CN114053482A - Preparation method of bionic artificial bone with natural spatial structure - Google Patents
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Abstract
The invention discloses a preparation method of a bionic artificial bone with a natural spatial structure, and particularly relates to the technical field of bionic manufacturing of bone tissue engineering, which comprises the following steps: s1, obtaining bone materials S1.1, cutting a dense bone from a large bone of a large bovine or porcine animal, and cutting the cancellous bone into small bone pieces with the length of less than 2cm, the width of less than 2cm and the height of less than 1 cm; s1.2, placing cancellous bones in 0.3-1mol/L sodium citrate solution to soak for 24-48H, wherein the bionic artificial bones are bionic artificial bones with natural spatial structures, are prepared by bionic dense bones of large bones of cattle or pigs, have natural spatial structures, can effectively guide and induce the generation of new tissues, and have high biological safety performance; secondly, the bionic artificial bone adopts compact bone of large bones of cattle or pigs as a raw material, has wide sources and lower cost, and the preparation steps of the bionic artificial bone are simpler and more effective, thereby being convenient for macro preparation and being capable of meeting different bone defect repairs.
Description
Technical Field
The invention relates to the technical field of bionic manufacturing of bone tissue engineering, in particular to a preparation method of a bionic artificial bone with a natural spatial structure.
Background
The loss or dysfunction of human tissue is a major health-threatening problem and is the leading cause of illness and death in humans. Particularly, with the aging of population and the development of industries, transportation, sports, etc., in recent years, there is an increasing demand for repair and replacement of bone tissue defects. For a long time, the bone graft used in clinic mainly comprises autogenous bone and allogeneic bone, but the autogenous bone graft cannot meet the requirements of large-section bone graft and cases of large-range bone defects due to limited material resources, and the allogeneic bone graft slowly influences the functional recovery of patients due to the replacement process, so people are eagerly expected to obtain a bionic artificial bone which can assist the regeneration of new bone and has sufficient source and short healing time;
the ideal bionic artificial bone not only provides structural support for the defect part, but also more importantly creates a microenvironment which is beneficial to cell adhesion, proliferation and function exertion, induces the generation of osteocyte, and gradually degrades and absorbs to finally form new bone. The bionic artificial bone scaffold is required to have good biocompatibility and bioactivity, a micro porous structure suitable for cell growth and proliferation, an appearance matched with a defect part, sufficient mechanical strength and the like;
many biomimetic artificial bone materials have been used in bone reconstruction in the prior art and can be classified as: (1) inorganic materials: such as bioceramics, are biocompatible but relatively inert, and are hard but brittle; (2) organic materials: such as collagen, polyester, etc., which are osteoinductive but have poor mechanical properties; (3) the composite material comprises the following components: such as calcium phosphate-collagen composite artificial bone, PLGA-BMP artificial bone, etc., have both mechanical property and biological activity, but the osteogenesis activity is still not ideal.
However, the prepared structure of the bionic artificial bone used in the prior art is a bionic structure, the biological safety is low, and the preparation raw materials are expensive; secondly, the preparation steps are complex and inconvenient for macro preparation, and therefore, a preparation method of the bionic artificial bone with the natural spatial structure is provided for solving the problems.
Disclosure of Invention
The invention aims to provide a preparation method of a bionic artificial bone with a natural spatial structure, which aims to solve the problems of low biological safety, expensive preparation raw materials, complicated preparation steps and inconvenience in macro preparation in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a preparation method of a bionic artificial bone with a natural spatial structure comprises the following steps:
s1 obtaining of bone material in preparation of bionic artificial bone
S1.1, cutting a large bone of a large bovine or pig animal to remove compact bone, and cutting the cancellous bone into small bone blocks with the length of less than 2cm, the width of less than 2cm and the height of less than 1 cm;
s1.2, placing the cut cancellous bone in a 0.3-1mol/L sodium citrate solution for soaking for 24-48H, wherein the sodium hydroxide solution is replaced every 2 hours, and the whole body needs to be placed in a refrigeration mechanism for refrigeration in the soaking process;
s1.3, after soaking, cleaning with hot water, taking out cancellous bone, placing the cancellous bone in a 10% sodium hydroxide solution containing 0.03% of EDTA disodium, and soaking for 6-12H;
s1.4, after hot water cleaning, placing the cancellous bone in trypsin to be soaked for 18-24H, dissolving the protease, adding sodium chloride, and preparing 0.9% sodium chloride;
s1.5, removing grease of the cancellous bone after cleaning with hot water, then, drying in an oven at 70-100 ℃ for 12H, and obtaining a bone material after drying;
s2 preparation of bionic artificial bone
S2.1, taking out the dried cancellous bone, and adding 5% of NH according to the mass ratio of the cancellous bone to 10 in the bone material table 1:14H2PO4Soaking in the solution for 24-48 hours, and draining the cancellous bone after soaking;
s2.2, placing the calcined bone material into a simulated mineralization solution SBF, continuously soaking the material for 5 days, and enabling mineralized substances HA to enter pores of the calcined bone block;
s2.3, starting the high-temperature furnace, putting bone materials into the high-temperature furnace after the high-temperature furnace is heated to 300 ℃, then heating the temperature to 400 ℃, keeping the temperature for 3H, simultaneously vacuumizing the high-temperature furnace, then introducing nitrogen to keep the temperature for 2H, then heating the temperature to 800 ℃, keeping the temperature for 3H, and finally heating the temperature to 1000 ℃ and keeping the temperature for 3H;
s3 obtaining the natural space structure bionic artificial bone
S3.1, taking out the calcined artificial bone, placing the calcined artificial bone in liquid nitrogen, and fixing for 12-24H;
and S3.2, taking out and packaging, and performing 25KGY irradiation sterilization to obtain the natural spatial structure bionic artificial bone.
As a preferable technical scheme of the invention, the mass ratio of the spongy bone in S1.2 to the 0.3-1mol/L sodium citrate solution is 1 g: 2g of the total weight.
As a preferable technical scheme of the invention, the refrigerating temperature in S1.2 is 2-8 ℃.
As a preferable technical scheme of the invention, the mass ratio of the spongy bone in S1.3 and the 10% sodium hydroxide solution containing 0.03% of EDTA disodium is 1 g: 2g of the total weight.
In a preferred embodiment of the present invention, the mass ratio of cancellous bone to trypsin in S1.4 is 100 g: 1g of the total weight of the composition.
As a preferable technical scheme of the invention, the soaking temperature in the S2.1 is controlled at 37 ℃.
As a preferable technical scheme of the invention, the soaking temperature in the S2.2 is constant temperature of 37 ℃.
Compared with the prior art, the invention has the beneficial effects that:
1. the bionic artificial bone is a natural space structure bionic artificial bone, is prepared by the compact bone of the large bone of a large bovine or porcine animal in a bionic manner, has a natural space structure, can effectively guide and induce the generation of a new tissue, and has high biological safety performance;
2. the bionic artificial bone adopts compact bone of large bones of cattle or pigs as a raw material, has wide sources and lower cost;
3. and the preparation step of bionical artificial bone is simple more effective, at first, carry out bone material and obtain, soak to bone material, wash and detach grease and drying process, afterwards, carry out bionical artificial bone preparation, soak again the cancellous bone of drying and calcine and obtain artificial bone, finally, place the artificial bone of calcining in liquid nitrogen and cool and carry out 25KGY irradiation sterilization, whole preparation step is simple more effective to be convenient for carry out the macro preparation, can satisfy different bone loss and restore.
Drawings
FIG. 1 is a schematic diagram of the steps of preparing a biomimetic artificial bone according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example (b): as shown in figure 1, the invention provides a preparation method of a bionic artificial bone with a natural space structure, which comprises the following steps:
s1 obtaining of bone material in preparation of bionic artificial bone
S1.1, cutting a large bone of a large bovine or pig animal to remove compact bone, and cutting the cancellous bone into small bone blocks with the length of less than 2cm, the width of less than 2cm and the height of less than 1 cm; the compact bone of the large bovine or porcine animal is selected as the raw material, so that the bionic artificial bone has wide material sources, and the material cost of the bionic artificial bone is low, thereby facilitating the macro preparation;
s1.2, placing the cut cancellous bone in a 0.3-1mol/L sodium citrate solution for soaking for 24-48H, wherein the sodium hydroxide solution is replaced every 2 hours, and the whole body needs to be placed in a refrigeration mechanism for refrigeration in the soaking process;
s1.3, after soaking, cleaning with hot water, taking out cancellous bone, placing the cancellous bone in a 10% sodium hydroxide solution containing 0.03% of EDTA disodium, and soaking for 6-12H; the soaked cancellous bone is taken out and washed by hot water, so that the damage of the cancellous bone space structure can be effectively prevented, and the quality of the cancellous bone is improved;
s1.4, after hot water cleaning, placing the cancellous bone in trypsin to be soaked for 18-24H, dissolving the protease, adding sodium chloride, and preparing 0.9% sodium chloride;
s1.5, removing grease of the cancellous bone after cleaning with hot water, then, drying in an oven at 70-100 ℃ for 12H, and obtaining a bone material after drying; after the spongy bone is cleaned by hot water, grease of the spongy bone needs to be removed, so that the grease in the spongy bone is prevented from influencing the preparation quality of the spongy bone, and the overall quality of the spongy bone is further improved;
s2 preparation of bionic artificial bone
S2.1, taking out the dried cancellous bone, and adding 5% of NH according to the mass ratio of the cancellous bone to 10 in the bone material table 1:14H2PO4Soaking in the solution for 24-48 hours, and draining the cancellous bone after soaking;
s2.2, placing the calcined bone material into a simulated mineralization solution SBF, continuously soaking the material for 5 days, and enabling mineralized substances HA to enter pores of the calcined bone block;
s2.3, starting the high-temperature furnace, putting bone materials into the high-temperature furnace after the high-temperature furnace is heated to 300 ℃, then heating the temperature to 400 ℃, keeping the temperature for 3H, simultaneously vacuumizing the high-temperature furnace, then introducing nitrogen to keep the temperature for 2H, then heating the temperature to 800 ℃, keeping the temperature for 3H, and finally heating the temperature to 1000 ℃ and keeping the temperature for 3H; the bone material is calcined in stages, wherein the calcining temperature is 400 ℃ of 3H, the calcining temperature is 800 ℃ of 3H, and the calcining temperature is 1000 ℃ of 3H, so that the bone material can be completely and effectively calcined, and the overall quality of the cancellous bone is improved;
s3 obtaining the natural space structure bionic artificial bone
S3.1, taking out the calcined artificial bone, placing the calcined artificial bone in liquid nitrogen, and fixing for 12-24H; cooling the calcined artificial bone by liquid nitrogen;
s3.2, taking out and packaging, and performing 25KGY irradiation sterilization to obtain the natural spatial structure bionic artificial bone;
the bionic artificial bone obtained by the invention is a bionic artificial bone with a natural spatial structure, is prepared by biomimetically compacting bone of large bones of large cattle or pigs, has a natural spatial structure, can effectively guide and induce the generation of new tissues, has high biological safety performance, and is simple and effective in preparation steps, thereby facilitating the macro preparation and meeting the requirements of different bone defect repairs.
Further, the mass ratio of the spongy bone in S1.2 to the 0.3-1mol/L sodium citrate solution is 1 g: 2g of the total weight.
Further, the refrigerating temperature in the S1.2 is 2-8 ℃.
Further, in the S1.3, the mass ratio of the spongy bone to the 10% sodium hydroxide solution containing 0.03% disodium EDTA is 1 g: 2g of the total weight.
Further, the mass ratio of the cancellous bone to the trypsin in the S1.4 is 100 g: 1g of the total weight of the composition.
Further, the soaking temperature in S2.1 is controlled at 37 ℃.
Further, the soaking temperature in the S2.2 is constant temperature of 37 ℃.
The bionic artificial bone is a natural space structure bionic artificial bone, is prepared by the compact bone of the large bone of a large bovine or porcine animal in a bionic manner, has a natural space structure, can effectively guide and induce the generation of a new tissue, and has high biological safety performance; secondly, bionic artificial bone adopts the compact bone of big bone of ox or pig macrozoo as the raw materials, the source is extensive, the cost is lower, and the preparation step of bionic artificial bone is more simple effective, firstly, carry out bone material and obtain, soak to bone material, wash and detach grease and drying process, afterwards, carry out the preparation of bionic artificial bone, soak again and calcine the cancellous bone of stoving and obtain artificial bone, finally, place the artificial bone of calcining in the liquid nitrogen and cool off and carry out 25KGY irradiation sterilization, whole preparation step is more simple effective, thereby be convenient for carry out the macro preparation, can satisfy different bone loss and restore.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A preparation method of a bionic artificial bone with a natural spatial structure is characterized by comprising the following steps:
s1, obtaining bone materials in the preparation of the bionic artificial bone; s2, preparing a bionic artificial bone; s3, obtaining a natural space structure bionic artificial bone;
the S1 includes:
s1.1, cutting a large bone of a large bovine or pig animal to remove compact bone, and cutting the cancellous bone into small bone blocks with the length of less than 2cm, the width of less than 2cm and the height of less than 1 cm;
s1.2, placing the cut cancellous bone in a 0.3-1mol/L sodium citrate solution for soaking for 24-48H, wherein the sodium hydroxide solution is replaced every 2 hours, and the whole body needs to be placed in a refrigeration mechanism for refrigeration in the soaking process;
s1.3, after soaking, cleaning with hot water, taking out the cancellous bone, placing the cancellous bone into a 10% sodium hydroxide solution containing 0.03% of EDTA disodium, soaking for 6-12H, after cleaning with hot water, placing the cancellous bone into trypsin, soaking for 18-24H, dissolving the protease, adding sodium chloride, and preparing into 0.9% sodium chloride;
s1.4, removing grease of the cancellous bone after cleaning with hot water, then, drying in an oven at 70-100 ℃ for 12H, and obtaining a bone material after drying;
the S2 includes:
s2.1, taking out the dried cancellous bone, and adding 5% of NH according to the mass ratio of the cancellous bone to 10 in the bone material table 1:14H2PO4Soaking in the solution for 24-48 hours, and draining the cancellous bone after soaking;
s2.2, placing the calcined bone material into a simulated mineralization solution SBF, continuously soaking the material for 5 days, and enabling mineralized substances HA to enter pores of the calcined bone block;
s2.3, starting the high-temperature furnace, putting bone materials into the high-temperature furnace after the high-temperature furnace is heated to 300 ℃, then heating the temperature to 400 ℃, keeping the temperature for 3H, simultaneously vacuumizing the high-temperature furnace, then introducing nitrogen to keep the temperature for 2H, then heating the temperature to 800 ℃, keeping the temperature for 3H, and finally heating the temperature to 1000 ℃ and keeping the temperature for 3H;
the S3 includes:
s3 obtaining the natural space structure bionic artificial bone
S3.1, taking out the calcined artificial bone, placing the calcined artificial bone in liquid nitrogen, and fixing for 12-24H;
and S3.2, taking out and packaging, and performing 25KGY irradiation sterilization to obtain the natural spatial structure bionic artificial bone.
2. The method for preparing the bionic artificial bone with the natural spatial structure according to claim 1, which is characterized in that: the mass ratio of the spongy bone in S1.2 to the 0.3-1mol/L sodium citrate solution is 1 g: 2g of the total weight.
3. The method for preparing the bionic artificial bone with the natural spatial structure according to claim 1, which is characterized in that: and the refrigerating temperature in the S1.2 is 2-8 ℃.
4. The method for preparing the bionic artificial bone with the natural spatial structure according to claim 1, which is characterized in that: the mass ratio of the spongy bone in S1.3 to the 10% sodium hydroxide solution containing 0.03% disodium EDTA is 1 g: 2g of the total weight.
5. The method for preparing the bionic artificial bone with the natural spatial structure according to claim 1, which is characterized in that: the mass ratio of the cancellous bone to the trypsin in the S1.4 is 100 g: 1g of the total weight of the composition.
6. The method for preparing the bionic artificial bone with the natural spatial structure according to claim 1, which is characterized in that: the soaking temperature in the S2.1 is controlled at 37 ℃.
7. The method for preparing the bionic artificial bone with the natural spatial structure according to claim 1, which is characterized in that: and the soaking temperature in the S2.2 is constant 37 ℃.
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| 王海彬: "EDTA在骨脱钙中的应用", 《中山大学学报》 * |
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