RANKL inhibitors; osteoanabolic agents; osteogenic agents; vitamin D or synthetic vitamin D analogs; androgens or androgen receptor modulators; SOST inhibitors; platelet-derived growth factors; pharmaceutically acceptable salts thereof; and mixtures thereof.

[23] The method according to

[22] , wherein the organic bisphosphonate is selected from the group consisting of alendronate, sodium alendronate, ibandronate, risedronate, zoledronate, zoledronic acid, etidronate, pamidronate, tildronate, neridronate, and olpadronate.

[24] The method according to

[22] , wherein the bone morphogenetic protein is selected from the group consisting of BMP-2, BMP-4 and BMP-7.

[25] The method according to any one of

[17] to

[24] , wherein the at least one therapeutic agent comprises a sodium channel blocker, a TRPV1 antagonist, an endothelin antagonist, a bradykinin antagonist, an ASIC inhibitor, a TrkA inhibitor, or a radionuclide.

[26] The method according to any one of

[17] to

[25] , wherein the at least one therapeutic agent comprises an anti-inflammatory agent selected from the group consisting of NSAIDs, corticosteroids and cytokine inhibitors.

[27] The method according to any one of

[17] to

[26] , wherein at least one therapeutic agent comprises an antibacterial agent and / or an antifungal agent.

[28] The method according to

[27] , wherein the antibacterial agent is a cephalosporin, a quinolone antibiotic and / or a macrolide.

[29] The method according to

[27] , wherein the antifungal agent is fluconazole, clotrimazole and / or itraconazole.

[30] The method according to any one of

[17] to

[29] , wherein at least one therapeutic agent comprises an anticancer agent.

[31] The method according to

[30] , wherein the anticancer agent is vincristine, doxorubicin, etoposide, gemcitabine and / or methotrexate.

[32] A method for repairing bone, comprising the step of implanting an artificial periosteum comprising a functionalized collagen-containing membrane and a drug-carrier mixture, wherein the drug-carrier mixture comprises BMP-2 and zoledronic acid.

[33] The method according to

[32] , wherein the functionalized collagen-containing membrane is a hydroxyapatite functionalized collagen-containing membrane.

[34] A method for repairing bone defects: (i) The step of implanting graft material into the bone defect; and (ii) The step of coating the graft with a functional collagen-containing membrane. A method that includes this.

Claims

[Claim 1] An artificial periosteum comprising a functionalized collagen-containing membrane and a drug-carrier mixture, wherein the drug-carrier mixture comprises bone morphogenetic protein-2 (BMP-2) and a mixture of zoledronic acid (ZA) and a calcium-containing carrier, and the functionalized collagen-containing membrane is a collagen-containing membrane functionalized with hydroxyapatite nanoparticles.

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