Patent Application
20190151418
Disclosed is a combination of a calcium phosphate-containing porous composite and PTH, and the use thereof.
Calcium phosphates, such as hydroxyapatite, β-tricalcium phosphate (β-TCP), and octacalcium phosphate (OCP), are used as bone regeneration materials (e.g., PTL 1 to PTL 5). Moreover, PTL 5 proposes incorporating OCP into a porous body made of collagen so as to impart shape-retaining properties to the OCP. Furthermore, NPL 1 reports that a collagen porous body containing OCP has a higher bone-regeneration effect than OCP alone.
NPL 2 reports that local bone formation was significantly promoted by subcutaneous administration of parathyroid hormone (PTH). NPL 2 also reports that bone formation by β-TCP is limited, and that the bone-forming effect by PTH is inhibited by the combined use of PTH and β-TCP.
An object of the present invention is to provide a novel means effective in bone regeneration or bone augmentation.
A combined use of a calcium phosphate-containing porous composite and PTH was confirmed to enable more effective bone regeneration or bone augmentation. Based on this finding, inventions represented by the following items are provided.
A: Method of bone regeneration or augmentation
A1. A method of bone regeneration or bone augmentation, comprising:
E1. A kit for bone regeneration or bone augmentation, comprising:
The present invention provides an effective means for bone regeneration and/or bone augmentation.
Group A: OCP/Col/PTH 1.0 (a freeze-dried composite containing 1.0 μg of PTH per disc of OCP/Col)
Group B: OCP/Col/PTH 0.1 (a freeze-dried composite containing 0.1 μg of PTH per disc of OCP/Col)
Group C: OCP/Col
Group D: β-TCP/Col/PTH 1.0 (a freeze-dried composite containing 1.0 μg of PTH per disc of β-TCP/Col)
Group E: β-TCP/Col/PTH 0.1 (a freeze-dried composite containing 0.1 μg of PTH per disc of β-TCP/Col)
Group F: β-TCP/Col.
Group A: OCP/Col/PTH 1.0
Group B: OCP/Col/PTH 0.1
Group C: OCP/Col
Group D: β-TCP/Col/PTH 1.0
Group E: β-TCP/Col/PTH 0.1
Group F: β-TCP/Col
Group A: OCP/Col/PTH 1.0
Group B: OCP/Col/PTH 0.1
Group C: OCP/Col
Group D: β-TCP/Col/PTH 1.0
Group E: β-TCP/Col/PTH 0.1
Group F: β-TCP/Col
Group A: OCP/Col/PTH 0.01 (a freeze-dried composite containing 0.01 μg of PTH per disc of OCP/Col)
Group B: OCP/Col/PTH 0.001 (a freeze-dried composite containing 0.001 μg of PTH per disc of OCP/Col)
In one embodiment, the method of bone regeneration or bone augmentation preferably comprises:
step (I) of implanting a porous composite containing calcium phosphate at a site in need of bone regeneration or bone augmentation; and
step (II) of administering parathyroid hormone (PTH) to a subject in need of bone regeneration or bone augmentation. Either step (I) or step (II) may be previously performed, or both steps may be performed at the same time. In the method of bone regeneration or bone augmentation according to one embodiment, step (II) is preferably performed after step (I). For example, PTH can be administered after the porous composite is implanted at a site in need of bone regeneration or bone augmentation. In the method of bone regeneration or bone augmentation according to another embodiment, step (I) is preferably performed after step (II). For example, PTH can be administered (e.g., by dropping) to a site in need of bone regeneration or bone augmentation, and then the porous composite can be implanted at the site. In another preferable embodiment, PTH is added to the porous composite beforehand.
PTH is generally called parathormone, parathyroid hormone, or parathyroid gland hormone, and is a human-derived polypeptide hormone comprising 84 amino acids secreted from the parathyroid (parathyroid gland). In this specification, the PTH includes not only wild-type PTH, but also derivatives having the same physiological function as wild-type PTH and pharmacologically acceptable salts thereof. Examples of such derivatives include teriparatide corresponding to a 34-amino-acid region of the N-terminal of wild-type PTH. Teriparatide is sold as a therapeutic agent for osteoporosis under the name of Forteo or Teribone, and has the amino acid sequence of SEQ ID NO: 1. In one embodiment, PTH is preferably teriparatide. The teriparatide mentioned herein also includes a pharmacologically acceptable salt thereof.
It is preferable that the porous composite contains calcium phosphate. Preferred calcium phosphate has a bone regeneration or bone augmentation effect. Examples of calcium phosphate include octacalcium phosphate (OCP), β-tricalcium phosphate (β-TCP), hydroxyapatite (HAP), amorphous calcium phosphate, dibasic calcium phosphate anhydrous (DCPA), and dibasic calcium phosphate dihydrate (DCPD). In one embodiment, the calcium phosphate is preferably at least one member selected from the group consisting of OCP, β-TCP, and HAP, and more preferably OCP. These calcium phosphates can be prepared by any method, or commercial products can be purchased for use.
OCP can be prepared by various known methods. For example, OCP can be prepared by a drop method (LeGeros R Z, Calcif Tissue Int 37:194-197, 1985) or the method using a synthesizing apparatus (three-way pipe) disclosed in PTL 6. Alternatively, OCP can be prepared by a mixing method in which a sodium dihydrogen phosphate aqueous solution and a calcium acetate aqueous solution are mixed under appropriate conditions, and the produced precipitate is collected. The OCP obtained from the precipitate is preferably used after it is dried and ground with an electric mill, etc., into a granular powder. The particle size is preferably in the range of 10 to 1,000 μm, more preferably 100 to 500 μm, and even more preferably 300 to 500 μm. The particle size can be classified, for example, by sieving based on the opening size of the sieve.
β-TCP can be prepared by various known methods. For example, the following method can be used. First, a calcium hydroxide powder and a calcium hydrogen phosphate powder are mixed so that the molar ratio of calcium to phosphorus (Ca/P) is 1.45 to 1.72 to prepare a raw material powder. The obtained raw material powder is mixed and ground, and a calcium phosphate precursor is prepared by soft mechanochemical synthesis. The resulting precursor is heated at a temperature of 600° C. or more, thereby obtaining β-TCP. There is another method in which a mixed slurry containing calcium hydrogen phosphate and calcium carbonate is ground and reacted in a pot mill for 24 hours, and the slurry is dried, followed by firing at 750° C. for 1 hour, thereby obtaining a β-TCP powder.
HAP can be prepared by various known methods. For example, a hydrothermal synthesis method, a dry synthesis method, or a wet synthesis method can be used. The hydrothermal synthesis method is as follows. The pH of an aqueous suspension of dibasic calcium phosphate anhydrous (CaHPO4) is adjusted to 4 using phosphoric acid (H3PO4). Then, the suspension is reacted in a hydrothermal synthesis apparatus at 350° C. at 8,800 Ib/in2 for 48 hours, thereby obtaining HAP. The dry synthesis method is as follows. HAP can be obtained by reacting calcium pyrophosphate (Ca2P2O7) or tricalcium phosphate (Ca3(PO4)2), which are poorly-soluble calcium phosphates, with calcium carbonate (CaCO3) in a steam atmosphere at 900 to 1,300° C. for 1 to 24 hours. The wet synthesis method is as follows. HAP can be obtained by reacting an aqueous solution of soluble phosphate or phosphoric acid, and an aqueous solution of soluble calcium salt at 70 to 100° C. for 24 to 48 hours while maintaining alkalinity. In another wet synthesis method, HAP can be obtained by suspending a water-insoluble calcium phosphate salt in water, adding water-insoluble calcium carbonate (CaCO3) to the suspension, and reacting the suspension by heating while stirring. In still another wet synthesis method, HAP can be obtained by mixing a calcium carbonate powder and a powder of calcium hydrogen phosphate or a dihydrate thereof so that the atomic ratio of calcium atoms to phosphoric acid is within the range of 1.5 to 1.67, and grinding and mixing the mixture using a wet grinding mill.
The material of the porous composite is any material and is not particularly limited, as long as it is suitable for bone regeneration or bone augmentation. For example, the porous composite may be calcium phosphate formed in a porous state. In one embodiment, when the porous composite is formed of calcium phosphate, the calcium phosphate is preferably β-TCP or HAP. In one embodiment, the porous composite is preferably formed of collagen and calcium phosphate.
The source, properties, etc., of the collagen constituting the porous composite are not particularly limited, as long as it is suitable as a material for bone regeneration or bone augmentation, and any collagen can be used. In one embodiment, the collagen is preferably enzyme-soluble collagen obtained by solubilizing collagen with protease (e.g., pepsin or pronase) to remove telopeptide. Such collagen is less allergenic. Moreover, the collagen is preferably fibrous collagen, i.e., type-I, type-II, type-III, or type-IV collagen. In one embodiment, the collagen is preferably type-I collagen, which is contained in a large amount in a living organism, or a mixture of type-I collagen and type-III collagen. Examples of the raw material of collagen include skin, bone, or tendon of pigs or cows, and scales of fish. Collagen is an organism-derived component, and is thus highly safe. Commercially available collagen can also be used.
When the porous composite is formed from collagen, the proportion of calcium phosphate and collagen can be suitably adjusted in consideration of the desired shape-retaining properties, operability, biocompatibility, etc. For example, the mixing ratio of calcium phosphate to 1 part by weight of collagen is 0.5 to 35 parts by weight, preferably 1 to 20 parts by weight, and more preferably 2 to 10 parts by weight. If the amount of calcium phosphate is less than 0.5 parts by weight per part by weight of collagen, the obtained composite may have an inferior bone regeneration function. If the amount of calcium phosphate is more than 35 parts by weight, shape-retaining properties may be reduced. The calcium phosphate used herein is preferably OCP.
In one embodiment, the porous composite preferably has a pore size of 3 to 90 μm. If the pore size exceeds 90 μm, the strength of the porous composite tends to decrease. If the pore size is less than 3 μm, bone metabolism cells, such as osteoblastic cells, are less likely to pass through the pores, and the effect of promoting bone regeneration may be reduced. The pore size of the porous composite is more preferably 5 to 40 μm, and even more preferably 7 to 36 μm.
The pore size of the porous composite is determined by pore distribution measurement with a mercury porosimeter. The specific measurement method is as follows.
As a pretreatment, samples (porous composites) are dried at a constant temperature of 120° C. for 4 hours. Each of the pretreated samples is measured for the distribution of the pore size (0.0018 to 200 μm) by a mercury penetration method using the following measuring apparatus under the following conditions: Measuring apparatus: AutoPore IV9520 (produced by Micromeritics) Measurement condition: contact angle between mercury and a sample: 140 deg
Surface tension of mercury: 0.48 N/m (converted based on 1 dyne=10−5 N)
In the pore distribution curve obtained by the mercury penetration method, the value of the pore size (diameter) showing the maximum peak having the largest area is the value of the pore size in this specification.
In one embodiment, in the pore distribution measured by the mercury penetration method, the proportion of pores of a size of 71 to 200 μm in all of the pores of a size of 200 μm or less is preferably 8% or less, and more preferably 3 to 8%. The pore ratio is represented by the following formula using the cumulative pore volume and entire pore volume measured by the mercury porosimeter:
Pore ratio (%)=cumulative pore volume/entire pore volume×100
The porosity (void ratio) of the porous composite is preferably 80 to 98%, more preferably 85 to 95%, and still more preferably 85 to 90%. The porosity is determined by the following formula using the entire pore volume and apparent density measured by the mercury penetration method:
Porosity (%)=entire pore volume/{(1/apparent density)+entire pore volume}×100
The shape of the porous composite can be freely determined in consideration of the shape, size, etc., of the affected part in which the porous composite is placed. For example, the porous composite is preferably in the form of a rectangular parallelepiped (block), a cube, a cylinder, tablets, or granules. In one embodiment, the size of the porous composite having a rectangular parallelepiped shape is preferably 5 mm×5 mm×5 mm or more. In general, the upper limit is preferably 100 mm×100 mm×100 mm. When the porous composite has a cylindrical shape, the size thereof is preferably such that the diameter is 5 to 50 mm, and the height is in the range of 1 to 50 mm. When the porous composite has a granular shape, the shape is not limited to spherical, and may be amorphous; however, the diameter is preferably 0.1 to 10 mm. The diameter of the granular porous composite is determined by a sieve analysis method.
The manner of implanting the porous composite is any manner, and is not particularly limited. For example, the porous composite is implanted by filling a site in need of bone regeneration or bone augmentation with the porous composite of a suitable size. When sufficient blood or body fluid is present in the site in need of bone regeneration or bone augmentation (e.g., bone defect site), the porous composite can be filled therein as it is or after being cut into a suitable shape. When sufficient blood, etc., is not observed in the site, or when the porous composite in the original shape cannot be filled in the site, the porous composite can be filled in the bone defect site after the porous composite is immersed in blood or physiological saline, and confirmed to have sponge-like elasticity.
The manner of administering PTH is any manner, and is not particularly limited. For example, PTH is administered topically or systemically by any method. In one embodiment, PTH is preferably administered topically to a site in need of bone regeneration or bone augmentation (e.g., bone defect site). Topical administration can be performed, for example, by dropping a solution containing PTH on the site. When PTH is topically administered after the porous composite is implanted, for example, PTH can be administered on (or into) the porous composite implanted in a site in need of bone regeneration or bone augmentation. PTH can be topically administered to the site, for example, by surgically exposing the site during bone regeneration or bone augmentation surgery, and dropping a solution containing PTH thereon. Alternatively, PTH can be topically administered to the site by inserting an injection needle into the site, and injecting a solution containing PTH. In one embodiment, PTH is administered systemically by injection. For example, PTH is administered by subcutaneous injection, intramuscular injection, or intravenous injection. In one embodiment, PTH is preferably administered systemically by subcutaneous injection.
The amount of PTH administered is freely determined and is not particularly limited. For example, the amount of PTH can be suitably determined depending on the degree of bone defect, the body weight and age of the subject, etc. When a liquid containing PTH is dropped on a site in need of bone regeneration or bone augmentation, the concentration of PTH in the liquid is 0.01 μg/mL to 500 μg/mL, for example. Moreover, the amount of the PTH-containing solution dropped on the site is 0.01 mL to 500 mL, for example. The PTH-containing solution can be prepared, for example, by adding a commercially available PTH (e.g., teriparatide) to physiological saline. When PTH is administered systemically by injection, its dosage can be set as 0.01 μg/kg/day to 50 μg/kg/day, for example. The PTH administration may be performed once or multiple times. In one embodiment, when PTH is systemically administered, the PTA is preferably administered multiple times for a certain period of time. For example, the administration frequency is once a day, once a week, 2 times to 6 times a week, or 1 time to 3 times a month. The dosing period is, for example, one week, one month, two to eleven months, one year, one and a half year, or two to five years.
The site in need of bone regeneration includes a bone defect site, for example. Bone defects can be caused by various factors. Examples of factors of bone defects include bone fracture, post-traumatic bone loss, bone surgery, congenital bone loss, and post-infectious bone loss; bone deficit conditions associated with bone chemotherapy treatment, allograft incorporation, and bone radiotherapy treatment; periodontal disease; and the like. Examples of bone surgery include spinal fusion surgery, arthrodesis including extremity arthrodesis, etc., post-traumatic bone surgery, post-prosthetic joint surgery, post-plastic bone surgery, dental surgery, and the like. In one embodiment, examples of bone defect sites include a cystic cavity, an atrophic alveolar ridge, a cleft jaw, a tooth-extraction cavity, and the like.
The site in need of bone augmentation is not particularly limited. Examples thereof include sites in need of at least one treatment selected from the group consisting of sinus lift, bone graft, and ridge expansion. In one embodiment, the site in need of bone augmentation can be a site that shows a pathological reduction in bone mineral content associated with osteoporosis, spondylosis deformans, rheumatoid arthritis, malignant tumors, trauma, etc. In another embodiment, examples of the site in need of bone augmentation include oral surgery pathological conditions associated with dysphemia, dysmasesis, and/or aesthetic defects.
Examples of the subject include, but are not particularly limited to, humans, dogs, cats, monkeys, rats, and like animals. In one embodiment, the subject is preferably a human.
In one embodiment, the porous composite preferably contains calcium phosphate and PTH. In other words, in one embodiment, PTH is preferably administered in a state of being incorporated in the porous composite. The porous composite containing PTH can be produced by any method. For example, a PTH-containing porous composite can be obtained by preparing a porous composite that does not contain PTH, and dropping a PTH-containing solution thereon, or by immersing a porous composite that does not contain PTH in a PTH-containing solution. Such a PTH-containing porous composite may be prepared immediately before the implantation of the porous composite, or may be prepared beforehand and stored until use. In an embodiment, the calcium phosphate in the porous composite containing calcium phosphate and PTH is preferably OCP.
In one embodiment, the porous composite containing calcium phosphate and PTH preferably has a moisture content of 5 wt. % or less, 4 wt. % or less, 3.5 wt. % or less, or 3 wt. % or less. As used herein, a moisture content of 5 wt. % or less means that when a porous composite containing 1 g of PTH is dried at 105° C. for 3 hours under reduced pressure at 2.0 kPa or less, the composite exhibits a loss in weight on drying of 50 mg or less (i.e., the value measured by the loss-on-drying test). Such a PTH-containing porous composite having a low moisture content is preferable from the standpoint of ease in handling and storage stability.
A porous composite that contains calcium phosphate and PTH and that has a moisture content of 5 wt. % or less can be obtained by any method. For example, a calcium phosphate-containing porous composite is impregnated with a PTH-containing solution, and then the composite is dried to prepare a porous composite having a low moisture content. Many known drying methods are available for use. For example, constant-temperature drying, infrared-ray drying, blow drying, hot-air drying, reduced-pressure drying, vacuum drying, suction drying, spin drying, and/or freeze drying may be used. In one embodiment, the drying method is preferably freeze drying. The conditions for freeze drying are not particularly limited, and may be suitably determined depending on the size, shape, etc. of the sample. For example, a porous composite is frozen at −20° C. to −90° C., and then dried with a freeze dryer at −25 to −40° C. for 12 to 72 hours. The temperature for freeze drying may be constant, or increased stepwise. After freeze drying, the porous composite is preferably sterilized with electron-beam irradiation.
The amount of PTH contained in the porous composite is any amount, and is not particularly limited. For example, the porous composite can contain 0.1 ng/g to 25 mg/g of PTH, preferably 1 ng/g to 1 mg/g, and more preferably 5 ng/g to 100 μg/g.
In one embodiment, a kit for bone regeneration or bone augmentation comprising the porous composite described above and PTH is provided. The kit may comprise any of the above-described porous composites. In addition to the porous composite and PTH, the kit may further comprise any components, reagents, instructions, etc.
The porous composite can be produced by any method. For example, the porous composite can be produced by the following methods (a) to (c). The following methods are described using OCP for calcium phosphate; however, when other calcium phosphate is used, a porous composite can also be obtained in the same manner. When β-TCP is used for calcium phosphate, method (a) or (b) is preferred.
OCP is added to a collagen solution whose concentration, pH, etc., are adjusted to a range in which gelation can occur, and they are sufficiently kneaded to prepare a mixture of OCP and collagen. Then, the mixture is molded in a suitable mold, frozen, and freeze-dried to obtain a composite. The obtained composite is preferably subjected to heat dehydration crosslinking treatment, and sterilized by a conventional sterilization method (e.g., γ-ray irradiation, electron beam irradiation, or ethylene oxide gas).
The pH of a collagen acidic solution with a suitable concentration is aseptically adjusted to 5.5 to 7.5 using a suitable buffer (e.g., phosphate buffer, tris buffer, or sodium acetate buffer). OCP is added thereto before the collagen is gelled, thereby preparing a suspension of collagen and OCP. Thereafter, the suspension is poured into a mold while maintaining a neutral to weakly alkaline pH. After molding, the suspension is gelled at a suitable temperature (e.g., 37° C.), and water washing is repeated to remove salts, such as of buffers. A composite can be thus obtained. Thereafter, the composite is preferably subjected to freeze drying and sterilization, as in method (a) described above.
The pH of a collagen acidic solution with a suitable concentration is aseptically adjusted to 5.5 to 7.5 using a suitable buffer (e.g., phosphate buffer, tris buffer, or sodium acetate buffer). A calcium solution and a phosphoric acid solution are added thereto before the collagen is gelled, thereby depositing the OCP on the collagen. Subsequently, while maintaining a neutral to slightly alkaline pH, the deposited OCP is poured into a mold, and molded. Gelation is performed at a suitable temperature (e.g., 37° C.), and water washing is repeated to remove salts, such as of buffers. A composite can be thus obtained. Thereafter, the composite is preferably subjected to freeze drying and sterilization, as in method (a) described above.
The deposition of OCP is based on the concentrations of Ca2+ and PO43−, and the degree of supersaturation (ionic product/solubility product) determined by pH, etc. Therefore, the OCP can be deposited by pouring a Ca2+ solution and a PO43− solution into a collagen solution whose pH is adjusted, under conditions in which OCP is oversaturated. The OCP is spontaneously deposited in the collagen gap, or deposited using the surface of the collagen fiber as the core.
In one embodiment, the method of producing the porous composite preferably comprises the step of freeze-drying after a gel, sol, or liquid containing OCP and collagen is frozen by immersion in a liquid refrigerant. The phrase “a gel, sol, or liquid containing OCP and collagen is frozen by immersion in a liquid refrigerant” includes, for example, an embodiment in which a container containing the gel, sol, or liquid is sealed, and then immersed in a liquid refrigerant to freeze the gel, sol, or liquid.
The liquid refrigerant is a liquid having a temperature lower than the freezing point of the gel, sol, or liquid containing OCP and collagen. Examples include methanol, ethanol, acetone, acetonitrile, liquid nitrogen, and the like. The temperature of the liquid refrigerant is preferably −20° C. or less, more preferably −40° C. or less, and even more preferably −80° C. or less. It is considered that the pore size of the porous composite to be obtained can be reduced by rapidly freezing the gel, sol, or liquid containing OCP and collagen by immersion in the liquid refrigerant.
In one embodiment, the porous composite is preferably subjected to heat treatment. Due to the heat treatment, part of the OCP molecular structure is broken, and bone-forming cells are more likely to enter the structure. Thus, bone regeneration is promoted, and the collagen is crosslinked to improve shape-retaining properties.
The temperature of heat treatment is preferably 50 to 200° C., and more preferably 60 to 180° C. Moreover, the heat treatment is preferably performed at reduced pressure. The pressure is preferably 0 to 3,000 Pa, and more preferably 0 to 300 Pa. The time of heat treatment is preferably 0.1 to 10 days, and more preferably 0.5 to 5 days.
The porous composite using HAP can be obtained, for example, by the following method. First, a phosphoric acid (salt) aqueous solution and a calcium salt aqueous solution or suspension are prepared. A collagen/phosphate aqueous solution is prepared using collagen, and phosphoric acid or phosphate, such as disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, or potassium dihydrogen phosphate. A calcium salt aqueous solution or suspension is prepared using calcium carbonate, calcium acetate, calcium hydroxide, or the like. Water in an amount almost the same as the amount of the calcium salt aqueous solution or suspension to be added is previously placed in a reaction vessel, and heated to about 40° C. The collagen/phosphate aqueous solution and the calcium salt aqueous solution or suspension are simultaneously dropped thereon at room temperature, thereby forming a fibrous HAP/collagen composite. After completion of dropping, the mixture of the water and HAP/collagen composite in the form of slurry is freeze-dried. Freeze drying is carried out by performing vacuum suction in a frozen state at −10° C. or lower, and performing rapid drying. Thereafter, a crosslinking treatment is performed to thereby obtain a porous composite containing HAP and collagen.
In one embodiment, the porous composite may contain any components other than the above-mentioned components. Examples of such components include bioabsorbable polymers (polyglycolic acid, polylactic acid, polylactic acid-polyethylene glycol copolymers, etc.).
The porous composites described above can be used as a bone regeneration material. Such bone regeneration materials are useful for bone defect recovery in the dental oral surgery field and orthopedic surgery field, and for bone defect recovery after craniotomy or thoracotomy. For example, in the dental oral surgery field, when a bone defect caused by periodontal disease, a cystic cavity, an atrophic alveolar ridge, a cleft jaw, a tooth-extraction cavity, or the like is filled with a bone regeneration material containing the porous composite, an excellent bone regeneration effect can be confirmed after several weeks to several months. In the orthopedic surgery field, for example, in the case of a bone defect after resection of a bone tumor or a bone defect caused by trauma, such as a bone fracture, the bone defect site can be filled with the bone regeneration material to thereby promote bone regeneration.
The present invention is described in more detail below with reference to Examples; however, the present invention is not limited thereto.
Liquid 1 and liquid 2 for preparation of OCP were prepared in the following manner. Sodium dihydrogen phosphate dihydrate (31.2 g) was dissolved in 2,500 g of distilled water to prepare liquid 1. Calcium acetate monohydrate (35.2 g) was dissolved in 2,500 g of distilled water to prepare liquid 2. Liquid 1 was placed in a separable flask, and heated to 70° C. using a mantle heater. While stirring liquid 1 at a rate of 250 rpm using a stirrer (MAZELA Z, produced by Tokyo Rikakikai Co., Ltd.) to which a stirring blade (blade diameter: 12 cm) was attached, liquid 2 was dropped at a rate of about 28 mL/min. After completion of dropping, the mixture of liquid 1 and liquid 2 was further stirred at 70° C. at 250 rpm for 2 hours.
The precipitate produced in the above mixture was filtered through a membrane filter (pore size: 3 μm; A300A293C, produced by Advantec Toyo Kaisha, Ltd.), and collected. The collected precipitate was dispersed in 1,500 mL of distilled water, and washed by stirring for 15 minutes. The process of filtering and washing was further repeated 3 times. The washed precipitate was dried in a constant temperature dryer at 30° C. for 24 hours. After the dried precipitate was ground with an electric mill, the ground product was classified using a sieve to a particle size of 300 to 500 μm. Thus, a powder was obtained. Finally, the obtained powder was subjected to dry sterilization at 120° C. for 2 hours, thereby obtaining OCP.
Pig dermis-derived collagen containing type-I collagen and type-III collagen (NMP Collagen PS, produced by NH Foods Ltd.; 1 part by weight) was dissolved in 200 parts by weight of distilled water that was cooled to 4° C., thereby obtaining an about 0.5 wt. % collagen solution. While maintaining the solution temperature at 4° C., a sodium hydroxide aqueous solution was added to the collagen aqueous solution, and the pH was adjusted to about 7.4 to obtain a collagen suspension. The ionic strength of the collagen suspension at this time was about 0.01. The OCP (particle size: 300 to 500 μm) obtained in Production Example 1 was added to the collagen suspension so that the weight ratio of OCP to collagen was 77:23. The mixture was stirred at room temperature, thereby obtaining an OCP/collagen suspension.
The OCP/collagen suspension was placed in a centrifuge bottle, and centrifuged by a centrifugal separator (GRX-250, produced by Tomy Seiko Co., Ltd.) at a centrifugal force of 7,000×g for 20 minutes. After the supernatant was discarded so that the amount of collagen in the OCP/collagen suspension was 3 wt. %, the content was mixed with a drug spoon for about 2 minutes to obtain an OCP/collagen composite gel. The gel was placed in a plastic container (inner diameter: 9.0 mm, volume: about 3.0 cm3) having a cylindrical inner space, and centrifuged at a centrifugal force of 230×g for 1 minute, followed by defoaming.
The sealed plastic container was immersed in methanol cooled to −80° C. in a large excess amount relative to the volume of the container for rapid freezing. After the container was opened, the frozen product was dried with a freeze dryer (−10° C., 48 hours) and molded. Subsequently, the molded product was heated at 150° C. for 24 hours at reduced pressure to perform thermal-dehydration crosslinking. The resulting product was cut into a disc shape with a thickness of 1.0 mm (used in Test Example 1) or 1.5 mm (used in Test Example 2, Test Example 4, and Production Example 7) with a scalpel. The cut pieces were sterilized by electron-beam irradiation to obtain OCP/col porous composites (OCP/Col).
A β-TCP/Col porous composite (β-TCP/Col) was obtained in the same manner as in Production Example 2, except that β-TCP (OSferion, produced by Olympus Terumo Biomaterials Corp.) granulated to a diameter of 300 to 500 μm was used in place of the OCP.
Physiological saline (1.13 mL) was added to 56.5 μg of a subcutaneous injection formulation of Teribone (produced by Asahi Kasei Pharma Corporation) to obtain a 50 μg/mL teriparatide (PTH) solution. The teriparatide solution (20 μL) was taken in a PCR tube (0.5 mL), and 80 μL of physiological saline was added thereto to obtain a 1.0 μg/0.1 mL (10 μg/mL) teriparatide solution. This solution was further diluted 10 times with physiological saline to obtain a 0.1 μg/0.1 mL (1.0 μg/mL) teriparatide solution. These were stored in a freezer at −20° C. until use.
Physiological saline (1.13 mL) was added to 56.5 μg of a subcutaneous injection formulation of Teribone (produced by Asahi Kasei Pharma Corporation) to obtain a 50 μg/mL teriparatide solution. The teriparatide solution (70 μL) was taken in a PCR tube (0.5 mL), and 280 μL of physiological saline was added thereto to obtain a 3.5 μg/0.35 mL (10 μg/mL) teriparatide solution. These were stored in a freezer at −20° C. until use.
Freeze-dried recombinant human Fibroblast growth factor-2 (FGF-2, produced by R & D Systems) was dissolved in a 0.01 M phosphate buffer (containing 0.1% or more bovine serum albumin and 1 mM DTT (dithiothreitol)). FGF-2 was added to 15 mg of dry-sterilized OCP granules (diameter: 300 to 500 μm) at three different concentrations (10 ng, 100 ng, and 1 μg). After immersion at −20° C. for 20 minutes, the resultant was freeze-dried to prepare OCP/FGF-2 granules. As a control sample, OCP granules to which FGF-2 was not added were used.
1.0 mL of injectable water was added to 56.5 μg of a subcutaneous injection formulation of Teribone (produced by Asahi Kasei Pharma Corporation) to obtain a 56.5 μg/mL teriparatide (PTH) solution. OCP/Col prepared in Production Example 2 was arranged in a 48-well plate, one disc for each well, and 17.7 μL of a PTH solution was added per well. These were frozen at −78° C. in a deep freezer, and then the frozen product was dried with a freeze dryer (−10° C., 24 hours). Finally, the dried product was sterilized with electron-beam irradiation, thereby obtaining a composite containing 1.0 μg of PTH per disc of OCP/Col (OCP/Col/PTH 1.0 (dry)). The same procedure was repeated using a solution prepared by diluting a 56.5 μg/mL PTH solution with injectable water 10 times, thereby obtaining a composite containing 0.1 μg of PTH per disc of OCP/Col (OCP/Col/PTH 0.1 (dry)). Additionally, the dilution rate of the PTH solution was changed, thereby preparing a composite containing 0.01 μg of PTH per disc of OCP/Col (OCP/Col/PTH 0.01(dry)) and a composite containing 0.001 μg of PTH per disc of OCP/Col (OCP/Col/PTH 0.001 (dry)).
The procedure of Production Example 7 was repeated, except that β-TCP/Col was replaced with OCP/Col, thereby preparing a composite containing 1.0 μg of PTH per disc of β-TCP/Col (β-TCP/Col/PTH 1.0 (dry)) and a composite containing 0.1 μg of PTH per disc of β-TCP/Col (β-TCP/Col/PTH 0.1 (dry)).
Male Wistar rats (12 weeks old) were each intraperitoneally anesthetized, and a skin incision and a periosteal incision were made in the calvarium part to expose the calvarium. A standardized full-thickness bone defect (diameter: about 9 mm), for which spontaneous recovery could not be expected, was created. Then, one disc-shaped OCP/Col produced in Production Example 2 or one disc-shaped β-TCP/Col produced in Production Example 3 was implanted therein. As the control, rats in which only a bone defect was created, and no sample was implanted (untreated group) were prepared. After the sample was implanted, the periosteum and skin were sutured, and the surgery was completed. The OCP/Col group, the β-TCP/Col group, and the untreated group were each divided into a teriparatide subcutaneous injection group (subcutaneous injection group) and a group that did not undergo teriparatide subcutaneous injection (non-injection group); they were divided into 6 groups in total. In the rats of the teriparatide subcutaneous injection group, 10 μg/kg of the teriparatide prepared in Production Example 5 was subcutaneously injected immediately after surgery and every day thereafter. Subcutaneous injection was performed in such a manner that the thawed teriparatide solution was sucked by a 1-mL syringe (27G injection needle), the back skin was sterilized with a dilute Hibitane solution, and then a considerable amount of teriparatide solution was injected subcutaneously in the back. The observation period after surgery was 8 weeks for each group, and 5 rats were used in each period.
Four weeks and eight weeks after surgery, tomography was carried out on the rats in the living state using an X-ray CT scanner for experimental animals (Latheta LCT-200, produced by Hitachi Aloka Medical, Ltd.) at a low X-ray tube voltage, and the state of new bone formation in the bone defect site was carefully examined. Similarly, after CT photography 8 weeks after surgery, the rats were euthanized under anesthesia with an excess amount of pentobarbital. The calvarium and surrounding tissue were taken, and immersed and fixed in 0.1 M phosphate-buffered 4% paraformaldehyde (pH 7.4). The extracted samples were photographed (20 kV, 5 mA) with a soft X-ray camera (CMBW-2, produced by Softex Co., Ltd.), and then decalcified with 10% EDTA to prepare paraffin samples. Then, tissue sections (thickness: 6 μm) cut in the frontal plane were prepared, and a histological search was performed by hematoxylin-eosin staining. Further, using the multiple tissue sections corresponding to the center of the defect of each sample, the ratio of new bone (n-Bone %) in the created defect was examined by histological quantification. Variance analysis, etc., were conducted using the average value and standard deviation of n-Bone % in each group, and a significance test was conducted. The significance level was set at 5%.
Using the multiple tissue sections corresponding to the center of the defect of each sample, the ratio of new bone (n-Bone %) in the defect was examined by histological quantification (
As described above, the results of
Male Wistar rats (12 weeks old) were intraperitoneally anesthetized, and a skin incision and a periosteal incision were made in the calvarium part to expose the calvarium, thereby creating a standardized full-thickness bone defect (diameter: about 9 mm), for which spontaneous recovery was not expected. Then, one disc-shaped OCP/Col produced in Production Example 2 or one disc-shaped β-TCP/Col produced in Production Example 3 was implanted therein. In this case, the teriparatide solution (1.0 μg/0.1 mL or 0.1 μg/0.1 mL) prepared in Production Example 4 was dropped on the OCP/Col in an amount of 0.1 mL per disc, and the same teriparatide solution (1.0 μg/0.1 mL) was dropped on the β-TCP/Col in an amount of 0.1 mL per disc. As the control, a group for which the teriparatide solution was not dropped on the OCP/Col or β-TCP/Col was prepared. After the sample was implanted, the periosteum and skin were sutured, and the surgery was completed. The observation period after surgery was 12 weeks for each group, and 6 rats were used in each period. The same analysis as in Test Example 1 was conducted 4 weeks and 12 weeks after surgery.
As described above, the results shown in
Male Wistar rats (12 weeks old) were each intraperitoneally anesthetized, and a skin incision and a periosteal incision were made in the rat calvarium part to expose the calvarium, thereby creating a standardized full-thickness bone defect (diameter: about 9 mm), for which spontaneous recovery was not expected. Then, the OCP/FGF-2 granules (15 mg) or OCP granules (15 mg) obtained in Production Example 6 were implanted therein. After the sample was implanted, the periosteum and skin were sutured, and the surgery was completed. Each group consisted of 4 or 5 rats, and was evaluated 4 weeks after surgery. Further, in the OCP/FGF-2 (100 ng) administration group and the OCP administration group (5 rats in each group), bone regeneration ability was examined 8 weeks after surgery.
After the observation period was over, the rats were euthanized under anesthesia with an excess amount of pentobarbital. The calvarium and surrounding tissue were taken, and immersed and fixed in 0.1 M phosphate-buffered 4% paraformaldehyde (pH 7.4). The extracted samples were photographed (20 kV, 5 mA) with a soft X-ray camera (CMBW-2, produced by Softex Co., Ltd.), and then decalcified with 10% EDTA to prepare paraffin samples. Then, tissue sections (thickness: 6 μm) cut in the frontal plane were prepared, and a histological search was performed by hematoxylin-eosin staining. Further, using the multiple tissue sections corresponding to the center of the defect of each sample, the ratio of new bone (n-Bone %) in the created defect was examined by histological quantification. Variance analysis, etc., were conducted using the average value and standard deviation of n-Bone % in each group, and a significance test was conducted. The significance level was set at 5%.
In the obtained soft X-ray photographs (
Male Wistar rats (12 weeks old) were divided into the following 6 groups (5 rats for each group).
OCP/Col/PTH 1.0 (dry) and OCP/Col/PTH 0.1 (dry) were those prepared in Production Example 7. β-TCP/Col/PTH 1.0 (dry) and β-TCP/Col/PTH 0.1 (dry) were those prepared in Production Example 8. OCP/Col was the one prepared in Production Example 2. β-TCP/Col was the one prepared in Production Example 3.
The rats were intraperitoneally anesthetized, and a skin incision and a periosteal incision were made in the calvarium part to expose the calvarium, thereby creating a standardized full-thickness bone defect (diameter: about 9 mm), for which spontaneous recovery was not expected. One of the implantation samples shown in Table 1 was implanted in the bone defect. Thereafter, the periosteum and skin were sutured, and the surgery was completed. The observation period after surgery was 12 weeks for all groups.
Four weeks and twelve weeks after surgery, tomography was carried out on the rats while alive, using an X-ray CT scanner for experimental animals (Latheta LCT-200 produced by Hitachi Aloka Medical, Ltd.) at a low X-ray tube voltage, and the state of new bone formation in the bone defect site was carefully examined. In the same manner, CT photography was performed 12 weeks after surgery, and the rats were euthanized under anesthesia with an excess amount of pentobarbital. The calvarium and surrounding tissue were taken, and immersed and fixed in 0.1 M phosphate-buffered 4% paraformaldehyde (pH 7.4). The extracted samples were photographed (20 kV, 5 mA) with a soft X-ray camera (CMBW-2, produced by Softex Co., Ltd.), and then decalcified with 10% EDTA to prepare paraffin samples. Then, tissue sections (thickness: 6 μM) cut in the frontal plane were prepared, and a histological search was performed by hematoxylin-eosin staining. Further, using multiple tissue sections corresponding to the center of the defect of each sample, the ratio of new bone (n-Bone %) in the created defect was examined by histological quantification. Statistical analysis was conducted using the average value and standard deviation of n-Bone % in each group, and a significance test was conducted. The significance level was set at 5%.
Table 2 shows the ratio of new bone for each group. As seen in Table 2, group A and group B were confirmed to have a significantly higher ratio of new bone than all other groups.
In accordance with Test Example 4, male Wistar rats (12 weeks old) were divided into the following two groups.
OCP/Col/PTH 0.01 (dry) and OCP/Col/PTH 0.001 (dry) were those prepared in Production Example 7.
The rats were intraperitoneally anesthetized, and a skin incision and a periosteal incision were made in the calvarium part to expose the calvarium, thereby creating a standardized full-thickness bone defect (diameter: about 9 mm), for which spontaneous recovery was not expected. One of the implantation samples shown in Table 3 was then implanted into the defect of each rat. After the sample was implanted, the periosteum and skin were sutured, and the surgery was completed. The observation period after surgery was 12 weeks for each group
Four weeks and twelve weeks after surgery, tomography was carried out on the rats while alive, using an X-ray CT scanner for experimental animals (Latheta LCT-200 produced by Hitachi Aloka Medical, Ltd.) at a low X-ray tube voltage, and the state of new bone formation in the bone defect site was carefully examined. Twelve weeks after surgery, the rats were subjected to a computed tomography scan in the same manner.
From the results, a porous composite containing PTH and OCP was confirmed to enable significant bone regeneration, compared with a PTH-free, OCP-containing porous composite, a porous composite containing PTH and β-TCP, and a PTH-free, β-TCP-containing porous composite.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2016-157877 | Aug 2016 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2017/007046 | 2/24/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15094724 | Apr 2016 | US |
| Child | 16091632 | US |
