Fibrous non-demineralized bone graft material and method for producing same

Abstract

One embodiment of the present invention provides a method of producing a fibrous non-demineralized bone graft material, the method comprising: a step of removing soft tissue, blood, and bone marrow from bone; a dehydration step of immersing, in an aqueous ethanol solution, the bone from which the soft tissue, blood, and bone marrow have been removed, and stirring same; a primary defatting step of immersing and stirring the dehydrated bone in diethyl ether; a step of crushing the bone subjected to the primary defatting into fibrously processed bone; a step of screening the fibrously processed bone; a hydration step of immersing and stirring the screened fibrously processed bone in ethanol; a secondary defatting step of immersing and stirring the hydrated fibrously processed bone in hydrogen peroxide; a washing step of washing the fibrously processed bone subjected to the secondary defatting; and a freeze-drying step.

Description

Fibrous demineralized bone graft material and method for manufacturing the same

[0001] The present invention relates to a fibrous demineralized bone graft material and a method for manufacturing the same.

[0002] Bone grafts are used to enhance the natural regenerative process when bone is damaged or injured, and must be biocompatible. Furthermore, they must possess osteoconductive or osteoinductive properties, which are fundamental factors for bone formation, and be easily manipulated during surgery.

[0003] In this regard, U.S. Patent No. 7,323,193 discloses a method of manufacturing a fibrous demineralized bone graft material by cutting bone separated from the body into an appropriate size, decalcifying it in an acidic solution for 6 hours, then performing decalcification in an acidic solution for another 2 days, and then pulverizing it.

[0004] Demineralized bone is an allogeneic bone graft material that contains only the inorganic components of bone, leaving behind organic components and various growth factors, including bone morphogenetic proteins. However, because demineralized bone is manufactured using a softer form of bone tissue compared to the original bone tissue, it has the disadvantage of being difficult to use as a support for bone defects.

[0005] Furthermore, in the case of powder-type bone graft materials used in existing bone grafts, they easily disperse in the body after transplantation and migrate to areas other than the defect site. Furthermore, existing powder-type bone graft materials have a relatively small surface area per unit mass, requiring a long hydration time. This necessitates prior hydration of the bone graft material. If this process is not performed in advance, the surgical site must remain open, which can lead to contamination and infection of the graft material or the patient during this process.

[0006] The present invention is intended to solve the problems of the prior art described above, and therefore, an object of the present invention is to provide a method for producing fibrous bone tissue for cortical bone tissue to which a demineralization process has not been applied, and a fibrous non-demineralized bone graft material produced thereby.

[0007] One aspect of the present invention provides a method for manufacturing a fibrous non-decalcified bone graft material, comprising: a step of removing soft tissue, blood, and bone marrow from a bone; a dehydration step of immersing the bone from which the soft tissue, blood, and bone marrow have been removed in an aqueous ethanol solution and stirring it; a first defatting step of immersing the dehydrated bone in diethyl ether and stirring it; a step of crushing the first defatted bone into fibrous bone; a step of selecting the fibrous bone; a hydration step of immersing the selected fibrous bone in an aqueous ethanol solution and stirring it; a second defatting step of immersing the hydrated fibrous bone in hydrogen peroxide and stirring it; a washing step of washing the second defatted fibrous bone; and a freeze-drying step.

[0008] In one embodiment, in the dehydration step, the bones are placed in an ethanol solution of 50% or more and 99.5% or less, stirred for 12 hours, and dried for 2 hours or more after stirring is completed.

[0009] In one embodiment, in the first degreasing step, the mixture may be stirred in diethyl ether of 80% or more and 100% or less for 6 hours, and after stirring, the mixture may be dried for 2 hours or more.

[0010] In one embodiment, in the step of selecting the bone processed into a fiber shape, a sieve may be used to select the bone processed into a fiber shape of 400 μm or more.

[0011] In one embodiment, in the hydration step, the bone processed into a fiber may be placed in an ethanol solution of 50% or more and 99.5% or less and stirred for 3 hours or more.

[0012] In one embodiment, in the second degreasing step, the bone processed into a fiber may be placed in hydrogen peroxide solution of 1% or more and 10% or less and stirred for 1 hour or more.

[0013] In one embodiment, in the crushing step, the bone may be crushed into fibrous bone having a major axis length in the range of 400 to 150,000 μm.

[0014] Another aspect of the present invention provides a fibrous non-demineralized bone graft material manufactured by a method for manufacturing a fibrous non-demineralized bone graft material, and having a volume per unit mass of 1.20 to 1.60 cc / g.

[0015] According to one aspect of the present invention, the fibrous non-decalcified bone graft material has properties similar to living bone tissue through its hardness compared to demineralized bone graft materials, and thus, when applied to a bone defect site, it not only promotes bone regeneration but also acts as a bone support.

[0016] In particular, fibrous demineralized bone grafting materials minimize dispersal from defect sites after in vivo implantation due to their inter-fiber aggregation properties. Therefore, they are highly applicable in the dental bone graft market, where support and shape must be maintained in the defect site where a pre-existing tooth once existed.

[0017] In addition, the fibrous demineralized bone graft material has a larger surface area per unit mass than the existing powder-type bone graft material, enabling rapid hydration.

[0018] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the composition of the invention described in the claims.

[0019] Figure 1 is a step diagram of a method for manufacturing a fibrous demineralized bone graft material according to one embodiment of the present invention.

[0020] Figure 2 (a) is a photograph of a fibrous non-decalcified bone graft, and (b) is a photograph of a fibrous demineralized bone graft.

[0021] Figure 3 is a scanning electron microscope photograph of a fibrous, non-calcified bone graft material.

[0022] Figure 4 is a scanning electron microscope photograph of a fibrous demineralized bone graft material.

[0023] Figure 5 (a) is a photo of a volume measurement test of a fibrous non-demineralized bone graft material, and (b) is a photo of a volume measurement test of a fibrous demineralized bone graft material.

[0024] Figure 6 shows the results of a compressive strength test for a fibrous non-demineralized bone graft material and a fibrous demineralized bone graft material.

[0025] Figure 7 shows the results of a moisture absorption test for a fibrous demineralized bone graft and a powdered cortical bone graft.

[0026] Hereinafter, the present invention will be described with reference to the attached drawings. However, the present invention can be implemented in various different forms and is therefore not limited to the embodiments described herein. In the drawings, irrelevant parts have been omitted for clarity of description, and similar parts have been designated with similar reference numerals throughout the specification.

[0027] Throughout the specification, when a part is said to be "connected" to another part, this includes not only "directly connected" but also "indirectly connected" with other elements intervening. Furthermore, when a part is said to "include" a component, this does not exclude other components, but rather implies that the other components may be included, unless otherwise specifically stated.

[0028] Terms containing ordinal numbers, such as "first" or "second," used herein may be used to describe various components or steps, but such components or steps are not limited by the ordinal numbers. Terms containing ordinal numbers should be interpreted only to distinguish one component or step from other components or steps.

[0029] Hereinafter, a method for manufacturing a fibrous demineralized bone graft material according to one embodiment of the present invention is described.

[0030] Figure 1 is a step diagram of a method for manufacturing a fibrous demineralized bone graft material according to one embodiment of the present invention.

[0031] Referring to FIG. 1, a method for manufacturing a fibrous non-demineralized bone graft material according to one embodiment of the present invention may include a step of removing soft tissue, blood, and bone marrow of a bone (S1), a dehydration step (S2), a first defatting step (S3), a step of crushing bone into fibrous processed bone (S4), a step of selecting the fibrous processed bone (S5), a hydration step (S6), a second defatting step (S7), a washing step (S8), and a freeze-drying step (S9).

[0032]

[0033] Step 1: Removal of soft tissue, blood, and bone marrow from the bone (S1)

[0034] The soft tissue attached to the raw material is removed using surgical instruments. The bone, from which the soft tissue has been removed, is then cut into appropriate sizes using a bone cutter. The remaining soft tissue is then completely removed using surgical instruments. The bone, completely free of soft tissue, is placed in a tray, moved to the sink, and placed in a cleaning box. Then, distilled water is used to remove blood, bone marrow, and other debris from the bone surface.

[0035]

[0036] Dehydration stage (S2)

[0037] Bones from which soft tissue has been removed contain a large amount of moisture. This moisture causes phase separation with the delipidating solution, reducing the delipidating effect. Therefore, prior to delipidation, bones from which soft tissue, blood, and marrow have been removed are immersed in an ethanol solution containing at least 50% and no more than 99.5% ethanol. The sealed sterilized bottle containing the bones is stirred at 100 ± 10 rpm for at least 12 hours. After stirring, the bones are dried on an aseptic workbench for at least 2 hours.

[0038]

[0039] 1st degreasing stage (S3)

[0040] Place the dried bones in diethyl ether (80% to 100%) and stir at 100 rpm ± 10 rpm for at least 6 hours. After stirring, dry in a sterile workbench for 12 to 72 hours.

[0041]

[0042] Step of crushing the bone into fibrous bone (S4)

[0043] The dried bone is pulverized using a bone crusher. Since powdered bone grafting materials easily disperse in the body after transplantation and migrate to other areas other than the defect site, the bone is crushed into fibrous bone in the present invention. The fibrous shape refers to a shape in which the major axis of the processed bone is longer than the minor axis and thickness. Furthermore, the fibrous shape refers to a shape in which the processed bone is not a spherical or streamlined particle, but rather a thin and long fiber-like shape like a thread.

[0044] Preferably, the crushing can be performed so as to obtain a bone processed into a fiber having a major axis length in the range of 400 to 150,000 μm, which can be obtained by setting appropriate crushing conditions depending on the type of crusher.

[0045] Preferably, the short axis length and thickness of the bone processed into a fiber can be formed within a range of 100 to 3,000 ㎛.

[0046]

[0047] Step of selecting bone processed into fibers (S5)

[0048] The crushed material is classified into 1,500 ㎛ or more, 850 to 1,500 ㎛, 400 to 850 ㎛, and 400 ㎛ or less through sieves of 400, 850, and 1,500 ㎛. Among the crushed material classified as 400 ㎛ or more, 850 ㎛ or more, and 1,500 ㎛ or more, any non-fibrous bones are removed.

[0049]

[0050] Hydration stage (S6)

[0051] The crushed, fibrous bone is immersed in an ethanol solution of 50% or more and 99.5% or less and stirred at 100 rpm ± 10 rpm for 3 hours or more.

[0052]

[0053] Secondary degreasing step (S7)

[0054] The crushed, fibrous bone is immersed in hydrogen peroxide solution containing 1% or more and 10% or less, and stirred at 100 rpm ± 10 rpm for 1 hour or more.

[0055]

[0056] Washing step (S8)

[0057] The crushed, fibrous bone is placed in sterile purified water and washed three times for 10 minutes each at 100 rpm ± 10 rpm.

[0058]

[0059] Freeze-drying step (S9)

[0060] The crushed, fibrous bone is packaged in an EO (Ethylene Oxide) gas sterilization pouch and then placed in a freeze dryer for freeze-drying.

[0061]

[0062] The fibrous demineralized bone graft material manufactured by the above manufacturing method may be composed of inorganic substances including about 60% hydroxyapatite (including calcium, phosphate, etc.), organic substances including about 30% type 1 collagen, and about 10% water.

[0063] The fibrous demineralized bone graft material can be formed so that the major axis length of the processed bone is more than twice the minor axis length and thickness.

[0064] The fibrous demineralized bone graft material may have a major axis length of 400 to 150,000 ㎛ and a minor axis to major axis ratio of 1:2 to 1:1,500.

[0065]

[0066] Hereinafter, the embodiments of this specification will be described in more detail. However, the experimental results below represent only representative experimental results among the above embodiments, and the scope and content of this specification cannot be interpreted as being reduced or limited by the embodiments, etc. The effects of each of the various implementation examples of this specification that are not explicitly presented below will be specifically described in the relevant sections.

[0067]

[0068] Example 1: Fibrous demineralized bone graft material

[0069] A fibrous demineralized bone graft material was manufactured through steps S1 to S9 of the above-described example.

[0070]

[0071] Comparative Example 1: Fibrous demineralized bone graft material

[0072] Steps S1 to S8 of the manufacturing process of Example 1 were performed in the same manner. After the washing step (S8), the bone was immersed in HCl having a concentration of 0.5 to 2.0 N and stirred for more than 3 hours, the crushed, fibrous bone was placed in sterile purified water, washed three times at 100 rpm ± 10 rpm for 10 minutes each, neutralized with a neutralizing solution (PBS: Phosphate buffered saline), and the crushed, fibrous bone was placed in sterile purified water, washed three times at 100 rpm ± 10 rpm for 10 minutes each, and freeze-dried. Through this, a fibrous demineralized bone graft material was manufactured.

[0073]

[0074] Comparative Example 2: Powdered demineralized bone graft material

[0075] A commercially available product was used as a powder (granular) demineralized bone graft material.

[0076]

[0077] Test Example 1: Changes in the shape of bone graft material depending on the presence or absence of demineralization

[0078] Referring to Fig. 2(a), the fibrous demineralized bone graft material was confirmed to have a long fibrous shape with the naked eye, but referring to Fig. 2(b), the fibrous demineralized bone graft material was confirmed to have a relatively cotton-like shape.

[0079]

[0080] Test Example 2: Changes in bone structure depending on the presence or absence of demineralization

[0081] To measure changes in bone structure according to the demineralization process, scanning electron microscope images of bone graft materials of examples and comparative examples are compared. Figure 3 is a scanning electron microscope image of a fibrous non-demineralized bone graft material, and Figure 4 is a scanning electron microscope image of a fibrous demineralized bone graft material.

[0082] As can be seen in Figures 3 and 4, it was confirmed that the demineralized bone graft material changed the shape of the bone surface.

[0083]

[0084] Test Example 3: Changes in bone graft material properties depending on the presence or absence of demineralization

[0085] The volume per 1 g of the bone graft material manufactured according to Example 1 and Comparative Example 1 was measured 9 times.

[0086]

[0087]

[0088] No. Classification Volume (cc) 1 Example 11.421.431.441.451.461.471.481.491.310 Comparative Example 12.3112.3122.4132.2142.2152.1162.2172182.3

[0089]

[0090] As shown in Fig. 5 and Table 1, the volume (cc) per 1g of the fibrous non-decalcified bone graft material may be 1.2cc or more and 1.6cc or less, preferably 1.3cc or more and 1.5cc or less, and more preferably approximately 1.4cc. It was confirmed that the volume per unit mass of the fibrous non-decalcified bone graft material was reduced compared to the fibrous demineralized bone graft material.

[0091]

[0092] In addition, test specimens of the same size were made from each of the bone graft materials of Example 1 and Comparative Example 1, and the compressive strength was measured three times and the average was calculated. The compressive strength was measured using a universal testing machine (UTM).

[0093]

[0094] Maximum compressive strength (N)Average compressive strength (N)Example 1804112Comparative example 113514

[0095] As shown in Fig. 6 and Table 2, it was confirmed that the compressive strength of the fibrous non-demineralized bone graft material was significantly increased compared to the fibrous demineralized bone graft material.

[0096]

[0097] Test Example 4: Comparison of moisture absorption between fibrous demineralized bone graft and powdered cortical bone graft

[0098] 0.5 g of the fibrous non-decalcified bone graft material according to Example 1 and 0.5 g of the powdered non-decalcified bone graft material according to Comparative Example 2 were each rehydrated with 2.5 g of sterile saline solution and dehydrated as much as possible. Then, the mass of the bone graft materials of Example 1 and Comparative Example 2 before and after rehydration was compared to determine the moisture absorption, and this was repeated 5 times.

[0099]

[0100] Figure 7 and Table 3 show the results of moisture absorption tests for fibrous demineralized bone graft materials and powdered cortical bone graft materials.

[0101]

[0102] Example 1 Comparative Example 21217.55%82.65%2178.77%72.82%3203.35%62.41%4187.43%67.82%5178.32%67.34%Average 193.08%70.61%Standard Deviation 15.22%6.86%

[0103] As shown in Figure 7 and Table 3, it was confirmed that the moisture absorption capacity of the fibrous demineralized bone graft material increased by an average of approximately 2.73 times compared to the powdered demineralized bone graft material.

[0104]

[0105] That is, the fibrous non-decalcified bone graft material according to the present invention has properties similar to living bone tissue through its harder properties compared to demineralized bone graft materials, and thus, when applied to a bone defect site, it not only promotes bone regeneration but also functions as a bone support. Accordingly, after implantation in the body, it can function as a support and cell growth support for the bone defect site, providing a space where bone regeneration cells can migrate and grow within the tissue along the fibers.

[0106] In addition, the fibrous demineralized bone graft material has a larger surface area per unit mass than the existing powder-type demineralized bone graft material, and thus has good moisture absorption, enabling rapid hydration.

[0107] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0108] The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. Step of removing soft tissue, blood and bone marrow from the bone; A dehydration step in which bones from which soft tissue, blood, and bone marrow have been removed are immersed in an ethanol solution and stirred; The first degreasing step involves immersing the dehydrated bones in diethyl ether and stirring them; A step of crushing the first degreasing process into a fibrous bone; A step of selecting bones processed into fibers; A hydration step in which the selected fibrous bone is immersed in ethanol and stirred; A secondary degreasing step in which the processed bone into a fibrous form is immersed in hydrogen peroxide solution and stirred; A washing step for washing the processed fibrous bone that has been subjected to secondary degreasing; and A method for manufacturing a fibrous demineralized bone graft material, comprising a freeze-drying step.

2. In paragraph 1, In the above dehydration step, A method for manufacturing a fibrous demineralized bone graft, characterized in that the bone is placed in an ethanol solution of 50% or more and 99.5% or less, stirred for 12 hours or more, and dried for 2 hours or more after stirring is complete.

3. In paragraph 1, In the first degreasing step above, A method for manufacturing a fibrous demineralized bone graft material, characterized by stirring in diethyl ether of 80% or more and 100% or less for 6 hours, and drying for 2 hours or more after stirring is complete.

4. In paragraph 1, In the step of selecting the bone processed into the above fiber, A method for manufacturing a fibrous demineralized bone graft material, characterized in that bone processed into a fiber size of 400㎛ or more is selected using a sieve.

5. In paragraph 1, In the above hydration step, A method for manufacturing a fibrous demineralized bone graft material, characterized in that the fibrous bone is placed in an ethanol solution of 50% or more and 99.5% or less and stirred for 3 hours or more.

6. In paragraph 1, In the above second degreasing step, A method for manufacturing a fibrous demineralized bone graft material, characterized in that bone processed into a fiber is placed in hydrogen peroxide solution of 1% or more and 10% or less and stirred for 1 hour or more.

7. In paragraph 1, In the above crushing step, A method for producing a fibrous demineralized bone graft material, characterized in that the bone is crushed into fibrous bone having a major axis length in the range of 400 to 150,000 ㎛.

8. A fibrous non-demineralized bone graft material manufactured by the manufacturing method of paragraph 1 and having a volume per unit mass of 1.20 to 1.60 cc / g.

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