Bone graft material composition
阅读说明：本技术 骨移植材料组合物 (Bone graft material composition ) 是由 朴庭馥 于 2020-09-03 设计创作，主要内容包括：本发明涉及骨移植材料组合物,更加详细地,涉及包括羟丙基甲基纤维素(hydroxypropyl methylcellulose)的骨移植材料组合物及其制造方法。并且,涉及所述骨移植材料组合物具有羟丙基甲基纤维素的溶解率优异的最优的组成比的骨移植材料组合物。并且,涉及包括用于具有形状维持性的比率的羟丙基甲基纤维素的骨移植材料及其制造方法。并且,更加详细地,涉及包括用于具有最优的渗透性以及形状维持性的比率的羟丙基甲基纤维素的骨移植材料组合物及其制造方法。(The present invention relates to a bone graft composition, and more particularly, to a bone graft composition including hydroxypropylmethylcellulose (hydroxypropyl methylcellulose) and a method for producing the same. Also disclosed is a bone graft material composition having an optimal composition ratio that is excellent in the dissolution rate of hydroxypropylmethylcellulose. Also, the present invention relates to a bone graft material comprising hydroxypropylmethylcellulose for a ratio having shape-retaining property and a method for producing the same. More particularly, the present invention relates to a bone graft material composition including hydroxypropylmethylcellulose for a ratio having an optimal permeability and a shape-maintaining property, and a method for manufacturing the same.)
1. A bone graft material composition comprising:
porous bone graft material and hydroxypropyl methylcellulose,
wherein the content of the hydroxypropyl methylcellulose is 0.15 to 6 parts by weight with respect to 1 part by weight of the porous bone graft material.
2. A bone graft material composition comprising:
porous bone graft material and hydroxypropyl methylcellulose,
wherein the content of the hydroxypropyl methylcellulose is 0.2 to 5 parts by weight with respect to 1 part by weight of the porous bone graft material.
3. A bone graft material composition comprising:
porous bone graft material and hydroxypropyl methylcellulose,
wherein the content of the hydroxypropyl methylcellulose is 0.25 to 4 parts by weight with respect to 1 part by weight of the porous bone graft material.
4. A bone graft material composition comprising:
porous bone graft material and hydroxypropyl methylcellulose,
wherein the content of the hydroxypropyl methylcellulose is 0.3 to 3 parts by weight with respect to 1 part by weight of the porous bone graft material.
Technical Field
The present invention relates to a bone graft material composition having excellent Hydroxypropyl methylcellulose (Hydroxypropyl methylcellulose) dissolution rate and excellent shape retention, and a method for producing the same.
Background
For the reconstruction of defective bone, various materials and various means can be used, for example, bone graft materials such as bone powder, bone chips, bone blocks, etc. or methods such as autograft, allograft, xenograft, etc. for the reconstruction of defective bone can be used.
Bone graft materials used for reconstructing a defective bone can be used in orthopedics, neurosurgery, dentistry, and the like, and for example, they can be used for guiding bone regeneration in a bone defect portion in intervertebral disc treatment, dental implant (implant) treatment, repair of a frontal bone defect, and the like.
Further, korean patent laid-open No. 10-0401941 discloses a technique regarding a bone graft material and a method for manufacturing the same, and in the case of using a mesh bone composed of bioceramic powder and having a three-dimensionally connected interconnected pore structure as such, the bone graft effect may be limited in terms of biocompatibility, mechanical properties, toxicity, and the like.
Disclosure of Invention
The present invention aims to provide a bone graft material composition comprising hydroxypropylmethylcellulose (Hydroxypropyl methylcellulose) having a dissolution rate suitable for bone formation and having excellent shape-retaining properties, and a method for producing the same.
Further, the present invention relates to a bone graft material composition including hydroxypropylmethyl cellulose (Hydroxypropyl methylcellulose) suitable for bone formation, and particularly, it is desirable to provide a bone graft material composition and a composition of the bone graft material composition that forms an optimum osmotic pressure with other solutions.
One embodiment of the present invention includes a bone graft material and hydroxypropylmethylcellulose capable of forming a dissolution rate of 50% or more within 48 hours.
In one example of the present invention, the bone graft material composition may include 0.15 to 6 parts by weight, 0.2 to 5 parts by weight, 0.25 to 4 parts by weight, or 0.3 to 3 parts by weight of hydroxypropylmethylcellulose with respect to 1 part by weight of the bone graft material.
In one embodiment of the present invention, a bone graft material composition is provided, wherein the bone graft material is a natural bone graft material and comprises a porous structure.
One embodiment of the present invention provides a method for producing a bone graft material composition, comprising the steps of: (1) mixing a solvent and a bone morphogenetic protein to prepare a bone morphogenetic protein solution; (2) mixing the bone morphogenetic protein and the graft material powder, and adsorbing the bone morphogenetic protein to the graft material powder; (3) mixing and stirring the graft material powder having the bone morphogenetic protein adsorbed thereon and hydroxypropyl methylcellulose powder, and forming a gel so that the dissolution rate of the hydroxypropyl methylcellulose powder becomes 50% or more within 48 hours; and (4) freeze-drying the gel in a vacuum state to form a structure including a plurality of pores.
One embodiment of the present invention provides a method for producing a bone graft material composition, wherein the bone morphogenetic protein can be at least one selected from the group consisting of: BMP-2, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, recombinant bone morphogenetic proteins thereof, and bone morphogenetic proteins equivalent thereto.
One embodiment of the present invention provides a method for producing a bone graft material composition, wherein the concentration of the bone morphogenetic protein in the bone morphogenetic protein solution is 0.05 to 0.15 mg/ml.
One embodiment of the present invention provides a method for preparing a bone graft material composition, in which acidity is adjusted to a pH of 4.6 to 5 using phosphate buffer saline (phosphate buffer saline).
One embodiment of the present invention provides a method for producing a bone graft material composition, wherein the volume ratio of the graft material powder to which the bone morphogenetic protein is adsorbed in the step (3) to the hydroxypropyl methylcellulose powder is 1:0.2 to 1: 0.8.
One embodiment of the present invention provides a method for producing a bone graft material composition, further comprising a step of sterilizing the bone graft material composition by Ethylene Oxide Gas (Ethylene Oxide Gas) or gamma ray irradiation.
One embodiment of the present invention provides a method for manufacturing a bone graft material composition, wherein the concentration of the ethylene oxide gas is 450mg/l to 1200mg/l, or the irradiation dose of the gamma ray is 10kGy to 25 kGy.
An example of the present invention provides a bone graft material composition, wherein when a force that causes a shape change of a spherical object is a maximum breaking force, and a reduction ratio of a short axis after the shape change with respect to a diameter of the spherical object is a short axis change rate, a value obtained by dividing the maximum breaking force (Nmax) by the short axis change rate is a shape retentivity, and the shape retentivity is 50 or more.
Provided is a bone graft material composition, wherein 0.3 to 3 parts by weight of hydroxypropyl methylcellulose is mixed with respect to 1 part by weight of a bone graft material.
One embodiment of the present invention provides a bone graft material composition, wherein the porous bone graft material is a natural bone graft material and has excellent shape retention.
One embodiment of the present invention provides a method for producing a bone graft material composition having excellent shape retention properties, comprising the steps of: (1) mixing a solvent and a bone morphogenetic protein to prepare a bone morphogenetic protein solution; (2) mixing the bone morphogenetic protein and the graft material powder, and adsorbing the bone morphogenetic protein to the graft material powder; (3) mixing and stirring the bone graft material powder having the bone morphogenetic protein adsorbed thereon and a hydroxypropyl methylcellulose powder to form a gel, thereby imparting shape-retaining properties to the bone graft material composition; and (4) freeze-drying the gel in a vacuum state to form a structure including a plurality of pores.
One example of the present invention provides a method for producing a bone graft material composition having excellent shape-retaining properties, wherein the bone morphogenetic protein can be at least one selected from the group consisting of: BMP-2, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, recombinant bone morphogenetic proteins thereof, and bone morphogenetic proteins equivalent thereto.
One embodiment of the present invention provides a method for producing a bone graft material composition having excellent shape-retaining properties, wherein the concentration of the bone morphogenetic protein in the bone morphogenetic protein solution is 0.05 to 0.15 mg/ml.
One embodiment of the present invention provides a method for producing a bone graft material composition having excellent shape-retaining properties, in which the acidity is adjusted to a pH of 4.6 to 5 using phosphate buffer saline (phosphate buffer saline).
One embodiment of the present invention provides a method for producing a bone graft material composition having excellent shape-retaining properties, wherein the volume ratio of the graft material powder to which the bone morphogenetic protein is adsorbed in the step (3) to the hydroxypropyl methylcellulose powder is 1:0.2 to 1: 0.6.
One embodiment of the present invention provides a method for producing a bone graft material composition having excellent shape retention, further comprising a step of sterilizing the bone graft material composition by Ethylene Oxide Gas (Ethylene Oxide Gas) or gamma ray irradiation.
One embodiment of the present invention provides a method for manufacturing a bone graft material composition having excellent shape-retaining properties, wherein the concentration of the ethylene oxide gas is 450mg/l to 1200mg/l, or the irradiation amount of the gamma ray is 10kGy to 25 kGy.
One embodiment of the present invention provides a bone graft material composition, wherein the osmotic pressure of saline is standardized to 100%, and 104 to 112% of the osmotic pressure is formed within 12 to 48 hours after the saline is administered into a bone graft material lysate.
One embodiment of the present invention provides a bone graft material composition, wherein the bone graft material solution is formed by mixing 1 part by weight of a bone graft material including hydroxypropyl methylcellulose and 0.5 to 2 parts by weight of a solvent, and the bone graft material including hydroxypropyl methylcellulose is formed by mixing 0.3 to 3 parts by weight of hydroxypropyl methylcellulose with respect to 1 part by weight of the bone graft material.
In one embodiment of the present invention, a bone graft material composition is provided, wherein the bone graft material is a natural bone graft material.
One embodiment of the present invention provides a bone graft material composition, wherein the solvent is water.
One embodiment of the present invention provides a method for producing a bone graft material composition, comprising the steps of: (1) mixing a solvent and a bone morphogenetic protein to prepare a bone morphogenetic protein solution; (2) mixing the bone morphogenetic protein and the graft material powder, and adsorbing the bone morphogenetic protein to the graft material powder; (3) mixing and stirring a graft material powder having the bone morphogenetic protein adsorbed thereon and a hydroxypropyl methylcellulose powder to form a gel, thereby imparting osmotic pressure retention; and (4) freeze-drying the gel in a vacuum state to form a structure including a plurality of pores.
One embodiment of the present invention provides a method for producing a bone graft material composition, wherein the bone morphogenetic protein can be at least one selected from the group consisting of: BMP-2, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, recombinant bone morphogenetic proteins thereof, and bone morphogenetic proteins equivalent thereto.
One embodiment of the present invention provides a method for producing a bone graft material composition, wherein the concentration of the bone morphogenetic protein in the bone morphogenetic protein solution is 0.05 to 0.15 mg/ml.
One embodiment of the present invention provides a method for preparing a bone graft material composition, wherein the acidity is adjusted to a pH of 4.6 to 5 using phosphate buffer saline (phosphate buffer saline).
One embodiment of the present invention provides a method for producing a bone graft material composition, wherein the volume ratio of the graft material powder to which the bone morphogenetic protein is adsorbed in the step (3) to the hydroxypropyl methylcellulose powder is 1:0.2 to 1: 0.6.
One embodiment of the present invention provides a method for producing a bone graft material composition, further comprising a step of sterilizing the bone graft material composition by Ethylene Oxide Gas (Ethylene Oxide Gas) or gamma ray irradiation.
One embodiment of the present invention provides a method for manufacturing a bone graft material composition, wherein the concentration of the ethylene oxide gas is 450mg/l to 1200mg/l, or the irradiation dose of the gamma ray is 10kGy to 25 kGy.
An example of the present invention provides a bone graft material composition produced by the method for producing a bone graft material composition.
The bone graft material composition according to the present invention, which includes hydroxypropylmethylcellulose (Hydroxypropyl methylcellulose), has an excellent dissolution rate of hydroxypropylmethylcellulose (Hydroxypropyl methylcellulose) within a predetermined time, a strong rigidity and non-restorability, and an excellent shape-maintaining property, and forms an optimal osmotic pressure with other solutions, thereby having effects of bone formation activation, biocompatibility, and convenience in use.
Drawings
Fig. 1 is a view schematically illustrating a sequence of a method for manufacturing a bone graft material composition according to an embodiment of the present invention.
Fig. 2 is a graph showing the weight ratio and the residual rate with time according to hydroxypropylmethyl cellulose (Hydroxypropyl methylcellulose) reflecting the result data (table 1) regarding experimental example 1 of the present invention.
Fig. 3 is a graph showing the volume reduction rate according to the weight ratio of hydroxypropylmethylcellulose (Hydroxypropyl methylcellulose) reflecting the result data (table 2) on experimental example 2 of the present invention.
Fig. 4 is a graph showing "maximum force of failure (N) of bone graft material composition sphere (sphere)/short axis change rate of bone graft material composition sphere (sphere)" with respect to a weight ratio of hydroxypropylmethylcellulose (Hydroxypropyl methylcellulose), and is a graph showing a weight ratio of hydroxypropylmethylcellulose (Hydroxypropyl methylcellulose) for a bone graft material composition having excellent shape-maintaining properties.
Fig. 5 is data of results of experimental examples according to the present invention, and is a graph showing changes in osmotic pressure of a mixed saline solution to a pure saline solution according to time in an osmotic ratio after mixing a bone graft material lysate formed by changing parts by weight of a solvent and the saline solution.
Detailed Description
An example of an embodiment of the present invention relates to a bone graft material composition which can have excellent bone formation activation, biocompatibility, and convenience in use by including a bone graft material and hydroxypropylmethylcellulose (Hydroxypropyl methylcellulose).
However, for the sake of more clear and concise explanation, the description of the present embodiment will omit the overlapping portions with other embodiments, and the omission of the description does not mean that the portion is excluded from the present invention, and the scope of the right thereof should be recognized as the same as other embodiments.
In explaining the present invention, a detailed description thereof will be omitted in a case where a detailed description about a known technology related to the present invention is judged to possibly bring unnecessary confusion about the gist of the present invention. Also, the terms described later are terms defined in consideration of functions in the present invention, and may be different according to the intention of a user or a custom. Therefore, its definition should be determined based on the entire contents throughout the present specification.
The technical idea of the present invention is defined by the claims, and the following embodiments are only a means for effectively explaining the technical idea of the present invention to a person having ordinary knowledge in the technical field to which the present invention pertains.
In the present invention, in the case where an isomer exists in the repeating unit, compound or resin represented by the formula, the corresponding formula representing the repeating unit, compound or resin represents a representative formula including the isomer.
Specific examples of the present invention are explained below. However, it is merely an example, and the present invention is not limited thereto.
The bone graft material composition of the present invention includes a porous bone graft material and hydroxypropylmethylcellulose (Hydroxypropyl methylcellulose). The bone graft material composition can be transplanted to a bone defect and filled therein for repair of the bone defect.
Here, "filling" includes both a case of being applied to a bone defect portion in a state without rigidity and a case of being applied to a bone defect portion in a state with rigidity. The application of the bone graft material composition to the bone defect portion by imparting rigidity thereto may mean a case where the bone graft material composition is applied to the bone defect portion in a state of being manufactured into a shape corresponding to the bone defect portion by a shape forming apparatus (for example, a three-dimensional printer or the like) and having rigidity.
The bone graft material composition may have adhesiveness to a bone defect by including hydroxypropylmethylcellulose. Meanwhile, the bone graft material composition may be imparted with shape-maintaining properties by HPMC. In the case of excellent shape retention, the bone graft material composition can be applied to the palate without flowing down and matching the bone defect, and even if an impact due to masticatory movement or the like occurs, the bone graft material composition can be assisted not to come off the bone defect.
In the bone graft material composition according to an embodiment of the present invention, the composition ratio of the composition for optimizing the solubility of the hydroxypropylmethylcellulose is 0.1 to 6 parts by weight, more preferably, 0.3 to 3 parts by weight with respect to 1 part by weight of the porous bone graft material.
In the case where the content of the hydroxypropylmethylcellulose is less than 0.3 parts by weight with respect to 1 part by weight of the porous bone graft material, although the content of the hydroxypropylmethylcellulose is small and rapidly dissolved in a short time, since the amount of binding with the bone graft material is too small, it is easily precipitated, and there is a problem in that it is difficult to function as a bone graft material. Also, in the case where the content of the hydroxypropylmethylcellulose exceeds 3 parts by weight with respect to 1 part by weight of the porous bone graft material, since the content of the hydroxypropylmethylcellulose is high, solidification of the hydroxypropylmethylcellulose of the bone graft material occurs, and thus dissolution is very slow, which may not be suitable for a bone formation rate. In contrast, in the case where a portion of the hydroxypropyl methylcellulose is contained in an amount of more than 3 parts by weight relative to 1 part by weight of the porous bone graft material, the volume of the bone graft material may increase with the lapse of time since the hydroxypropyl methylcellulose increases to form a shape in which the hydroxypropyl methylcellulose wraps the bone graft material, thereby possibly absorbing moisture before dissolution of the hydroxypropyl methylcellulose occurs in a humid environment in the oral cavity. Therefore, in order to optimize the solubility of hydroxypropylmethylcellulose, the content of hydroxypropylmethylcellulose is determined to be 0.1 to 6 parts by weight, more preferably 0.3 to 3 parts by weight, with respect to 1 part by weight of the porous bone graft material.
In the dissolution, water may be used as a solvent, and the dissolution rate of the bone graft material composition formed by hydration with a dissolution time may be expressed in% by way of example. The water is hydrated using 1 to 1.5 parts by weight, more preferably, 1.2 to 1.5 parts by weight, compared to the hydrated bone graft material composition. This is an example of an optimal hydration amount for hydration of a bone graft material composition.
A bone graft material composition kit according to another embodiment of the present invention includes the aforementioned bone graft material composition and a syringe (syring) for installing the same. By providing a syringe including the bone graft material composition, convenience of use can be ensured, and the possibility of contamination occurring during use can be effectively reduced.
However, for the sake of more clear and concise explanation, the description of the present embodiment will omit the overlapping portions with other embodiments, and the omission of the description does not mean that the portion is excluded from the present invention, and the scope of the right thereof should be recognized as the same as other embodiments.
A method for producing a bone graft material composition according to another embodiment of the present invention includes the steps of:
adding a bone morphogenetic protein to a solvent or adding a solvent to a bone morphogenetic protein, and dissolving the bone morphogenetic protein in the solvent to produce a bone morphogenetic protein solution;
mixing and stirring the bone morphogenetic protein-adsorbed graft material powder and hydroxypropyl methylcellulose powder to form a viscous gel having a dissolution rate of hydroxypropyl methylcellulose of 50% or more within 48 hours; and
the mixture of the graft material powder and the hydroxypropyl methylcellulose powder, which is mixed and stirred, is frozen and dried at a low temperature in a vacuum state to form a sponge form including a structure including a plurality of pores, and thus, it can have excellent effects of bone formation activation, biocompatibility, and convenience in use.
However, for the sake of more clear and concise explanation, the description of the present embodiment will omit the overlapping portions with the aforementioned embodiment, and the omission of the description does not mean that the portions are excluded from the present invention, and the scope of the right thereof should be recognized as the same as the aforementioned embodiment.
Fig. 1 is a view schematically illustrating a sequence of a method for manufacturing a bone graft material composition according to an embodiment of the present invention.
Fig. 2 is a graph showing the weight ratio and the residual rate with time according to hydroxypropylmethyl cellulose (Hydroxypropyl methylcellulose) reflecting the result data (table 1) regarding experimental example 1 of the present invention.
First, a bone morphogenetic protein solution is prepared by dissolving a bone morphogenetic protein in a solvent. The bone morphogenetic protein solution can be prepared by dissolving the bone morphogenetic protein in a solvent by adding the bone morphogenetic protein to the solvent or by adding the solvent to the bone morphogenetic protein.
The bone morphogenic protein can be at least one selected from the group consisting of: BMP-2, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, a recombinant bone morphogenetic protein thereof, and a bone morphogenetic protein equivalent thereto may preferably be rhBMP-2 in the bone forming effect of the present invention.
The bone morphogenetic protein concentration of the bone morphogenetic protein solution according to an embodiment of the present invention can be 0.05 to 0.15mg/ml, and can preferably be 0.08 to 0.12 mg/ml. Bone formation of bone morphogenetic proteins can be activated by satisfying the concentration range. In the case where the bone morphogenetic protein concentration is less than 0.05, the new bone forming ability may be decreased, and in the case where it exceeds 0.15, side effects may be caused.
Also, for example, the acidity of the bone morphogenic protein solution according to an embodiment of the present invention can be pH 4.6 to 5. By satisfying the acidity range, bone formation of bone morphogenetic protein can be activated, and in the case where the acidity of the bone morphogenetic protein solution is lower than pH 4.6, the new bone forming ability may be decreased, and in the case where the acidity exceeds 5, the expression of the new bone forming ability may be decreased. For example, acidity can be adjusted using phosphate buffered saline (phosphate buffer saline), which may have the effect of new bone formation.
Next, the graft material powder is impregnated with the bone morphogenetic protein solution to adsorb the bone morphogenetic protein to the graft material powder. The bone-forming proteins can be adsorbed to the graft material powder by dipping the graft material powder into the bone-forming protein solution by dropping the bone-forming protein solution into the graft material powder prepared in advance or by sprinkling the graft material powder into the bone-forming protein solution.
The graft material powder can be autologous bone, allogeneic bone, or xenogeneic bone. For example, the graft material powder may be manufactured by injecting a micro tube (Snap tube).
The average particle diameter (D50) of the graft material powder may be 200 μm to 5,000 μm, and may preferably be 250 μm to 1,000 μm. In the case where the average particle diameter of the powder is less than 200 μm, the graft material may be rapidly absorbed to result in insufficient bone conduction required for bone formation, and in the case where it exceeds 5,000 μm, it may be difficult to perform precision processing upon application to a patient.
The step of adsorbing the bone-forming proteins to the graft material powder according to an embodiment of the present invention may include a step of adsorption using a refrigerated centrifuge.
In some cases, the bone morphogenetic protein can be suspended in the solution, and when the bone morphogenetic protein is adsorbed by rotation in a centrifuge, the bone morphogenetic protein can be prevented from being suspended in the solution, and the bone morphogenetic protein can be favorably adsorbed on the surface or in the pores of the graft material powder. The bone morphogenic proteins can be resuspended without falling out of the graft powder by rapid rotation and adsorption. If the speed is slow, the bone morphogenetic proteins may be suspended and not well adsorbed. The bone morphogenetic proteins can be rapidly adsorbed onto the surface of the graft material powder or into the pores.
The rotational speed of the refrigerated centrifuge according to an embodiment of the present invention may be 4000rpm or more. In the case of adsorption using a centrifuge, the higher the rotation speed, the better the adsorption, for example, the rotation speed of the centrifuge may be 4000rpm or more, and in the case of satisfying the above range, the bone morphogenetic protein can be prevented from being suspended in the solution.
The step of performing adsorption using a refrigerated centrifuge according to an embodiment of the present invention may be performed at a refrigerated temperature of 5 ℃ or less. By performing the adsorption step using a refrigerated centrifuge at a refrigerated temperature of 5 ℃ or lower, it is possible to prevent the temperature of the solution from rising due to rotation and maximize the adsorption effect on the surface or pores of the graft material powder of bone morphogenetic proteins by the rotation while preventing the deformation of the bone morphogenetic proteins which are weak to heat. The refrigeration may be at a temperature at which the solution is not frozen, for example, 5 ℃ or less, and may preferably be 0.5 ℃ to 1.5 ℃.
Next, the graft material powder having the bone morphogenetic protein adsorbed thereon and the hydroxypropyl methylcellulose powder were mixed and stirred to form a gel. Thereby, a gel having viscosity can be formed, and the formed gel can improve the adhesiveness of the graft material powder. For example, the agitation may be performed by a stirrer. A product of uniform quality can be obtained by stirring graft material powder with hydroxypropylmethylcellulose in powder form.
The volume ratio of the bone morphogenetic protein-adsorbed graft material powder to the hydroxypropyl methylcellulose powder according to an embodiment of the present invention may be 1:0.2 to 1: 0.8. In the case where the volume ratio of the hydroxypropylmethylcellulose powder is less than 0.2, it is difficult to form a gel, and in the case where it exceeds 0.8, the volume of the gel becomes larger than that of the graft material powder, and it may be difficult to form a bone graft material composition. In terms of the effect of the invention, preferably, the volume ratio of the graft material powder adsorbing bone morphogenetic proteins and the hydroxypropyl methylcellulose powder may be 1:0.6 to 1: 0.7.
Next, the mixed and stirred graft material powder and hydroxypropyl methylcellulose powder mixture were vacuum freeze-dried to form a sponge form including a porous structure. The mixture of the graft material powder and the hydroxypropyl methylcellulose powder, which is mixed and stirred, may also be frozen and dried at a low temperature in a vacuum state to form a sponge form having a structure including a plurality of pores.
A sponge form including a porous structure may be formed by performing a vacuum freeze-drying process. The formation of a sponge form including a porous structure can be formed by allowing the gel to be absorbed by the graft material powder, and the formation of a sponge form including a porous structure is judged to be a major contribution by the vacuum treatment.
The method of manufacturing a bone graft material composition according to an embodiment of the present invention may further include a packaging step.
The method for manufacturing a bone graft material composition according to an embodiment of the present invention may further include the step of mounting a bone graft material composition including a sponge form into a micro tube of a size that can be inserted into a syringe, wherein the sponge form includes a structure including the plurality of pores manufactured. By further including the step of installing a micro tube to a size capable of being inserted into a syringe, since it has a size capable of being inserted into a syringe, it is possible to directly insert the syringe without a separate process, and thus it is possible to easily operate in the process of manufacturing the bone graft material composition.
The method for manufacturing a bone graft material composition according to an embodiment of the present invention may further include the step of placing the bone graft material composition including a sponge form into a syringe and sealing the syringe, wherein the sponge form includes a structure including the plurality of pores installed in a micro tube. By providing the bone graft material composition in the syringe, convenience in use can be ensured, and the possibility of contamination during use can be effectively reduced.
The method for manufacturing a bone graft material composition according to an embodiment of the present invention may further include a step of performing a sterilization treatment.
An embodiment of the present invention may sterilize a sponge-shaped bone graft material composition including a structure including a plurality of pores by Ethylene Oxide Gas (Ethylene Oxide Gas). For example, the concentration of Ethylene Oxide Gas (Ethylene Oxide Gas) may be 450mg/l to 1200 mg/l.
When the concentration of Ethylene Oxide Gas (Ethylene Oxide Gas) is less than 450mg/l, sterilization may be insufficient, and when it exceeds 1200mg/l, bone morphogenetic protein may be deformed.
In one embodiment of the present invention, the sponge-shaped bone graft material composition including a structure including a plurality of pores may be sterilized by irradiating gamma rays. For example, the irradiation amount of the gamma ray may be 10kGy to 25 kGy. In the case where the gamma irradiation dose is less than 10kGy, sterilization may be insufficient, and in the case where the dose exceeds 25kGy, bone morphogenetic protein may be deformed.
The bone graft material manufactured according to the method of manufacturing a bone graft material as described above has a predetermined dissolution rate of a predetermined condition for application to a human body, for example, to a tooth. Such dissolution rate can be determined by the content of HPMC or the like included in the bone graft material.
Taking the case of applying the bone graft material to the tooth as an example, when the operator applies the bone graft material to the tooth defect, Hydroxypropylmethylcellulose (HPMC) contained in the bone graft material needs to have a predetermined dissolution rate or more for a predetermined time to have an adhesive property to be applied to the tooth, and can be treated to match the defect shape of the tooth. In the treatment, the bone graft material cannot flow to the periphery or be detached when applied to the tooth defect, or Hydroxypropyl methylcellulose (HPMC) cannot be dissolved in the bone graft material and the bone graft material cannot be bonded to the bone defect, and the treatment cannot be performed. Therefore, the bone graft material requires critical dissolution conditions of HPMC, and its function may be differentiated according to the dissolution conditions of HPMC.
When the bone graft material is used for treatment, the HPMC used as the additive can achieve the effective function of the bone graft material only when the dissolution rate reaches more than 50 percent within 48 hours. In the case where 50% or more of the HPMC is not dissolved within 48 hours, the undissolved HPMC may be solidified, and the space in which blood can flow into the bone graft material is reduced as the solidification rate is increased, thereby making it difficult to function as the bone graft material.
When the dissolution rate of HPMC reaches 89% or more (the residual amount is 11% or less) within 48 hours, the bone graft material cannot maintain its shape and cannot aggregate due to a low HPMC content in the bone graft material, and thus the bone graft material cannot be treated. Further, even if the treatment is performed, the adhesion of the bone graft material is reduced due to physical activities such as salivary gland activity, masticatory movement due to eating, respiration, and conversation of the patient, and side effects such as detachment of a shape object that is treated so as to match the shape of the defect may occur.
In the bone graft material composition according to an embodiment of the present invention, in order to ensure shape retentivity, the content of the hydroxypropylmethylcellulose may be formed to be 0.3 to 3 parts by weight, more preferably, 0.4 to 2 parts by weight with respect to 1 part by weight of the porous bone graft material, in which case the shape retentivity can be further enhanced. As will be understood from the experimental examples described later, the short axis change rate increases as the content of the hydroxypropylmethylcellulose increases, and the maximum breaking force tends to increase and then decrease. As shown in fig. 4, such increase and decrease rapidly occurred, and it was confirmed that the content of hydroxypropyl methylcellulose was 0.3 parts by weight or more and 3 parts by weight or less with respect to 1 part by weight of the bone graft material in order to manufacture the bone graft material composition having shape-maintaining property. In the case where the content of the hydroxypropylmethylcellulose is less than 0.3 parts by weight with respect to 1 part by weight of the bone graft material, the addition of HPMC is weak due to an excessively low specific gravity of HPMC, and the HPMC is easily squeezed and broken at a low pressure (force), and the shape-maintaining property thereof is not suitable as a bone graft material, and in the case where the content of the hydroxypropylmethylcellulose exceeds 3 parts by weight with respect to 1 part by weight of the porous bone graft material, the HPMC is easily squeezed even with a small force due to an excessively high content thereof, and the short axis change rate is also increased, and the shape-maintaining property thereof is not suitable as a bone graft material.
A bone graft material composition kit according to another embodiment of the present invention includes the aforementioned bone graft material composition and a syringe (syring) for installing the same. By providing a syringe directly including the bone graft material composition, convenience of use can be ensured, and the possibility of contamination during use can be effectively reduced.
However, for the sake of more clear and concise explanation, the description of the present embodiment will omit the overlapping portions with other embodiments, and the omission of the description does not mean that the portions are excluded from the present invention, and the scope of the right thereof should be recognized as the same as other embodiments.
A method for producing a bone graft material composition according to another embodiment of the present invention includes the steps of:
adding a bone morphogenetic protein to a solvent or adding a solvent to a bone morphogenetic protein, and dissolving the bone morphogenetic protein in the solvent to produce a bone morphogenetic protein solution;
mixing and stirring the bone graft material powder having the bone morphogenetic protein adsorbed thereon and the hydroxypropyl methylcellulose powder to form a viscous gel, thereby imparting shape-retaining properties to the bone graft material composition; and
the mixture of the graft material powder and the hydroxypropyl methylcellulose powder, which is mixed and stirred, is frozen and dried at a low temperature in a vacuum state to form a sponge form including a structure including a plurality of pores, and thus, it can have excellent effects of bone formation activation, biocompatibility, and convenience in use.
However, for the sake of more clear and concise explanation, the description of the present embodiment will omit the overlapping portions with the aforementioned embodiment, and the omission of the description does not mean that the portions are excluded from the present invention, and the scope of the right thereof should be recognized as the same as the aforementioned embodiment.
Fig. 1 is a view schematically showing a sequence of a method for manufacturing a bone graft material composition according to an embodiment of the present invention.
Fig. 4 is a graph showing "maximum force of failure (N) of bone graft material composition sphere (sphere)/short axis change rate of bone graft material composition sphere (sphere)" with respect to a weight ratio of hydroxypropylmethylcellulose (Hydroxypropyl methylcellulose), and is a graph showing a weight ratio of hydroxypropylmethylcellulose (Hydroxypropyl methylcellulose) for a bone graft material composition having excellent shape-maintaining properties.
First, a bone morphogenetic protein solution is prepared by dissolving a bone morphogenetic protein in a solvent. The bone morphogenetic protein solution can be prepared by dissolving the bone morphogenetic protein in a solvent by adding the bone morphogenetic protein to the solvent or by adding the solvent to the bone morphogenetic protein.
The bone morphogenic protein can be at least one selected from the group consisting of: BMP-2, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, a recombinant bone morphogenetic protein thereof, and a bone morphogenetic protein equivalent thereto may preferably be rhBMP-2 in the bone forming effect of the present invention.
The bone morphogenetic protein concentration of the bone morphogenetic protein solution according to an embodiment of the present invention can be 0.05 to 0.15mg/ml, and can preferably be 0.08 to 0.12 mg/ml. Bone formation of bone morphogenetic proteins can be activated by satisfying the concentration range. In the case where the bone morphogenetic protein concentration is less than 0.05, the new bone forming ability may be decreased, and in the case where it exceeds 0.15, side effects may be caused.
Also, for example, the acidity of the bone morphogenic protein solution according to an embodiment of the present invention can be pH 4.6 to 5. By satisfying the acidity range, bone formation of bone morphogenetic protein can be activated, and in the case where the acidity of the bone morphogenetic protein solution is lower than pH 4.6, the new bone forming ability may be decreased, and in the case where the acidity exceeds 5, the expression of the new bone forming ability may be decreased. For example, the acidity can be adjusted by phosphate buffer saline (phosphate buffer saline), and the adjustment of the acidity by phosphate buffer saline (phosphate buffer saline) may have the effect of the capacity for new bone formation.
Next, the graft material powder is impregnated with the bone morphogenetic protein solution to adsorb the bone morphogenetic protein to the graft material powder. The bone-forming proteins can be adsorbed to the graft material powder by dipping the graft material powder into the bone-forming protein solution by dropping the bone-forming protein solution into the graft material powder prepared in advance or by sprinkling the graft material powder into the bone-forming protein solution.
The graft material powder can be autologous bone, allogeneic bone, or xenogeneic bone. For example, the graft material powder may be manufactured by injecting a micro tube (Snap tube).
The average particle diameter (D50) of the graft material powder may be 200 μm to 5,000 μm, and may preferably be 250 μm to 1,000 μm. In the case where the average particle diameter of the powder is less than 200 μm, the graft material may be rapidly absorbed to result in insufficient bone conduction required for bone formation, and in the case where it exceeds 5,000 μm, it may be difficult to perform precision processing upon application to a patient.
The step of adsorbing the bone-forming proteins to the graft material powder according to an embodiment of the present invention may include a step of adsorption using a refrigerated centrifuge.
In some cases, the bone morphogenetic protein can be suspended in the solution, and in the case where the bone morphogenetic protein is rapidly rotated and adsorbed by the centrifuge, the bone morphogenetic protein can be prevented from being suspended in the solution, and the bone morphogenetic protein can be favorably adsorbed on the surface of the graft material powder or in the pores. The bone morphogenic proteins can be resuspended without falling out of the graft powder by rapid rotation and adsorption. If the speed is slow, the bone morphogenetic proteins may be suspended and not well adsorbed. The bone morphogenetic proteins can be rapidly adsorbed onto the surface of the graft material powder or into the pores.
The rotational speed of the refrigerated centrifuge according to an embodiment of the present invention may be 4000rpm or more. In the case of adsorption using a centrifuge, the faster the rotation speed, the better the adsorption, for example, the rotation speed of the centrifuge may be 4000rpm or more, and if the above range is satisfied, the bone morphogenetic protein may be prevented from being suspended in the solution.
The step of performing adsorption using a refrigerated centrifuge according to an embodiment of the present invention may be performed at a refrigerated temperature of 5 ℃ or less. By performing the adsorption step using a refrigerated centrifuge at a refrigerated temperature of 5 ℃ or lower, it is possible to prevent the temperature of the solution from rising due to rotation and maximize the adsorption effect on the surface or pores of the graft material powder of bone morphogenetic proteins by the rotation while preventing the deformation of the bone morphogenetic proteins which are weak to heat. The refrigeration may be at a temperature at which the solution is not frozen, for example, 5 ℃ or less, and may preferably be 0.5 ℃ to 1.5 ℃.
Next, the graft material powder having the bone morphogenetic protein adsorbed thereon and the hydroxypropyl methylcellulose powder were mixed and stirred to form a gel. Thereby, a gel having viscosity can be formed, and the formed gel can improve the adhesiveness of the graft material powder. For example, the agitation may be performed by a stirrer. A product of uniform quality can be obtained by stirring graft material powder with hydroxypropylmethylcellulose in powder form.
The volume ratio of the bone morphogenetic protein-adsorbed graft material powder to the hydroxypropyl methylcellulose powder according to an embodiment of the present invention may be 1:0.2 to 1: 0.6. In the case where the volume ratio of the hydroxypropylmethylcellulose powder is less than 0.2, it is difficult to form a gel, and in the case where it exceeds 0.6, the volume of the gel becomes larger than that of the graft material powder, and it may be difficult to effectively form the bone graft material composition. In terms of the effect of the invention, preferably, the volume ratio of the graft material powder adsorbing bone morphogenetic proteins and the hydroxypropyl methylcellulose powder may be 1:0.25 to 1: 0.35.
Next, the mixed and stirred graft material powder and hydroxypropyl methylcellulose powder mixture were vacuum freeze-dried to form a sponge form including a porous structure. The mixture of the graft material powder and the hydroxypropyl methylcellulose powder, which is mixed and stirred, may also be frozen and dried at a low temperature in a vacuum state to form a sponge form having a structure including a plurality of pores.
A sponge form including a porous structure may be formed by performing a vacuum freeze-drying process. The formation of a sponge form including a porous structure can be formed by allowing the gel to be absorbed by the graft material powder, and the formation of a sponge form including a porous structure is judged to be a major contribution by the vacuum treatment.
The method of manufacturing a bone graft material composition according to an embodiment of the present invention may further include a packaging step.
The method for manufacturing a bone graft material composition according to an embodiment of the present invention may further include the step of mounting a bone graft material composition including a sponge form into a micro tube of a size that can be inserted into a syringe, wherein the sponge form includes a structure including the plurality of pores manufactured. By further including the step of installing a micro tube to a size capable of being inserted into a syringe, since it has a size capable of being inserted into a syringe, it is possible to directly insert the syringe without a separate process, and thus it is possible to easily operate in the process of manufacturing the bone graft material composition.
A method of manufacturing a bone graft material composition according to an embodiment of the present invention may include a structure including the plurality of air holes mounted to a micro tube. As an example of the same structure, a step of placing a bone graft material composition including a sponge form into a syringe and sealing it may be further included. By providing the bone graft material composition in the syringe, convenience of use can be ensured, and the possibility of contamination that may occur during use can be effectively reduced.
The method for manufacturing a bone graft material composition according to an embodiment of the present invention may further include a step of performing a sterilization treatment.
An embodiment of the present invention may sterilize a sponge-shaped bone graft material composition including a structure including a plurality of pores by Ethylene Oxide Gas (Ethylene Oxide Gas). For example, the concentration of Ethylene Oxide Gas (Ethylene Oxide Gas) may be 450mg/l to 1200 mg/l.
When the concentration of Ethylene Oxide Gas (Ethylene Oxide Gas) is less than 450mg/l, sterilization may be insufficient, and when it exceeds 1200mg/l, bone morphogenetic protein may be deformed.
In one embodiment of the present invention, the sponge-shaped bone graft material composition including a structure including a plurality of pores may be sterilized by irradiating gamma rays. For example, the irradiation amount of the gamma ray may be 10kGy to 25 kGy. In the case where the gamma irradiation dose is less than 10kGy, sterilization may be insufficient, and in the case where the dose exceeds 25kGy, bone morphogenetic protein may be deformed.
The bone graft material according to the embodiment of the present invention, which can be manufactured according to the manufacturing method of the bone graft material as described above, may have shape-maintaining properties for application to a human body, for example, to a tooth defect. Such shape-retaining property can be determined by the content of HPMC or the like included in the bone graft material.
Taking the case of applying a bone graft material to a tooth as an example, when an operator applies a bone graft material to a tooth defect, the bone graft material needs to have plasticity to a predetermined degree or more so as to be deformable in conformity with the defect shape of the tooth when applied to the tooth defect. Moreover, the phenomenon of flow around or detachment cannot occur when applied to a tooth defect. Therefore, the bone graft material needs to have a plasticity of a predetermined degree or more, and further needs to have a rigidity of a predetermined degree or more capable of withstanding gravity or movement of the bone (tooth) defect portion.
The degree of such rigidity and plasticity can be defined as shape-retaining property, which is defined as the rate of change of the minor axis that a sphere deforms into an ellipse and can be quantified when a force of XXX N is applied to a spherical test piece of XXX mm for the purpose of quantifying the objective degree of shape-retaining property.
The shape retention property for satisfying the plasticity and rigidity described earlier should be 50 or more. In the case of less than 50, it is difficult to apply to the bone defect due to the strong elastic property. That is, when the operator injects the bone defect, the bone graft material needs to be deformed to match the shape of the bone defect, but when the elastic property is strong, the restorability is large, and such deformation may be difficult to occur. Furthermore, when the shape-retaining property is less than 50, it has been confirmed that the actual treatment cannot be performed because the operator flows around during the hydration treatment or the like.
Therefore, in order to ensure the applicability of the bone graft material while ensuring the easiness of the actual treatment, it is possible to obtain a bone graft material composition which has "the maximum fracture force (N)/the minor axis change rate" of 50 or more and is excellent in shape retention, is easily deformed in conformity with the shape of the bone defect portion, and is free from problems such as flowing to the surroundings during the treatment such as hydration. Therefore, a bone graft material composition having a shape-retaining property of 50 or more can be formed when the content of the hydroxypropyl methylcellulose is 0.3 parts by weight or more and 3 parts by weight or less relative to 1 part by weight of the bone graft material.
In the bone graft material composition according to an embodiment of the present invention, the composition ratio of the composition for optimizing the bone graft material at the time of treatment is 0.15 to 6 parts by weight, more preferably, may be preferably formed to 0.3 to 3 parts by weight with respect to 1 part by weight of the hydroxypropyl methylcellulose of the bone graft material. In the case where the content of the hydroxypropylmethylcellulose is less than 0.3 parts by weight with respect to 1 part by weight of the bone graft material, the adhesiveness to the bone defect portion is insufficient, and thus the possibility of falling from the bone defect portion at the time of use is high, and the content thereof is minute, and thus the osmotic pressure characteristic of the bone graft material is hardly affected. When the amount of the hydroxypropyl methylcellulose exceeds 3 parts by weight based on 1 part by weight of the bone graft material, the hygroscopicity and wettability of the heterogeneous bone may be inhibited to inhibit bone formation, and the hydroxypropyl methylcellulose may be solidified and may not be dissolved well and flow out to the outside, thereby exerting a large influence on osmotic pressure.
The bone graft material composition comprising the hydroxypropylmethylcellulose is dissolved in a solvent and used in the form of a dissolved substance. A solvent that can be used in the art can be appropriately selected and used as the solvent, and water is used as an example. Since physical properties such as dissolution rate, concentration, osmotic pressure characteristics, shape retention property, and the like of the bone graft material composition vary depending on the conditions of the solvent, it is important to set appropriate solvent conditions for implementation.
The dissolved substance of a bone graft material according to an embodiment of the present invention may be manufactured by mixing 0.5 to 2 parts by weight of the solvent (water) with respect to 1 part by weight of the bone graft material composition including the hydroxypropylmethylcellulose in order to optimize the bone graft material at the time of treatment, specifically, in order to form an optimal osmotic pressure with other solutions. As will be understood from the experimental examples described later, the content of the solvent satisfies a predetermined range so that a proper permeation phenomenon with other solutions as a bone graft material can be formed. As shown in fig. 5, it was confirmed that 0.5 to 2 parts by weight of the mixed solvent (water) with respect to 1 part by weight of the bone graft material composition including hydroxypropylmethylcellulose was required in order to form a suitable permeation phenomenon.
In the case where the content of the solvent (water) is less than 0.5 parts by weight with respect to 1 part by weight of the bone graft material composition including hydroxypropylmethylcellulose, the amount of the solvent (water) is too small to be well dissolved, and thus hydroxypropylmethylcellulose is not well combined with the bone graft material, and thus the hydroxypropylmethylcellulose function is not well exerted. In general, the composition is too hard to be suitable for bone formation, and is formed to have a high osmotic pressure with other solutions, so that it is difficult to maintain the shape, and there is a possibility that the function as a bone graft material is impaired.
In the case where the amount exceeds 2 parts by weight with respect to 1 part by weight of the bone graft material composition including hydroxypropylmethylcellulose, the amount of the solvent (water) is too large and the dissolved matter does not have appropriate viscosity and has fluidity, so that it is difficult to have shape-maintaining property. Further, since the dissolution of hydroxypropylmethylcellulose proceeds rapidly due to the high content of the solvent (water), hydroxypropylmethylcellulose may be dissolved and flow to the outside, and thus its function may not be exhibited. Also, since the penetration phenomenon with other solutions is strong, it is difficult to maintain the shape, and there is a possibility that the function as a bone-maintaining material is impaired.
Therefore, in order to allow the bone graft material composition including hydroxypropylmethylcellulose to function well as hydroxypropylmethylcellulose and to provide a solvent having shape-maintaining properties as a bone graft material, it is preferable to produce a bone graft material dissolved substance by mixing 0.5 to 2 parts by weight of the solvent (water) with respect to 1 part by weight of the bone graft material composition including hydroxypropylmethylcellulose.
A bone graft material composition kit according to another embodiment of the present invention includes the aforementioned bone graft material composition and a syringe (syring) for installing the same. By providing a syringe directly including a bone graft material, convenience of use can be ensured, and the possibility of contamination occurring during use can be effectively reduced.
However, for the sake of more clear and concise explanation, the description of the present embodiment will omit the overlapping portions with other embodiments, and the omission of the description does not mean that the portion is excluded from the present invention, and the scope of the right thereof should be recognized as the same as other embodiments.
A method for producing a bone graft material composition according to another embodiment of the present invention includes the steps of:
adding a bone morphogenetic protein to a solvent or adding a solvent to a bone morphogenetic protein, and dissolving the bone morphogenetic protein in the solvent to produce a bone morphogenetic protein solution;
mixing and stirring a graft material powder having the bone morphogenetic protein adsorbed thereon and a hydroxypropyl methylcellulose powder to form a viscous gel; and
the mixture of the graft material powder and the hydroxypropyl methylcellulose powder, which is mixed and stirred, is frozen and dried at a low temperature in a vacuum state to form a sponge form including a structure including a plurality of pores, and thus, it can have excellent effects of bone formation activation, biocompatibility, and convenience in use.
However, for the sake of more clear and concise explanation, the description of the present embodiment will omit the overlapping portions with the aforementioned embodiment, and the omission of the description does not mean that the portions are excluded from the present invention, and the scope of the right thereof should be recognized as the same as the aforementioned embodiment.
Fig. 1 is a view schematically showing a sequence of a method for manufacturing a bone graft material composition according to an embodiment of the present invention.
Fig. 5 is data of results of experimental examples according to the present invention, and is a graph showing changes in osmotic pressure of a mixed saline solution to a pure saline solution according to time in an osmotic ratio after mixing a bone graft material lysate formed by changing parts by weight of a solvent and the saline solution.
First, a bone morphogenetic protein solution is prepared by dissolving a bone morphogenetic protein in a solvent. The bone morphogenetic protein solution can be prepared by dissolving the bone morphogenetic protein in a solvent by adding the bone morphogenetic protein to the solvent or by adding the solvent to the bone morphogenetic protein.
The bone morphogenic protein can be at least one selected from the group consisting of: BMP-2, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, a recombinant bone morphogenetic protein thereof, and a bone morphogenetic protein equivalent thereto may preferably be rhBMP-2 in the bone forming effect of the present invention.
The bone morphogenetic protein concentration of the bone morphogenetic protein solution according to an embodiment of the present invention can be 0.05 to 0.15mg/ml, and can preferably be 0.08 to 0.12 mg/ml. Bone formation of bone morphogenetic proteins can be activated by satisfying the concentration range. In the case where the bone morphogenetic protein concentration is less than 0.05, the new bone forming ability may be decreased, and in the case where it exceeds 0.15, side effects may be caused.
Also, for example, the acidity of the bone morphogenic protein solution according to an embodiment of the present invention can be pH 4.6 to 5. By satisfying the acidity range, bone formation of bone morphogenetic protein can be activated, and in the case where the acidity of the bone morphogenetic protein solution is lower than pH 4.6, the new bone forming ability may be decreased, and in the case where the acidity exceeds 5, the expression of the new bone forming ability may be decreased. For example, the acidity can be adjusted by phosphate buffer saline (phosphate buffer saline), and the adjustment of the acidity by phosphate buffer saline (phosphate buffer saline) may have the effect of the capacity for new bone formation.
Next, the graft material powder is impregnated with the bone morphogenetic protein solution to adsorb the bone morphogenetic protein to the graft material powder. The bone-forming proteins can be adsorbed to the graft material powder by dipping the graft material powder into the bone-forming protein solution by dropping the bone-forming protein solution into the graft material powder prepared in advance or by sprinkling the graft material powder into the bone-forming protein solution.
The graft material powder can be autologous bone, allogeneic bone, or xenogeneic bone. For example, the graft material powder may be manufactured by injecting a micro tube (Snap tube).
The average particle diameter (D50) of the graft material powder may be 200 μm to 5,000 μm, and may preferably be 250 μm to 1,000 μm. In the case where the average particle diameter of the powder is less than 200 μm, the graft material may be rapidly absorbed to result in insufficient bone conduction required for bone formation, and in the case where it exceeds 5,000 μm, it may be difficult to perform precision processing upon application to a patient.
The step of adsorbing the bone-forming proteins to the graft material powder according to an embodiment of the present invention may include a step of adsorption using a refrigerated centrifuge.
In some cases, the bone morphogenetic protein can be suspended in the solution, and in the case where the bone morphogenetic protein is rapidly rotated and adsorbed by the centrifuge, the bone morphogenetic protein can be prevented from being suspended in the solution, and the bone morphogenetic protein can be favorably adsorbed on the surface of the graft material powder or in the pores. The bone morphogenic proteins can be resuspended without falling out of the graft powder by rapid rotation and adsorption. If the speed is slow, the bone morphogenetic proteins may be suspended and not well adsorbed. The bone morphogenetic proteins can be rapidly adsorbed onto the surface of the graft material powder or into the pores.
The rotational speed of the refrigerated centrifuge according to an embodiment of the present invention may be 4000rpm or more. In the case of adsorption using a centrifuge, the faster the rotation speed, the better the adsorption, for example, the rotation speed of the centrifuge may be 4000rpm or more, and if the above range is satisfied, the bone morphogenetic protein may be prevented from being suspended in the solution.
The step of performing adsorption using a refrigerated centrifuge according to an embodiment of the present invention may be performed at a refrigerated temperature of 5 ℃ or less. By performing the adsorption step using a refrigerated centrifuge at a refrigerated temperature of 5 ℃ or lower, it is possible to prevent the temperature of the solution from rising due to rotation and maximize the adsorption effect on the surface or pores of the graft material powder of bone morphogenetic proteins by the rotation while preventing the deformation of the bone morphogenetic proteins which are weak to heat. The refrigeration may be at a temperature at which the solution is not frozen, for example, 5 ℃ or less, and may preferably be 0.5 ℃ to 1.5 ℃.
Next, the graft material powder having the bone morphogenetic protein adsorbed thereon and the hydroxypropyl methylcellulose powder were mixed and stirred to form a gel. Thereby, a gel having viscosity can be formed, and the formed gel can improve the adhesiveness of the graft material powder. For example, the agitation may be performed by a stirrer. A product of uniform quality can be obtained by stirring graft material powder with hydroxypropylmethylcellulose in powder form.
The volume ratio of the bone morphogenetic protein-adsorbed graft material powder to the hydroxypropyl methylcellulose powder according to an embodiment of the present invention may be 1:0.2 to 1: 0.6. In the case where the volume ratio of the hydroxypropylmethylcellulose powder is less than 0.2, it is difficult to form a gel, and in the case where it exceeds 0.6, the volume of the gel becomes larger than that of the graft material powder, and it may be difficult to effectively form the bone graft material composition. In terms of the effect of the invention, preferably, the volume ratio of the graft material powder adsorbing bone morphogenetic proteins and the hydroxypropyl methylcellulose powder may be 1:0.25 to 1: 0.35.
Next, the mixed and stirred graft material powder and hydroxypropyl methylcellulose powder mixture were vacuum freeze-dried to form a sponge form including a porous structure. The mixture of the graft material powder and the hydroxypropyl methylcellulose powder, which is mixed and stirred, may also be frozen and dried at a low temperature in a vacuum state to form a sponge form having a structure including a plurality of pores.
A sponge form including a porous structure may be formed by performing a vacuum freeze-drying process. The formation of a sponge form including a porous structure can be formed by allowing the gel to be absorbed by the graft material powder, and the formation of a sponge form including a porous structure is judged to be a major contribution by the vacuum treatment.
The method of manufacturing a bone graft material composition according to an embodiment of the present invention may further include a packaging step.
The method for manufacturing a bone graft material composition according to an embodiment of the present invention may further include the step of mounting a bone graft material composition including a sponge form into a micro tube of a size that can be inserted into a syringe, wherein the sponge form includes a structure including the plurality of pores manufactured. By further including the step of installing a micro tube to a size capable of being inserted into a syringe, since it has a size capable of being inserted into a syringe, it is possible to directly insert the syringe without a separate process, and thus it is possible to easily operate in the process of manufacturing the bone graft material composition.
The method for manufacturing a bone graft material composition according to an embodiment of the present invention may further include the step of placing and sealing a bone graft material composition including a sponge form, which is mounted in a micro tube, in a syringe, wherein the sponge form includes a structure including the plurality of pores. By providing the bone graft material composition in the syringe, convenience of use can be ensured, and the possibility of contamination that may occur during use can be effectively reduced.
The method for manufacturing a bone graft material composition according to an embodiment of the present invention may further include a step of performing a sterilization treatment.
An embodiment of the present invention may sterilize a sponge-shaped bone graft material composition including a structure including a plurality of pores by Ethylene Oxide Gas (Ethylene Oxide Gas). For example, the concentration of Ethylene Oxide Gas (Ethylene Oxide Gas) may be 450mg/l to 1200 mg/l.
When the concentration of Ethylene Oxide Gas (Ethylene Oxide Gas) is less than 450mg/l, sterilization may be insufficient, and when it exceeds 1200mg/l, bone morphogenetic protein may be deformed.
In one embodiment of the present invention, the sponge-shaped bone graft material composition including a structure including a plurality of pores may be sterilized by irradiating gamma rays. For example, the irradiation amount of the gamma ray may be 10kGy to 25 kGy. In the case where the gamma irradiation dose is less than 10kGy, sterilization may be insufficient, and in the case where the dose exceeds 25kGy, bone morphogenetic protein may be deformed.
The bone graft material manufactured according to the method for manufacturing a bone graft material as described above needs to secure hydration properties in order to be applied to a human body, for example, to teeth. In other words, it is required to have a critical osmotic pressure characteristic. The formation of a predetermined permeation ratio when mixed with another solution (e.g., saline solution) is an important factor for maintaining a predetermined concentration, thereby maintaining a predetermined shape and maintaining the function of the bone graft material. These physical properties can be determined depending on the content of HPMC and the like included in the bone graft material and the conditions of the solvent used for forming the solute.
Taking the case of applying a bone graft material to a tooth as an example, when an operator applies a bone graft material to a tooth defect portion, the bone graft material provided in the form of powder or the like is hydrated and applied to the bone defect portion. In such hydration process, if HPMC included in the bone graft material is eluted in a large amount (osmotic pressure of hydrate is increased), HPMC cannot be included in the bone graft material, not only the shape of the bone graft material cannot be maintained, but also the bone graft material cannot be aggregated and thus cannot be treated. Therefore, the bone graft material composition needs to have osmotic pressure characteristics within a predetermined degree to be able to maintain the shape in a state of not being affected by external conditions. Further, in the case where an external environment of water, saliva, or the like is created when the bone graft material is applied to the tooth defect portion, a phenomenon in which the bone graft material composition flows to the surroundings or is detached may occur, and thus, there is a problem in that foreign substances may flow into the defect portion. Therefore, the bone graft material is required to have osmotic pressure characteristics within a predetermined range, and further, to have physical properties corresponding to external conditions as well as internal physical properties.
In fact, when the osmotic pressure of saline is normalized to 100%, the saline needs to develop an osmotic pressure of 104% to 112% within 12 hours to 48 hours after the addition to the transplant material lysate. If the osmotic pressure exceeds 112%, the outflow of HPMC occurs during hydration, and the shape maintenance and adhesion with the bone defect of the bone graft material are significantly reduced to fail to apply to the bone graft material. Further, if the osmotic pressure is less than 104%, hydration is not performed well, and it is difficult to achieve plastic deformation corresponding to the bone defect, thereby adversely affecting bone regeneration.
Hereinafter, although preferred embodiments are set forth to aid in understanding the present invention, the embodiments are merely illustrative of the present invention and do not limit the scope of the claims, it is apparent to those skilled in the art that various changes and modifications can be made to the embodiments within the scope of the present invention and the technical idea, and such changes and modifications also fall within the scope of the claims.
Experimental example 1
1. Confirmation of the amount of residue based on the ratio of hydroxypropylmethylcellulose (HPMC: Hydroxypropylmethylcellulose) and the solubility
HPMC was added to bone graft material (0.25g) in different weight parts (0.1-6) as shown in Table 1. The mixture of bone graft material and HMPC was dissolved in a solvent (water), and the residual amount of HPMC was confirmed over time. The amount of HMPC added first was normalized to 100%, and the residual amount remaining after dissolution was compared to the standard value (100%) and shown in percent (%). The ratio of the residual amount was closer to 100% indicating almost no dissolution, and the ratio was closer to 0% indicating almost total dissolution. As another meaning, a ratio of the residual amount of 100% can be interpreted as a dissolution rate of 0%, and a ratio of the residual amount of 0% can be interpreted as a dissolution rate of 100%.
The amount of the solvent (water) is an optimum hydration amount for the mixture to be well dissolved, and is 1 to 1.5 times by weight, for example, 1.2 times by weight of the total weight of the mixture.
[ TABLE 1 ]
As shown in Table 1, it was confirmed that the residual amount became small and the dissolution rate was improved as the dissolution time elapsed. Also, it was confirmed that as the amount of HPMC increased, the residual amount increased in the same period of time, and the dissolution rate decreased.
Fig. 2 reflects the result data (table 1) on the experimental example 1 and shows the residual ratio according to the weight ratio of hydroxypropylmethylcellulose and time. The closer the value on the Y-axis is to 1, the closer the residual rate is 100% (dissolution rate 0%), and the closer to 0, the closer the residual rate is 0% (dissolution rate 100%).
The dissolution rate of hydroxypropylmethylcellulose in the bone graft material composition may require different conditions depending on the use, use environment, and use purpose thereof. The composition ratio of the porous bone graft material and the hydroxypropylmethylcellulose may be performed in consideration of dissolution time and dissolution rate according to the purpose.
Under the condition that 50% or more of hydroxypropyl methylcellulose needs to be dissolved within 48 hours, 0.1 to 3 parts by weight of hydroxypropyl methylcellulose may be mixed with 1 part by weight of the porous bone graft material to form a bone graft material composition, as another example, under the condition that 60% or more of hydroxypropyl methylcellulose needs to be dissolved within 48 hours, 0.1 to 2 parts by weight of hydroxypropyl methylcellulose may be mixed with 1 part by weight of the porous bone graft material to form a bone graft material composition, as another example, under the condition that 70% or more of hydroxypropyl methylcellulose needs to be dissolved within 48 hours, 0.1 to 1.2 parts by weight of hydroxypropyl methylcellulose may be mixed with 1 part by weight of the porous bone graft material to form a bone graft material composition, as another example, under the condition that 80% or more of hydroxypropyl methylcellulose needs to be dissolved within 48 hours, the bone graft material composition may be formed by mixing 0.1 to 0.6 parts by weight of hydroxypropylmethylcellulose with respect to 1 part by weight of the porous bone graft material. In view of the functionality of the bone graft material, it is preferable that the mixing ratio is 0.3 parts by weight or more of hydroxypropylmethylcellulose to 1 part by weight of the porous bone graft material.
As another example, the bone graft material composition may be formed by mixing 0.1 to 1 part by weight of hydroxypropylmethylcellulose with respect to 1 part by weight of the porous bone graft material under the condition that it is necessary to dissolve 50% or more of hydroxypropylmethylcellulose within 24 hours. Also, the bone graft material composition may be formed by constituting the mixing ratio to be 0.3 to 1.0 part by weight in consideration of the functionality of the bone graft material.
As another example, the bone graft material composition may be formed by mixing 0.1 to 0.6 parts by weight of hydroxypropyl methylcellulose with respect to 1 part by weight of the porous bone graft material under the condition that it is necessary to dissolve 50% or more of hydroxypropyl methylcellulose within 12 hours. Also, the bone graft material composition may be formed by constituting the mixing ratio to 0.3 to 0.6 parts by weight in consideration of the functionality of the bone graft material.
Experimental example 2
2. Volume reduction and solubility confirmation experiments according to HPMC ratio
HPMC was added to bone graft material (0.25g) in different weight parts (0.1-6) as shown in Table 2. A mixture of the bone graft material and HPMC was dissolved (hydrated) in a solvent (water) to manufacture a test piece, and the residual amount of HPMC according to the lapse of time was confirmed. Specifically, the hydrated test piece was put into a conical tube (a chemical tube) having a capacity of 15ml without any space, and then washed for the first time, and then the portion of the conical tube not occupied by the test piece was cut off. Then, the open part of the remaining truncated conical tube was covered with a mesh (mesh) so that only the dissolved test piece could pass through the open part.
Thereafter, the conical tube is positioned in an ultrasonic cleaner maintaining the temperature of the human body (for example, 37 degrees), and pure water is circulated at a predetermined speed to measure the volume of the test piece after 48 hours. Pure water remaining in the test piece may be removed before the volume of the test piece is measured.
The reduction rate of volume was measured in an environment similar to that of human body according to the amount of HPMC added. The higher the volume reduction rate, the higher the solubility of the reagent, and the lower the volume reduction rate, the lower the solubility.
The amount of the solvent (water) is an optimum hydration amount for the mixture to be well dissolved, and is 1 to 1.5 times or 1.2 to 1.5 times by weight of the total weight of the mixture, and is 1.2 as an example.
[ TABLE 2 ]
[ TABLE 3 ]
HPMC parts by weight
Initial weight (g)
Weight (g) after 48 hours
0.1
1.630
0.610
0.2
3.190
1.850
0.3
3.930
3.280
0.4
4.620
4.120
0.6
6.820
6.090
0.8
7.690
6.850
1.2
11.980
10.800
1.5
13.940
12.600
2
16.690
15.400
3
24.600
23.010
4
26.400
28.510
5
32.720
35.900
6
34.200
38.120
As shown in tables 2 and 3, it was confirmed that the volume and weight of the test piece were changed after 48 hours in human environment according to the weight ratio of HPMC. Further, it was confirmed that the amount of HPMC remained increased and the dissolution rate decreased as the amount of HPMC increased.
Fig. 3 reflects the result data (table 2) on the experimental example 2, showing the volume reduction rate according to the weight ratio of HPMC.
As shown in fig. 3, it was confirmed that the volume reduction rate was 40% when the weight part of HPMC in the bone graft material composition was 0.2, and the volume reduction rate was sharply reduced to 16.54% when the weight part of HPMC was 0.3. Further, it was confirmed that the volume reduction rate was 6.46% when the weight part of HPMC in the bone graft material composition was 3, and the volume reduction rate was negative when the weight part of HPMC was 4.
This indicates that HPMC does not promote or help bone formation because HPMC dissolves and flows out too quickly in a human environment and thus cannot maintain the volume of the test piece in the case where the weight part of HPMC is less than 0.3 relative to 1 weight part of bone graft material. In contrast, in the case where the weight part of HPMC relative to 1 weight part of bone graft material exceeds 3, the volume of HPMC is greater than that of bone graft material, so that it is formed in a shape in which HPMC wraps the bone graft material, and thus a phenomenon in which HPMC absorbs external moisture to increase the volume occurs. As can be seen from table 3, it was confirmed that when the amount of HPMC is more than 3 parts by weight based on 1 part by weight of the bone graft material, moisture is absorbed from the outside, and thus the weight of the test piece after the experiment is increased.
Therefore, in order to realize the functional side of the bone graft material, HPMC may be mixed in an amount of 0.3 parts by weight or more and 3 parts by weight or less with respect to 1 part by weight of the porous bone graft material.
Experimental example 3
3. Shape Retention test according to HPMC ratio
As shown in table 4, HPMC was added to a bone graft material (0.25g) in different parts by weight (0.1 to 6), mixed, dissolved in a solvent (DBS) to form a viscous material, and then manufactured so as to have a spherical shape. The dimensions of the initial sphere (sphere) are shown in table 4 as the length of the major axis and the minor axis. The ball was pressed with a press (push-push gage) to apply a pressing force to the ball, and the maximum breaking force (N) before the ball (sphere) was broken, i.e., the minor axis length at the maximum peak, was measured. The minor axis rate of change is shown in table 4 comparing the minor axis length at the initial minor axis length and at the maximum failure force.
Experimental example 4
4. Rigidity test according to HPMC ratio
The maximum breaking force (N), i.e., the maximum peak value, before the sphere (sphere) of experimental example 3 was broken was measured and shown in table 4. It can be explained that the higher the maximum breaking force, the greater the stiffness.
[ TABLE 4 ]
Experimental example 5
5. Confirmation of shape maintenance by maximum breaking force (N) with respect to short axis change rate
In order to form a bone graft material composition excellent in shape-retaining property, optimum rigidity and non-restorability are required. Table 5 below shows the maximum destructive power with respect to the short axis rate of change according to the HPMC ratio. The values in table 5 are reflected in fig. 4 to show "maximum breaking force (N) of the ball of bone graft material composition (sphere)/short axis change rate of the ball of bone graft material composition (sphere)" with respect to the weight ratio of hydroxypropylmethylcellulose.
[ TABLE 5 ]
The HPMC in 0.1 to 0.2 parts by weight has a high minor axis change rate, a small maximum breaking force (N), and a low value of "maximum breaking force (N)/minor axis change rate", and thus is not excellent in shape retention. This means that the shape is also easily changed by a small force, which has a very adverse effect on the treatment process of the bone graft material. This is understood to be a phenomenon that occurs due to the lack of HPMC that can increase the rigidity of the bone graft material.
In the case of a bone graft material including 4 parts by weight or more of HPMC, the shape retention property is not excellent because the change rate of the minor axis is high, the maximum breaking force (N) is low, and the numerical value of "maximum breaking force (N)/change rate of the minor axis" is low. That is, it is known that including at least 4 parts by weight of HPMC in a bone graft material, i.e., including an excessive amount of HPMC, does not improve the rigidity of the bone graft material, but rather reduces the shape-retaining property.
When the composition is applied to a bone graft material containing 0.3 to 3 parts by weight of HPMC per 1 part by weight of the bone graft material, a "maximum breaking force (N)/short axis change rate" of 50 or more can be obtained, and a bone graft material composition having excellent shape retention can be obtained. As another example, if the bone graft material is prepared to include 0.4 to 2 parts by weight of HPMC per 1 part by weight of the bone graft material, a "maximum fracture force (N)/minor axis change rate" of 60 or more can be formed, and a bone graft material composition having excellent shape retention can be obtained. As another example, if the bone graft material is prepared to include 0.6 to 1.5 parts by weight of HPMC per 1 part by weight of the bone graft material, a "maximum breaking force (N)/short axis change rate" of 70 or more can be formed, and a bone graft material composition having excellent shape retention can be obtained.
Experimental example 6
6. Osmolarity measurement of saline solution
The osmotic pressure means a pressure to which the semipermeable membrane is subjected when an osmotic phenomenon occurs, and is formed in proportion to a concentration difference of the solution. The osmotic pressure of the saline solution was measured by a Control experiment, and as shown in the following table 6, whether the osmotic pressure was changed according to the lapse of time was also measured. The value of the osmotic pressure can be determined by introducing the solution and a pressure sensor connected to the outflow opening. The osmolality value of the saline solution determined by this experiment was 286, which was normalized to 100%.
Experimental example 7
7. Manufacture of bone graft material hydrates including HPMC
Water was added to 1 part by weight of the bone graft material (0.5g) and 0.6 part by weight of HPMC to prepare a hydrate of the bone graft material including HPMC. The amount of the added water is 0.4 to 6 parts by weight of water to 1 part by weight of the mixture of the bone graft material and the HPMC.
Experimental example 8
8. Osmolality measurement of saline solution added to bone graft material hydrate including HPMC
The bone graft material hydrate manufactured by the 7 was put into a 15ml conical tube (conicaltube) and 5ml of saline was added. Saline solution 0.5ml was collected at each time node of the following Table 6 and the osmotic pressure was measured. The numerical values relative to the control experimental values of 6 are shown by "%".
[ TABLE 6 ]
1 hour
3 hours
6 hours
12 hours
24 hours
48 hours
Control
100％
100％
100％
100％
100％
100％
0.4
100％
108％
112％
113％
115％
117％
0.5
100％
104％
109％
110％
111％
112％
0.6
100％
103％
107％
109％
110％
111％
0.8
100％
103％
106％
107％
108％
109％
1.0
100％
102％
103％
106％
107％
108％
1.2
100％
103％
104％
105％
106％
108％
1.5
100％
101％
102％
105％
107％
108％
2.0
100％
102％
102％
104％
106％
107％
3.0
100％
109％
115％
116％
117％
117％
4.0
100％
109％
116％
117％
118％
118％
6.0
100％
110％
114％
116％
118％
118％
As shown in said table 6, the control experiment was set as 100% of the standard value. It is a value in which the osmotic pressure of pure saline solution according to time is measured and normalized.
As shown in Table 6, it was confirmed that the osmotic pressure increased with the lapse of time at the same weight part. This is because the contact time and contact amount of the saline solution with the hydrated bone graft material containing HPMC dissolved therein are increased with the lapse of time, and thus the osmotic pressure is increased.
As shown in table 6, it was confirmed that the osmotic pressure increased as the weight of water increased at the same time. This is because the greater the amount of water, the more rapidly the HPMC of the HPMC-dissolved bone graft material hydrate is dissolved, resulting in an increase in osmotic pressure.
As shown in fig. 5, it was confirmed that the osmotic pressure was greatly increased in a short time and the osmotic ratio was greatly changed when the weight part of water was 0.4 or less, and that the osmotic pressure was greatly increased in a short time and the osmotic ratio was greatly changed when the weight part of water was 3 or more. A large increase in osmotic pressure in a short time indicates that HPMC flows outside without being fused with the bone graft material, and shape maintenance is reduced in the case where the osmotic pressure is increased with an increase in concentration. Therefore, for optimal shape-maintaining properties, it is suitable to set the weight part of water to 0.5 to 2 parts by weight with respect to the weight part of the bone graft material composition including HPMC.
If the water is set to 0.5 to 2 parts by weight in comparison with the bone graft material composition, the osmotic pressure of the saline solution may be formed to 104% to 112% or less within 12 hours to 48 hours. The osmotic pressure value formed within 12 hours is not sufficient to grasp the performance of the bone graft material with respect to the maintenance of functionality, and the direct application thereof is slightly insufficient to verify the functionality and stability of the bone graft material. Therefore, the formation of osmotic pressure within 12 hours to 48 hours was confirmed, and the functionality, osmotic pressure, shape-retaining property, and the like of the bone graft material were evaluated. If the osmotic pressure is more than 115% compared with that of pure saline solution within 48 hours, hydration of the bone graft material composition including HPMC is not well performed and does not sufficiently function as an additive, and thus it is difficult to apply the bone graft material composition, and if it is close to 100%, it is not different from that of pure saline solution, and thus it is difficult to consider the bone graft material including HPMC to have its physical properties and functions. Therefore, it is preferable that the bone graft material composition has an osmotic pressure at which saline is formed within 12 to 48 hours after the addition, which is 104 to 112% or less of that of a control (control).
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