artificial periosteum

Abstract

An artificial periosteum that can also be used to locally deliver bone-active agents such as BMP-2 over an extended period of time to promote bone growth and repair bone defects, and a method for repairing bone, are provided. An artificial periosteum is provided, 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.

Description

[Technical Field]

[0001] The present invention relates to artificial periosteum, systems and methods for bone repair, and the use of artificial periosteum for the local delivery of therapeutic agents, such as bone-active agents. [Background technology]

[0002] Today, many medical procedures rely on regenerating bones that have deteriorated due to disease or aging, or that have been damaged (e.g., fractured). While a variety of surgical procedures are available, advances in modern medicine have enabled certain techniques to enhance and sometimes even replace these surgical procedures.

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

[0004] The development of an artificial periosteum with the biochemical and mechanical properties of autologous osteochondral grafts, with better integration properties and without the need to harvest osteochondral grafts, would be very attractive.

[0005] An additional advantage of artificial periosteum is that it can be used to deliver therapeutic agents, such as bone-active drugs like BMP-2, for cortical bone regeneration.

[0006] The existing material used to deliver recombinant human BMP-2 (rhBMP-2) is a porous collagen sponge approved by the FDA. Other rhBMP-2 carriers have been described in the literature (Morales et al., (2017), J Drug Delivery Sciences and Technology, Vol. 42). Current issues with approved biomaterials are their rapid degradation, resulting in rapid release of the protein, and secondary osteoclastogenic activity, which reduces overall net bone formation. While porous biomaterials have been used for general bone regeneration by delivering rhBMP-2, Horstmann and colleagues have reported that these materials tend to protrude into the cortical bone and delay cortical bone healing (Horstmann et al. (2018), Tissue Eng. Part A, Vol. 23). Therefore, from a clinical perspective, there is a need for a thin biomaterial-form membrane that can prevent cancellous bone void fillers from protruding into the cortical bone by providing a cell template and localized release of growth factors, while simultaneously promoting the natural process of cortical bone healing. The healing process of cancellous bone differs from that of cortical bone, and the present invention is primarily directed to cortical bone regeneration. While cancellous bone can be treated with any bone substitute, cortical bone requires specific biomaterial properties.

[0007] Thus, there remains a need for improved bone repair methods, particularly the development of an artificial periosteum that can also be used to promote bone growth and locally deliver bone-active agents such as BMP-2 over an extended period of time to repair bone defects. Summary of the Invention

[0008] The present invention provides artificial periosteum and methods for bone repair and delivery of therapeutic agents to bone, which may be bone-active agents that repair bone defects or promote bone growth, bone-active agents that treat bone-related pain, anti-inflammatory agents for treating inflammation-related conditions (e.g., arthritis), anti-cancer agents for treating bone cancer, anti-microbial agents for treating or preventing infection at the treatment site, or combinations 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 synovial membrane comprising a functionalized collagen-containing membrane and a drug-carrier mixture for use in a method of repairing bone, wherein the drug-carrier mixture comprises at least one therapeutic agent and a calcium-containing carrier mixture.

[0012] In certain embodiments 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] Thus, 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 certain embodiments of the present invention, the therapeutic agent is a bone-active agent comprising a compound that activates osteoblasts. In other embodiments, the therapeutic agent is a bone-active agent that inhibits osteoclasts. In yet other embodiments, the therapeutic agent is a bone-active agent comprising one or more of 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; inhibitors of osteoclast proton ATPase; inhibitors of HMG-CoA reductase; integrin receptor antagonists; RANKL inhibitors; bone anabolic agents; bone forming agents; vitamin D or synthetic vitamin D analogs; androgens or androgen receptor modulators; SOST inhibitors; platelet-derived growth factor; pharmaceutically acceptable salts thereof; and mixtures thereof.

[0015] One aspect of the present invention provides a method for bone repair, 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.

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

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

[0018] In one aspect of the invention, there is provided a method of repairing a bone defect in a patient, comprising: (i) implanting a bone graft material into the bone defect; and (ii) coating the implant with a hydroxyapatite-functionalized collagen-containing membrane; and The present invention provides a method comprising:

[0019] Also disclosed is a hydroxyapatite-functionalized collagen-containing membrane-coated bone graft material for use in a method for repairing a bone defect in a patient. [Brief explanation of the drawings]

[0020] [Figure 1] Figure 1 shows an overview of the structure of the material and the alignment of the collagen fibers. [Figure 2] Figure 2 shows the surgical procedure for the tibial defect model. [Figure 3] Figure 3 shows micro-CT quantification of the tibial defect experiment 8 weeks after surgical treatment. [Figure 4] Figure 4 shows the assessment of cortical bone healing using micro-CT. [Figure 5] FIG. 5 shows histological analysis of tibial defect healing. [Figure 6] FIG. 6 shows an X-ray of a sample taken from the abdominal pouch 4 weeks after surgery. [Figure 7] FIG. 7 illustrates the role of a collagen membrane as a containment device for ceramic or polymer biomaterials placed within a bone void. [Figure 8] Figure 8 shows a comparative study of an absorbable collagen sponge (ACS) manufactured by Medtronic and sold as INFUSE® Bone Graft, used with a solution containing rhBMP-2, and a collagen-containing membrane of the present invention containing rhBMP-2 and ZA. Data are CT data, and represent the mean ± standard deviation (shown at the top) for n=8 / group for ACS and n=5 / group for collagen membrane. DETAILED DESCRIPTION OF THE INVENTION

[0021] Repair of bone defects caused by trauma, infection, or tumor typically involves replacing the lost / removed material with allogeneic, autologous, or synthetic graft material. When bone defects involve cortical bone loss, even if healing occurs in the cancellous bone, it takes a considerable amount of 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 nonunion fractures, which account for up to 5% of all high-impact fractures.

[0022] Because periosteal cells have a powerful role in cortical bone healing, the periosteum is thought to be involved in the successful healing of bone defects. A particular method for treating cortical bone defects is to temporarily insert a spacer, create a periosteum-like soft tissue shell around the spacer (which is metabolically active), and then remove the spacer after several months and replace it with a bone graft. The temporary periosteum thus formed can be re-suturized, 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 a replacement material. The artificial periosteum of the present invention reduces swelling, leakage, and, when functionalized with biomolecules, forms a bridge for new cortical bone. It may be attached to or around the bone by adhesive, suture, or peripheral loop. It may also be applied by inserting it under the cortical bone using an onlay or inlay technique. When functionalized with a bone-activating agent, it will accelerate cortical bone regeneration.

[0024] In a broad aspect, the present invention relates to a collagen-containing membrane that is functionalized.

[0025] The term "collagen" as used herein refers to all collagens, including those that have been processed or otherwise modified. Preferred collagens are processed to remove immunogenic telopeptide regions ("atelopeptide collagens"), are soluble, and can be reconstituted into fibrils.

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

[0027] The collagen-containing membrane preferably has the following properties: a) pores that interconnect in a way that promotes tissue integration and vascularization; b) biodegradable and / or bioabsorbable, such that the collagen-containing membrane is eventually replaced by normal tissue; c) surface chemistry that promotes cell attachment, proliferation, and differentiation; d) strength and flexibility, and e) Low immunogenicity.

[0028] Collagen-containing membranes are typically prepared or manufactured from "collagen-containing tissue," which includes the dense connective tissue found in mammals. The term "collagen-containing tissue" refers to skin, muscle, and the like, which can be isolated from a mammal that contains collagen. The term "collagen-containing tissue" also includes "synthetically" produced tissue, where collagen or collagen-containing materials are assembled or manufactured outside the body.

[0029] In some embodiments, the collagen-containing tissue is isolated from a mammal, including but not limited to, an ovine, bovine, porcine, or human, hi other embodiments, the collagen-containing tissue is isolated from a human.

[0030] In some embodiments, the collagen-containing tissue is "autologous," ie, isolated from the body of the patient in need of treatment.

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

[0032] The collagen-containing membrane can be prepared by any method known in the art, although one preferred method includes the following steps (i) to (iv): (i) Isolating collagen-containing tissue and incubating the tissue in an ethanol solution; (ii) incubating the collagen-containing tissue of step (i) in a first solution comprising an inorganic salt and an anionic surfactant to denature non-collagenous proteins contained therein; (iii) incubating 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) incubating the collagen-containing tissue from step (iii) in a third solution comprising an inorganic acid with a simultaneous mechanical stimulus for a time sufficient to allow collagen bundles within said tissue to align, wherein said mechanical stimulus comprises applying cyclic tension to the collagen-containing tissue. It may also be produced by

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

[0034] Any anionic surfactant can be used for 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 comprises about 1% (v / v) SDS and about 0.2% (v / v) LiCl.

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

[0037] It will be understood by those skilled in the art that the incubation time for each step will vary 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) occurs at about 4° C. In other embodiments, the incubation in step (ii) occurs for at least 12 hours.

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

[0040] In some embodiments, the incubation in step (iii) is carried out for about 30 minutes. In other embodiments, the incubation in step (iii) is carried out with shaking.

[0041] In some embodiments, the third solution comprises about 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 comprises a neutralization step between steps (iii) and (iv), comprising incubating the collagen-containing tissue with about 0.5% (v / v) NaOH.

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

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

[0046] The glycerol may be allowed to contact the collagen-containing tissue for a time to facilitate removal of fat and / or blood vessels, hi some embodiments, the contact time is at least 10 minutes.

[0047] In some embodiments, the method further comprises 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 between steps (ii) and (iii) is to remove 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. If the collagen-containing tissue is washed with NaOH:NaCl, it is preferably further washed with sterile water.

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

[0051] The term "concurrent mechanical stimulation" as used in the methods herein refers to stretching a collagen-containing tissue during chemical treatment of the collagen-containing tissue. The collagen-containing tissue may be subjected to static and / or cyclic stretching. Thus, in some embodiments, the concurrent mechanical stimulation (i) stretching of collagen-containing tissue for a predetermined period of time; (ii) relaxation of collagen-containing tissue for a predetermined period of time; and (iii) Repeating steps (i) and (ii) n times (n is an integer of 1 or more). may also include:

[0052] When the collagen-containing tissue is stretched by mechanical stimulation, the collagen-containing tissue is preferably stretched along its longitudinal axis.

[0053] In some embodiments, the concurrent mechanical stimulation comprises cyclically applying tension to the collagen-containing tissue, the tension periodicity comprising a stretch time of about 10 seconds to about 20 seconds and a relaxation time of about 10 seconds resulting in a strain of about 10%, and continuing the mechanical stimulation until the collagen bundles within the collagen-containing tissue are aligned as described herein.

[0054] The resulting collagen-containing tissue comprises collagen fibers or bundles in a knitted structure. As used herein, the term "knitted structure" refers to a structure comprising first and second groups of fibers or bundles, with the first group of fibers or bundles extending primarily in a first direction and the second group of fibers or bundles extending primarily in a second direction, the first and second directions being different from each other and the fibers or bundles of the first group interleaved or otherwise interwoven with the fibers or bundles of the second group. The difference in direction may be approximately 90°.

[0055] Collagen-containing tissues produced by preferred methods have an "ultimate tensile load strength" of greater than 20 N. In some embodiments, collagen-containing tissues of the invention have an ultimate tensile load strength of greater than 25 N, 40 N, 60 N, 80 N, 100 N, 120 N, or 140 N.

[0056] Additionally, the knitted structure of the collagen-containing tissue embodiment is believed to increase the elastic modulus of the collagen-containing patch while exhibiting reduced elongation at maximum load.

[0057] As used herein, the term "elastic modulus" refers to Young's modulus, which is determined as the ratio of stress to strain, and is a measure of the stiffness of the collagen-containing tissue and / or patch.

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

[0059] As used herein, the term "elongation at maximum load" refers to the elongation of a collagen-containing tissue at its maximum tensile load strength relative to the original length of the collagen-containing tissue in an unloaded state, as opposed to the greater maximum extension.

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

[0061] After the collagen-containing tissue is generated, the collagen-containing membrane can be shaped for use. In some embodiments, the collagen-containing membrane is shaped into a membrane to facilitate in situ manipulation.

[0062] Preferably, the collagen-containing membrane of the present invention is thick enough to support the drug-carrier mixture, but not so thick that in situ operability of the collagen-containing membrane is impaired. Thus, in some embodiments, the collagen-containing membrane has a thickness of 25 μm to 200 μm. In some embodiments, the collagen-containing membrane has a thickness of 30 μm to 180 μm. In other embodiments, the collagen-containing membrane has a thickness of 35 μm to 170 μm. In still other embodiments, the collagen-containing membrane has a thickness of 40 μm to 160 μm. In still other embodiments, the collagen-containing membrane has a thickness of 45 μm to 150 μm. In still other embodiments, the collagen-containing membrane has a thickness of 50 μm to 140 μm. In still other embodiments, the collagen-containing membrane has a thickness of 50 μm to 100 μm. Also, in some embodiments, the collagen-containing membrane has a thickness of about 50 μm.

[0063] In one form of collagen-containing membrane, the membrane is perforated to allow natural osteoactive molecules or therapeutically active agents in the graft material to be transported across the membrane and to recruit 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 of loose collagen fibers. The rough surface is particularly suitable for promoting cell attachment, and in fact, when the membrane is overlaid with muscle, it is important that the rough surface faces the muscle. However, in the case of no overlaid muscle, for example, in the case of distal tibia repair, it is not so important that the rough surface be any particular surface.

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

[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, 0.96 M) is maintained at 90-100°C under constant stirring and then mixed with an aqueous solution of diammonium hydrogen orthophosphate ((NH4)2HPO4, 0.6 M). The pH of the system is constantly monitored and maintained at 10.0 by adding NH4OH solution. nHAP precipitates from the solution as white crystals. After completion of the reaction, 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 washed thoroughly with Milli-Q Type I water (DI-H2O). It is then dried at 120°C.

[0067] In some embodiments, the synthesized nHAP is heat treated to enhance its crystallinity, density, and phase purity, with the temperature conditions ranging from 500°C up to 1000°C, and the holding time being 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 the nHAP.

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

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

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

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

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

[0074] As described in more 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 combined with a suitable bone matrix material to form the artificial periosteum of the present invention, as described further below. Alternatively, the drug-carrier mixture may be applied to a functionalized collagen-containing membrane, similarly forming the artificial periosteum of the present invention. The artificial periosteum may then be applied to the treatment site, for example, by implantation.

[0075] Calcium phosphate cements (CPCs) that can be used in the carrier component of the 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 1:3 mixture of α-TCP and β-TCP. In some embodiments, calcium phosphate cements and mixtures thereof may contain added hydroxyapatite (e.g., 2.5% wt / w hydroxyapatite crystals). α-TCP and β-TCP may be used in various ratios. For example, in some embodiments, the CPC comprises a 1:3 mixture of α-TCP and β-TCP, optionally containing 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 an α-TCP cement with 2.5% hydroxyapatite added, which results in a harder cement when placed.

[0076] The drug-carrier mixture may comprise at least partially demineralized bone matrix. The bone matrix may be a demineralized bone matrix putty or may be partially or fully demineralized intact bone matrix. The intact bone matrix may be used in bone grafting to serve as a scaffold for delivering bone repair drugs.

[0077] Human demineralized bone matrix putty may be used as the carrier component of the drug-carrier mixture. It is commercially available, for example, as 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 and Dowell (Inductive Substratum for Osteogenesis in Pellets of Particulate Bone Matrix, Clin. Orthop. Relat. Res., 1968, 61, 61-78.). This method involves demineralizing bone and defatting the solid demineralized bone, cutting it into small pieces, and grinding it under liquid nitrogen to a coarse powder. The ground demineralized bone matrix has the consistency of a putty after thawing.

[0078] If required, setting solutions for tricalcium phosphate cement powders are well known in the art and include 2.5% wt / vol NaHPO solution or commercially available solutions. See Dorozhkin, Materials 2009, 2, 221-291.

[0079] Another example of a carrier component is one made by combining gelatin with calcium sulfate (CaS), optionally in the presence of hydroxyapatite (HA), using the cryogel formation technique of Kumar et al. (Mater. Today, 13, (2010), 42-44). An example using a gelatin-CaS-HA composite is described in Raina et al. (2018), J Control Release, Vol. 272, 83-96, and a similar composite of silk, chitosan, bioactive glass, and HA is described in Raina et al. (J Control Release, Vol. 235, 365-378. (2016)). Murphy et al. also described a porous collagen-hydroxyapatite-based carrier 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] The 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, wet the therapeutic agent-carrier mixture with a suitable hardening solution to produce the drug-carrier mixture.

[0081] Treatment method The artificial periosteum of the present invention is useful in the treatment of bone fractures and bone loss due to periodontal disease, surgical procedures, cancer, or trauma. Additional uses of the artificial periosteum of the present invention include use in increasing bone density in preparing bone to receive dental or orthopedic implants, as a coating for implants for enhanced osseointegration, and in all forms of spinal fusion.

[0082] The present invention relates to a method of treatment, which includes using the artificial periosteum of the present invention in a patient in need thereof, and the artificial periosteum may contain a therapeutically effective amount of a bone repair drug as described herein. The method of treatment generally includes stimulating, promoting, enhancing, or inducing bone formation or inhibiting bone resorption. The method of treatment also includes, for example, promoting bone remodeling, activating osteoblasts, promoting osteoblast differentiation, inhibiting osteoclasts, increasing the number and activity of osteoblasts, increasing average 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 method of treatment also includes treating one or more of osteoporosis, fractures, low bone density, or periodontal disease.

[0083] In one embodiment of the method of treatment, one or more bone-repairing drugs are released from a drug-carrier mixture described herein. In another embodiment, the bone-repairing drug is released from the drug-carrier mixture in combination with another therapeutic agent that is administered systemically (e.g., orally). For example, the bone-repairing drug may be released from the artificial periosteum in combination with one or more additional therapeutic agents for treating bone loss or osteoporosis that are administered systemically.

[0084] Therapeutic methods further include application of artificial periosteum to localized desired sites in humans, other mammals, and birds, for example, in bone gaps such as alveolar defects, adjacent sites of alveolar bone, or bone defects 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, anti-cancer, 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 administration of a single formulation containing one or more of the compounds described herein and one or more additional pharmaceutical agents, as well as administration of the compound and each additional pharmaceutical agent in its own separate formulation. For example, the compound described herein and one or more additional pharmaceutical agents can be administered to a patient together in an artificial periosteum having a fixed ratio of each active ingredient, or each agent can be administered as a separate formulation. For example, a patient may be treated with an active agent delivered locally to the site of a bone defect via an artificial periosteum in combination with another drug that is administered systemically. When separate formulations are used, the compound and one or more additional pharmaceutical agents can be administered essentially simultaneously (e.g., concurrently) or at staggered times (e.g., sequentially).

[0087] In one aspect of the invention, a bone void is filled with, for example, a bone graft or a stand-alone or composite substitute for a bone graft derived from natural or synthetic sources, and then coated with an artificial periosteum comprising a functionalized collagen-containing membrane and a drug-carrier mixture of the invention, or coated with a hydroxyapatite and / or bioactive agent-functionalized collagen-containing membrane of the invention, which acts as a crosslinker to regenerate bone by guided tissue regeneration.

[0088] One use of the artificial periosteum of the present invention is as an external covering for bone defects and / or gaps that have been filled with bone graft material. The artificial periosteum may be held in place using methods known in the art, including suturing, clamping, or fixation with medical adhesives. The artificial periosteum may also simply be placed on the endosteal side 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 body fluids. With respect to the artificial periosteum, medical adhesives can be used to promote fixation of the artificial periosteum or to structurally hold portions of the artificial periosteum together. For convenience, the term "adhesive" generally refers to the adhesive in its applied form and the adhesive composition after it has hardened 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 such as cyanoacrylates (e.g., 2-octyl cyanoacrylate, Ethicon Products' DERMABOND™), fibrin glues (e.g., Baxter's TISSUCOL®), and mixtures thereof, although a wide variety of suitable adhesives are available.

[0090] For repair of long bone segment defects, the bone cavity may be filled with any bone graft substitute (synthetic, natural, or natural), which may or may not include internal or external fixation. The artificial periosteum of the present invention can then be wrapped around the cortical bone to hold the bone graft substitute in place and avoid the two-stage Maskeillet procedure. The Maskeillet procedure is used in long bone trauma applications where there is a large intermediate defect, such as when a portion of the long bone is missing. The Maskeillet procedure typically involves two steps: a first step in which a spacer is placed and soft tissue forms around the spacer, and a second step in which the formed soft tissue is used to cover the bone graft. Thus, in some embodiments, the artificial periosteum of the present invention may be used to repair trauma in long bone segment defects. For example, a relatively large coverage may be provided with a material suitable for trauma repair provided therein, where the artificial periosteum is used to maintain the space of the long bone (free of soft tissue) and allow soft tissue to form around it. The second step of the Masquere procedure may be avoided because the graft material is provided when the artificial periosteum is originally placed.

[0091] Further desirable embodiments include cells seeded or placed on the artificial periosteum of the present invention. While any cells may be used, cells typically 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, unidifferentiated stem cells, and bone marrow stem cells. Other examples of cells that may be used in various embodiments include, but are not limited to, osteoblasts, fibroblasts, chondrocytes, and connective tissue cells.

[0092] It will be appreciated that there is also provided an artificial periosteum of the present invention for use in such methods of treatment.

[0093] It will be appreciated that there is also provided the use of a functionalized collagen-containing membrane and a drug-carrier mixture in the manufacture of an artificial periosteum for use in such methods of treatment.

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

[0095] The term "bone repair agent" or "bone active agent," as used herein, refers to an agent capable of stimulating, promoting, enhancing, or inducing bone formation or inhibiting bone resorption. Thus, a bone active agent may be a bone anabolic or anti-catabolic agent. A bone active agent may have one or more of the following effects: promoting bone remodeling, activating osteoblasts, promoting osteoblast differentiation, inhibiting osteoclasts, increasing osteoblast number and activity, 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. Bone-active agents include: prostaglandin E1 (PGE1); prostaglandin E2 (PGE2); EP2 receptor agonists; EP4 receptor agonists; EP2 receptor / EP4 receptor dual agonists; organic bisphosphonates (e.g., alendronate or alendronate sodium); cathepsin K inhibitors; estrogens or estrogen receptor modulators; calcitonin; inhibitors of osteoclast proton ATPase; inhibitors of HMG-CoA reductase (i.e., statins); αβ-integrin receptor antagonists; and RA inhibitors such as denosumab. Examples of bone-active agents include, but are not limited to, NKL inhibitors; bone 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. The bone-active agent preferably does not degrade to an inactive form when exposed to a pH of about 4-5.

[0096] The term "calcium phosphate cement" as used 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 to a bone repair composition made from any of the foregoing or a mixture thereof upon hardening. Calcium phosphate cements may also include hydroxyapatite incorporated therein along 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 that of a natural agonist. An agonist may have full potency (i.e., the same as the natural agonist), partial potency (lower maximal potency compared to the natural agonist), or supramaximal potency (higher maximal potency compared to the natural agonist). Agonists with partial potency are referred to as "partial agonists," and agonists with supramaximal potency are referred to as "superagonists." In one embodiment, the natural agonist may be PGE2.

[0099] Pain-relieving agents 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, zibotentan), bradykinin antagonists, ASIC inhibitors, TrkA inhibitors, and radionuclides ( 89 Sr, 153 Sm-lexidronam, 186 Re-etidronate).

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

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

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

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

[0104] The bone active agent may also be an organic bisphosphonate, such as alendronate, alendronate sodium, ibandronate, risedronate, zoledronate, zoledronic acid, etidronate, pamidronate, tiludronate, neridronate, and olpadronate.

[0105] The bone active agent may also be a cathepsin K inhibitor, such as the compounds disclosed and cited in Bromme in Expert Opin. Investig. Drugs 2009, 18(5) 585-600 (eg odanacatib).

[0106] The bone active agent may be an estrogen or estrogen receptor modulator, including, for example, raloxifene, bazedoxifene, and lasofoxifene, including the compounds described in http: / / en.wikipedia.org / wiki / Selective_estrogen-receptor_modulator.

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

[0108] The bone-active agent may be, for example, a compound described in Nyman in Potential of the Osteoclast's Proton Pump as a Drug Target in Osteoporosis, Annales Universitatis Turkuensis 2011, which is an inhibitor of osteoclast proton ATPase, including SB242784, bafilomycin (e.g., bafilomycin A1), concanamycin A, apicularen, archazolide, benzolactone enamides (salicylihalamide A, lobatamide A), FR167356, FR177995, and dyphylline.

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

[0110] The bone-active agent may be, for example, an αβ-integrin receptor antagonist, such as the compounds described in Millard et al. in Integrin Targeted Therapeutics, Theranostics 2011, 154-188, including cilengitide (EMD 121974), L000845704, SB2730005, etc.

[0111] The bone active agent may be a RANKL inhibitor such as denosumab.

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

[0113] The bone-active agent may be an EP2 receptor / EP4 receptor dual agonist, for example, as described in 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 representative EP2 receptor / EP4 receptor dual agonist is 2-((2-((R)-2-((S,E)-3-hydroxy-4-(m-tolyl)but-1-en-1-yl)-5-oxopyrrolidin-1-yl)ethyl)thio)thiazole-4-carboxylic acid (CAS#494223-86-8).

[0114] Bone-active agents may be prepared by any of the methods described in 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. WO 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 compound may be an EP4 receptor agonist, including but not limited to 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; Heterocycles, 2004, 64, 437-445.

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

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

[0117] In other embodiments, the bone-active agent contained in the artificial periosteum is one or more of an EP2 receptor agonist, an EP2 receptor / EP4 receptor dual agonist, an EP4 receptor agonist, an organic bisphosphonate, an estrogen receptor modulator, an inhibitor of HMG-CoA reductase, and strontium ranelate.

[0118] The present invention also provides an artificial periosteum comprising an agent, drug, or drug combination as described herein. For example, one or more agents / drugs that activate osteoblasts may be combined with one or more agents / drugs that inhibit osteoclasts.

[0119] Alternatively, multiple agents / drugs that activate osteoblasts and inhibit osteoclasts may be combined.

[0120] In some embodiments, the artificial periosteum may comprise an EP4 receptor agonist in combination with any one or more of: a bisphosphonate; a cathepsin K inhibitor; an estrogen or estrogen receptor modulator; calcitonin; an inhibitor of osteoclast proton ATPase; an inhibitor of HMG-CoA reductase (i.e., a statin); an αvβ3-integrin receptor antagonist; a RANKL inhibitor such as denosumab; a bone anabolic agent such as parathyroid hormone; a bone morphogenetic protein (e.g., BMP-2, BMP-4, BMP-7); vitamin D or a synthetic vitamin D analog such as ED-70; an androgen or androgen receptor modulator; an activator of Wnt / β-catenin signaling (e.g., a GSK-3 inhibitor, a sclerostin antagonist, a SOST inhibitor); bortezomib; strontium ranelate; or a platelet-derived growth factor.

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

[0122] In other embodiments, the EP4 receptor agonist is combined with one or more of raloxifene, bazedoxifene, and lasofoxifene.

[0123] In other embodiments, the EP4 receptor agonist is combined with a bone morphogenetic protein 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 (ribenprost) with BMP-2 or BMP-7. Yet another combination includes AE1-329 with BMP-2 or BMP-7. Yet another combination includes L-902,688 with 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-yn-1-yl)-2-oxopyridin-1-yl)heptanoic acid with BMP-2 or BMP-7. Another combination includes 7-((R)-2-((3S,4S,E)-3-hydroxy-4-methylnon-1-en-6-yn-1-yl)-5-oxopyridin-1-yl)heptanoic acid together with BMP-2 or BMP-7.

[0124] In still other embodiments, the EP4 receptor agonist is 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 the specification are incorporated by reference in their entirety, including any drawings and tables, to the extent not inconsistent with the explicit teachings of this specification.

[0126] Where prior art publications are referred to in the specification, it is understood that such references do not constitute an acknowledgment that they form part of the common general knowledge in the art in Australia or anywhere else.

[0127] The following are examples illustrating procedures for the practice of the present invention. These examples are not to be construed as limiting. [Example]

[0128] Example 1 In vivo experiments 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 the cortical bone and below the metaphysis of the proximal tibia of male Sprague-Dawley rats. Figure 2 shows an example of the surgical procedure for the tibial defect model. As a bone void filler, a gelatin-calcium sulfate-hydroxyapatite scaffold supplemented with ZA, with or without rhBMP-2, was filled into the cancellous bone cavity of the defect. This was done as described by Raina et al. (2018), J Control Release, Vol. 272. This was done to provide support for the overlying collagen-containing membrane, which would otherwise have been difficult to overlay. In some groups, the scaffold was also covered with a 6 mm piece of collagen membrane, with the remaining membrane inserted into the endosteum. This membrane prevented the scaffold from leaking outside the circular defect. A detailed description of the groups and bioactive molecule doses is shown in Table 1.

[0129] [Table 1]

[0130] Eight weeks after the surgical procedure, the animals were sacrificed and subjected to quantitative micro-CT and histological examination to assess defect healing.

[0131] In a second experiment, collagen membranes (4 mm round) alone or functionalized on both sides with 1 mg of hydroxyapatite nanoparticles were analyzed in the abdominal 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 per group were sacrificed 4 weeks after surgery, followed by radiography and micro-CT quantification.

[0133] result First experiment: micro-CT Region of interest 1 (ROI 1): For micro-CT analysis, we defined three ROIs. In ROI 1, the mineralized volume (MV) / tissue volume (TV)% within the defect excluding the cortical bone was measured. The diameter of ROI 1 varied, 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, regardless of the type and presence or absence of bioactive molecules, regenerated significantly higher volumes of mineralized tissue or MV / TV% compared to the empty group (Figure 3, top).

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

[0135] Figure 3 shows the micro-CT quantification of the tibial defect 8 weeks after the surgical procedure. * indicates a comparison with the empty group. * indicates p value compared with the S + ZA + (CM + rhBMP-2) group. δ indicates a comparison with the S + ZA + rhBMP-2 + (CM + rhBMP-2) group. * indicates comparison with the empty group, and δ indicates comparison with the scaffold-only group. * or δ indicates p<0.05; **or δδ indicates p<0.01; *** or δδδ indicates p<0.001.

[0136] Region of Interest 3 (ROI 3): ROI 3 was used to measure the complete defect as well as bone proximal and distal to the defect. This not only provided a measure of the regenerated bone within the defect, but also provided insight into the volume of mineralized tissue regenerated around the implanted scaffold and membrane. ROI 3 was 6.5 mm high and encompassed the 4.5 mm defect, as well as an area 1 mm proximal and 1 mm distal to the defect. Groups 3–8 showed significantly higher MV compared to Group 1. Furthermore, Groups 4, 5, 6, and 8 had significantly higher MV compared to Group 2. No significant differences were observed between Groups 1 and 2, or among any of Groups 3–8 (Figure 3, bottom).

[0137] First experiment: Cortical bone healing by micro-CT Figure 4 shows the evaluation of cortical bone healing using micro-CT. White arrows indicate the location of the cortical bone defect and the extent of cortical bone regeneration (images are for representational purposes only).

[0138] In the empty group, almost all animals healed on the cortical side, and the cortical bone at the defect site was very thin. Little to no bone formation was observed within the defect. Groups treated with scaffold only (2–4) had varying degrees of bone formation at the defect site, but no cortical bone regeneration occurred. In groups 5 and 6, a white radiopaque rim was visible along the membrane surface, further confirming 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 8 (7 / 10) showed significantly improved cortical bone bridge formation, and these groups were the only groups treated with either scaffold or membrane to form the greatest number of cortical bone bridges. See Figure 4 for details.

[0139] In Figure 5, the left image is a low-magnification overview of the defect healing, and the right image is a high-magnification view of the cortical bone healing. The box indicates the extent of the cortical bone defect, and the black arrow indicates the approximate center of the cortical bone defect.

[0140] Histological analysis fully confirmed the microCT imaging results. The empty group showed thin but healed cortical bone, with bone marrow infiltration in the metaphyseal region. Group 2 scaffolds showed some bone formation around the scaffold periphery, but the cortical bone did not heal. Groups 3–6 showed significant amounts of new cancellous bone around the defect, as well as some bone formation within the scaffold pores. However, cortical bone regeneration was only partial. Representative histological images show cortical bone bridging in Groups 7 and 8. Similar to Groups 3–6, the interior of the defect was filled with cancellous bone around the scaffold periphery, but bone formation within the scaffold was limited.

[0141] Second experiment: X-ray photography The X-ray images shown in Figure 6 demonstrate that the addition of rhBMP-2 to collagen-containing membranes with or without hydroxyapatite resulted in an increase in the radiopaque area in the samples compared to the collagen-containing membrane alone. The addition of both ZA and rhBMP-2 to collagen-containing membranes with or without nHA significantly increased the radiopaque area in the samples.

[0142] conclusion In Experiment 2, we demonstrated the true carrier properties of collagen-containing membranes by inducing bone formation in an abdominal muscle pouch model. The delivery of both rhBMP-2 and rhBMP-2 + ZA, regardless of the presence of nHA, induced bone formation to varying degrees, and the simultaneous delivery of rhBMP-2 and ZA induced greater bone formation than the rhBMP-2 group. The addition of nHA to the collagen membrane further increased the osteogenic potential of the collagen membrane 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 revealed the membrane's true potential in cortical bone healing through the phenomenon of guided tissue regeneration. Apart from the empty group, groups 2–4 failed to demonstrate complete cortical bone regeneration. In groups 5–8, adding a membrane over the scaffold prevented the scaffold from being forced out of the defect and interfering with cortical bone regeneration. This experiment also demonstrated that delivering ultra-low doses of rhBMP-2 through a collagen-containing membrane significantly increased cortical bone regeneration, as seen in both groups 7 and 8. Therefore, membranes functionalized with low doses of rhBMP-2 can be used to regenerate bone in demanding orthopedic environments.

[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 leakage of biomaterials filled in bone gaps into the cortical bone. The inventors believe that when biomaterials (ceramics or polymers) are placed in bone defects, they tend to be extruded 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 bone gap, particularly on the endosteal side, i.e., below the inner edge of the cortical bone (endosteum), the artificial periosteum prevents the biomaterial from being extruded into the cortical bone. Although this is not essential, we believe that placing the artificial periosteum on the endosteal side is important because it provides a strong grip that covers the implanted material during the experiment.

[0145] Figure 7 illustrates the role of the artificial periosteum of the present invention as a containment device for ceramic or polymer biomaterials placed within bone gaps. The dashed lines indicate the inner and outer edges of the cortical bone. The arrows in the upper left and upper right panels indicate the ceramic and polymer biomaterials protruding from and nested between the edges of the cortical bone, respectively, as indicated by the lower dashed lines. The lower arrow in the lower left panel indicates the ceramic material leaking into the cortical bone, while the upper arrow indicates the mineralization of the collagen membrane placed on the outer membrane side. The arrow in the lower right panel points to the artificial periosteum covering the polymer scaffold placed in the bone defect. Note that the membrane is mineralized to some extent, ensuring that the material remains beneath the cortical bone at all times, rather than between the cortical bone edges. All images are representative micro-CT slices taken after 8 weeks of in vivo treatment.

[0146] Naturally, another role of the artificial periosteum is to act as a cross-linking agent and regenerate cortical bone through guided tissue regeneration (see Figure 4). Experiments performed showed that when the membrane was placed on the outer membrane side, it did not tightly cover the defect and tended to be extruded from the cortex and mineralized by the overlying muscle. However, when the membrane was placed on the inner membrane side, with or without rhBMP-2, both the biomaterial was trapped in the bone gap and cortical bone regeneration was demonstrated (more pronounced when a low dose of rhBMP-2 was used in the membrane).

[0147] Example 3 Comparative Experiment Using a published abdominal pouch model (Raina et al. (2018), J. Control Release, Volume 272, Pages 83-96), we compared a commercially available ACS collagen sponge (Medtronic) with our functionalized collagen-containing membrane in the presence of BMP-2 and ZA.

[0148] Micro-CT data of the ACS group was acquired and compared with the data of the artificial periosteal muscle pouch of the present application.

[0149] Both experiments were performed in an abdominal muscle pouch model with the same doses of rhBMP-2 (10 μg / scaffold) and ZA (10 μg / scaffold). Direct comparison of these data is not entirely possible because the experiments were performed at two different time points and micro-CT phantom calibration was not available. However, we noted that the same micro-CT settings were used and the voxel size was the same (10 μm). Furthermore, X-ray images were acquired with the same settings and also show differences.

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

[0151] summary In light of the above examples, the present invention has several advantages over the prior art, such as: Obtaining a bone graft of appropriate size and shape, ease of use regardless of the pathology of the graft site, and integration with surrounding tissue; and Providing alternative bone repair methods, in particular the development of an artificial periosteum that can also be used to promote bone growth and locally deliver bone-active agents such as BMP-2 over an extended period of time to repair bone defects.

[0152] In this specification and claims (where applicable), the word "comprising" and its derivatives, such as "comprises" and "comprise," includes each of the listed integers but does not exclude the inclusion of one or more additional integers.

[0153] Throughout this specification, references to "one embodiment" or "an embodiment" mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more combinations.

[0154] It is to be understood that the invention is not limited to the specific features shown or described, since the means described in the specification include preferred modes of carrying out the invention. Accordingly, the invention may be claimed in any of its forms or modifications.

Claims

1. An artificial periosteum comprising a functionalized collagen-containing membrane and a drug-carrier mixture, said drug-carrier mixture comprising at least one therapeutic agent and a calcium-containing carrier mixture.

2. The artificial periosteum according to claim 1, wherein the functionalized collagen-containing membrane is a hydroxyapatite-functionalized collagen-containing membrane.

3. The artificial periosteum according to claim 1 or 2, wherein the therapeutic agent is a bone activating agent.

4. The artificial periosteum according to claim 3 , wherein the bone activator activates osteoblasts.

5. The artificial periosteum according to claim 3 , wherein the bone-activating agent inhibits osteoclasts.

6. The artificial periosteum of any one of claims 3 to 5, wherein the bone-active agent comprises one or more of 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; inhibitors of osteoclast proton ATPase; inhibitors of HMG-CoA reductase; integrin receptor antagonists; RANKL inhibitors; bone anabolic agents; bone forming 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 of claim 6, wherein the organic bisphosphonate is selected from the group consisting of alendronate, alendronate sodium, ibandronate, risedronate, zoledronate, zoledronic acid, etidronate, pamidronate, tiludronate, neridronate, and olpadronate.

8. The artificial periosteum according to claim 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 claims 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 claims 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 claims 1 to 10, wherein the drug-carrier mixture comprises an antibacterial agent and / or an antifungal agent.

12. The artificial periosteum according to claim 11, wherein the antibacterial agent is a cephem, a cephalosporin, a quinolone antibiotic and / or a macrolide.

13. The artificial periosteum according to claim 11, wherein the antifungal agent is fluconazole, clotrimazole and / or itraconazole.

14. The artificial periosteum according to any one of claims 1 to 13, wherein the drug-carrier mixture comprises an anticancer drug.

15. The artificial periosteum according to claim 14, wherein the anticancer drug 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. 18. The method of claim 17, wherein the functionalized collagen-containing membrane is a hydroxyapatite-functionalized collagen-containing membrane.

19. The method of claim 17 or 18, wherein the drug-carrier mixture comprises a bone-active agent.

20. 20. The method of claim 19, wherein the bone active agent activates osteoblasts.

21. 20. The method of claim 19, wherein the bone active agent inhibits osteoclasts.

22. 22. The method of any one of claims 19 to 21, wherein the bone-active agent comprises one or more of 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; inhibitors of osteoclast proton ATPase; inhibitors of HMG-CoA reductase; integrin receptor antagonists; RANKL inhibitors; bone anabolic agents; bone forming agents; vitamin D or synthetic vitamin D analogs; androgen or androgen receptor modulators; SOST inhibitors; platelet-derived growth factor; pharmaceutically acceptable salts thereof; and mixtures thereof.

23. 23. The method of claim 22, wherein the organic bisphosphonate is selected from the group consisting of alendronate, alendronate sodium, ibandronate, risedronate, zoledronate, zoledronic acid, etidronate, pamidronate, tiludronate, neridronate, and olpadronate.

24. 23. The method of claim 22, wherein the osteogenic protein is selected from the group consisting of BMP-2, BMP-4, and BMP-7.

25. 25. The method of any one of claims 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. 26. The method of any one of claims 17 to 25, wherein the at least one therapeutic agent comprises an anti-inflammatory agent selected from the group consisting of an NSAID, a corticosteroid, and a cytokine inhibitor.

27. The method of any one of claims 17 to 26, wherein the at least one therapeutic agent comprises an antibacterial agent and / or an antifungal agent.

28. 28. The method of claim 27, wherein the antibacterial agent is a cephem, cephalosporin, quinolone antibiotic and / or macrolide.

29. 28. The method of claim 27, wherein the antifungal agent is fluconazole, clotrimazole and / or itraconazole.

30. 30. The method of any one of claims 17 to 29, wherein the at least one therapeutic agent comprises an anti-cancer agent.

31. 31. The method of claim 30, wherein the anticancer drug 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. 33. The method of claim 32, wherein the functionalized collagen-containing membrane is a hydroxyapatite-functionalized collagen-containing membrane.

34. 1. A method for repairing a bone defect comprising: (i) implanting a graft material into the bone defect; and (ii) covering the implant with a functionalized collagen-containing membrane A method comprising:

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