CN116531566A - Calcined bone meal without adding chemical reagent and preparation method and application thereof - Google Patents
Calcined bone meal without adding chemical reagent and preparation method and application thereof Download PDFInfo
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- CN116531566A CN116531566A CN202310471568.0A CN202310471568A CN116531566A CN 116531566 A CN116531566 A CN 116531566A CN 202310471568 A CN202310471568 A CN 202310471568A CN 116531566 A CN116531566 A CN 116531566A
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- bone powder
- bone
- calcined
- chemical reagent
- pressure treatment
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- 238000002360 preparation method Methods 0.000 title claims abstract description 15
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- 239000002374 bone meal Substances 0.000 title claims description 11
- 210000000988 bone and bone Anatomy 0.000 claims abstract description 116
- 239000000843 powder Substances 0.000 claims abstract description 69
- 238000001354 calcination Methods 0.000 claims abstract description 12
- 238000005520 cutting process Methods 0.000 claims abstract description 11
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- 238000000498 ball milling Methods 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims abstract description 5
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- 238000001816 cooling Methods 0.000 claims abstract description 4
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- 238000000034 method Methods 0.000 claims description 24
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- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 2
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- HOBWAPHTEJGALG-JKCMADFCSA-N [(1r,5s)-8-methyl-8-azoniabicyclo[3.2.1]octan-3-yl] 3-hydroxy-2-phenylpropanoate;sulfate Chemical compound [O-]S([O-])(=O)=O.C([C@H]1CC[C@@H](C2)[NH+]1C)C2OC(=O)C(CO)C1=CC=CC=C1.C([C@H]1CC[C@@H](C2)[NH+]1C)C2OC(=O)C(CO)C1=CC=CC=C1 HOBWAPHTEJGALG-JKCMADFCSA-N 0.000 description 1
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- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 description 1
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- 239000011575 calcium Substances 0.000 description 1
- BRPQOXSCLDDYGP-UHFFFAOYSA-N calcium oxide Chemical compound [O-2].[Ca+2] BRPQOXSCLDDYGP-UHFFFAOYSA-N 0.000 description 1
- 239000000292 calcium oxide Substances 0.000 description 1
- ODINCKMPIJJUCX-UHFFFAOYSA-N calcium oxide Inorganic materials [Ca]=O ODINCKMPIJJUCX-UHFFFAOYSA-N 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
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- 235000019441 ethanol Nutrition 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
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- 238000002695 general anesthesia Methods 0.000 description 1
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- 238000003837 high-temperature calcination Methods 0.000 description 1
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- LSQZJLSUYDQPKJ-UHFFFAOYSA-N p-Hydroxyampicillin Natural products O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)C(N)C1=CC=C(O)C=C1 LSQZJLSUYDQPKJ-UHFFFAOYSA-N 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 1
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- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
- A61L27/3645—Connective tissue
- A61L27/365—Bones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/12—Materials or treatment for tissue regeneration for dental implants or prostheses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
Abstract
The invention discloses a calcined bone powder without adding chemical reagents, and a preparation method and application thereof. The preparation method of the calcined bone powder comprises the following steps: firstly, cutting the pretreated milk cow bone blocks into small blocks, putting the small blocks into a pressure cooker, carrying out high-pressure treatment, repeating the high-pressure treatment for a plurality of times, separating liquid in the pressure cooker after each high-pressure treatment, carrying out next high-pressure treatment, removing softened cortical bone when the heat is still hot after the last high-pressure treatment is finished, and washing to be neutral after ultrasonic cleaning; calcining the washed sample at 700-900 ℃ for 6-10 hours, naturally cooling, ball milling, sieving, and finally ultrasonic cleaning and drying to obtain the calcined bone powder without adding the chemical reagent. The invention does not use chemical reagent, solves the problem of chemical reagent residue in the existing bone powder preparation process, ensures that the bone powder meets the specification, has better bone cell growth promotion effect and has better application prospect.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to calcined bone meal without adding chemical reagents, and a preparation method and application thereof.
Background
With the continuous development of science and technology, more and more people realize the influence of oral health problems on the daily life of people. Dental bone powder is used as the most widely used biological material in stomatology, but the expensive price makes the dental bone powder prohibitively expensive, and delays treatment and even causes serious consequences. The most clinically cited materials in stomatology are Geistlich Bio-Oss under Geistlich or Geistlich Bio-Oss collagen, and the main production method is to subject cancellous bone of milk cow bone to degreasing and decellularization treatment and then calcine to remove immune sources. The main flow of the dental calcined bone powder at the present stage is divided into three types: alkali method, high pressure method and methanol chloroform method. The methanol chloroform method has been faded out of the field of view of people due to the uncontrollable cytotoxicity, and more people prefer to use an alkaline method, namely, the method of obtaining the product by respectively soaking the bovine spongy bone in NaOH solution and high-concentration hydrogen peroxide for degreasing and decellularizing treatment and then calcining at high temperature. In view of the problem that the chemical reagent residues and the high-concentration hydrogen peroxide are dangerous in the final product due to long-time soaking by using the chemical reagent, it is very necessary to provide a preparation method of dental calcined bone powder without introducing any chemical reagent.
Disclosure of Invention
Aiming at overcoming the defects of the prior art, the invention aims to provide calcined bone powder without adding chemical reagent, and a preparation method and application thereof.
The aim of the invention is achieved by the following technical scheme:
a preparation method of calcined bone powder without adding chemical reagent comprises the following steps:
(1) Firstly, cutting the pretreated milk cow bone blocks into small blocks, putting the small blocks into a pressure cooker, carrying out high-pressure treatment, repeating the high-pressure treatment for a plurality of times, separating liquid in the pressure cooker after each high-pressure treatment, carrying out next high-pressure treatment, removing softened cortical bone when the heat is still hot after the last high-pressure treatment is finished, and washing to be neutral after ultrasonic cleaning;
(2) Calcining the washed sample in the step (1) at 700-900 ℃ for 6-10 hours, naturally cooling, ball milling, sieving, ultrasonic cleaning and drying to obtain the calcined bone powder without adding the chemical reagent.
Preferably, the pretreatment in step (1) is performed in the following manner: taking femur and tibia of fresh dairy cow, freezing, cutting into bone blocks with width of about 1cm, placing into container, adding clear water cover without bone blocks, boiling for 40min, removing soft tissue and cartilage tissue attached to bone, cutting into appropriate size, packaging, and freezing in freezer (below-20deg.C).
Preferably, in the step (1), the pressure of the high-pressure treatment is 1.5 to 2 times of the standard atmospheric pressure.
Preferably, in the step (1), the time of the high-pressure treatment is 1-5 hours each time.
Preferably, in step (1), the washing to neutrality is performed in the following manner: washing with distilled water.
Preferably, in the step (2), the heating rate of calcination is 2-4 ℃/min, and the applicant tests that the heating rate of 1-10 ℃/min is used as a test range, so that the influence of the heating rate of 2-4 ℃/min on the product is small, and the condition of uneven combustion can occur when the heating rate is too large.
Preferably, in the step (2), the particle diameter selected by sieving is 0.335-8 mm.
Preferably, in the step (2), the ultrasonic cleaning mode is as follows: adopting absolute ethyl alcohol and deionized water to alternatively ultrasonically clean for a plurality of times.
The calcined bone powder is prepared by the preparation method of the calcined bone powder without adding chemical reagents.
The calcined bone powder is used as dental bone powder.
The principle or mechanism involved in the invention is as follows:
the components of the bovine spongiform bone are similar to those of human spongiform bone, but the heterogeneous bone has immunogenicity to human body, and if the heterogeneous bone cannot be completely inactivated, the human body can generate immune rejection reaction, and the human body can be infected with zoonosis and the like. The method generally adopts hydrogen peroxide and sodium hydroxide solution to remove organic components, adopts long-time high pressure to basically remove cells, proteins, lipids and the like existing in the cancellous bone of the dairy cow, and then completely inactivates the cells, proteins, lipids and the like through high-temperature calcination to ensure that the cells, proteins, lipids and the like do not exist immunogenicity any more, and only leaves inorganic components such as hydroxyapatite calcium sulfate and the like in the cancellous bone of the dairy cow. The mechanism of the calcined bone powder which shows better performance in cell experiments has not been fully explored, and the inventor speculates that the calcined bone powder does not convert certain components of the cancellous bone of the milk cow bone due to the fact that no chemical reagent is used, and certain component structures of animal bones are reserved; or the cells, proteins and lipids of the cancellous bone of the bovine bone are not completely removed under high pressure, but are only inactivated, and certain components suitable for the mesenchymal stem cells of the bone marrow are obtained through calcination.
Compared with the prior art, the invention has the beneficial effects that:
the preparation method of the calcined bone powder is simple in operation and simple in preparation process, and the main component of the bone powder prepared by the method is still hydroxyapatite by comparing the infrared and X-ray results of the existing bone powder and products sold in the market at home and abroad. And according to national standard GB23101.3-2010/ISO13779-3:2008, the bone powder prepared by the method is characterized by calcium-phosphorus ratio, purity, crystallinity and the like of hydroxyapatite, and the bone powder prepared by the method meets the specification and has industrialization possibility. And compared with the cell experimental results of the commercial products, the bone powder prepared by the invention has better effect of promoting the growth of bone cells.
Drawings
FIG. 1 is a photograph showing the calcined bone powder prepared in example 1 of the present invention.
FIG. 2 is a scanning electron microscope image of the calcined bone meal prepared in example 1 of the present invention.
FIG. 3 is an elemental mapping of the calcined bone meal prepared in example 1 of the present invention.
FIG. 4 is an EDS spectrum analysis of the calcined bone powder prepared in example 1 of the present invention.
FIG. 5 is an XRD contrast pattern of Bei Aolu bioceramic-artificial bone, straumann XenoGraft, geistlich Bio-Os and calcined bone powder prepared in example 1 of the present invention.
FIG. 6 is a chart showing the Fourier IR spectrum of the calcined bone powder prepared in example 1 of the present invention and the bone powder prepared in comparative examples 1 to 2.
FIG. 7 is a graph showing the IR spectrum of calcined bone powder, bei Aolu bioceramic-artificial bone, straumann XenoGraft and Geistlich Bio-Os prepared in example 1 of the present invention.
FIG. 8 is a graph showing comparison of the results of cytotoxicity test of CCK-8 on calcined bone powder, bei Aolu bioceramic-artificial bone, straumann XenoGraft and Geistlich Bio-Os prepared in example 1 of the present invention.
FIG. 9 is a graph showing the results of the experiment of the living and dead cells of the bone marrow mesenchymal stem cells of rat, which is prepared in example 1 of the present invention.
Fig. 10 is a CT image of the calcined bone powder prepared in example 1 of the present invention for rabbit tooth extraction, in which BP is an experimental group and sham is a blank group.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Pretreatment of bones:
purchasing femur and tibia of fresh cow, freezing in freezer (-20deg.C) and cutting into bone pieces with width of about 1cm, boiling in iron pan with clear water cover for 40min, removing soft tissue and cartilage tissue, cutting into suitable size, packaging, freezing in freezer (-20deg.C), and storing. The following examples and comparative examples used bone pieces after this pretreatment.
Example 1
A preparation method of calcined bone powder without adding chemical reagent comprises the following steps:
cutting the pretreated bone blocks into blocks with the size of 1 x 1cm as much as possible, placing the blocks into a container sleeved with a net bag, adding water, placing the container into a pressure cooker, treating the container for 1 hour at 1.5-2 times of normal pressure, repeating the process for 2-3 times according to the amount of each treatment, pouring out liquid converged at the bottom of the container, removing softened cortical bone when the container is hot after high pressure is finished, washing the cortical bone to PH=7 by using a large amount of distilled water after ultrasonic cleaning, taking out the container and placing the container into a muffle furnace, heating the container to 800 ℃ at 2 ℃/min, calcining the container for 2 hours, and naturally cooling the container to room temperature; the process must make the bone blocks spread in the container, the stacking condition can not occur, the calcination can not be covered, the heating speed can not be more than 4 ℃/min, and the uneven burning condition can not occur. Taking out a sample, ball milling, sieving, selecting particles with the diameter of 0.335-8 mm, alternately ultrasonically cleaning for three times by using absolute ethyl alcohol and deionized water, and drying to obtain the calcined bone powder.
Example 2
The procedure was as in example 1, except that the calcination temperature was changed to 700 ℃.
Example 3
The procedure was as in example 1, except that the calcination temperature was changed to 900 ℃.
Example 4
The procedure was as in example 1, except that the calcination temperature rise rate was changed to 3℃per minute.
Comparative example 1
A preparation method of bone powder comprises the following steps:
cutting the pretreated bone pieces into proper sizes of not more than 1 x 2cm, and then boiling and drying; soaking the processed bone blocks in 0.5M NaOH solution overnight, boiling for 15min to remove softened cortical bone, treating in 30% hydrogen peroxide overnight, ultrasonically cleaning, washing with a large amount of distilled water to reach pH=7, placing into a muffle furnace for high-temperature sintering at 800 ℃ for 2h (heating rate is 2 ℃/min), taking out for ball milling, sieving, selecting particles with the diameter of 0.2-1 mm, alternately ultrasonically cleaning with absolute ethyl alcohol and deionized water for three times, and drying to obtain bone powder.
Comparative example 2
The pre-treated bone pieces were cut to a suitable size of no more than 1 x 2cm, then boiled and dried. Placing the processed bone blocks in an autoclave for 2 hours at 3 times of normal pressure, peeling off cortical bone, soaking in 30% hydrogen peroxide for overnight treatment, ultrasonically cleaning, washing with a large amount of distilled water to reach PH=7, then placing in a muffle furnace for high-temperature sintering at 800 ℃ for 2 hours (the heating rate is 2 ℃/min), taking out for ball milling, sieving, selecting particles with the diameter of 0.2-1 mm, alternately ultrasonically cleaning for three times with absolute ethyl alcohol and deionized water, and drying to obtain bone powder.
Performance testing
The appearance of the aggregates prepared in examples 2-4 was substantially the same as in example 1, and therefore no additional comparison was made in the figures.
FIG. 1 is a photograph showing the calcined bone powder prepared in example 1 of the present invention. From figure 1, it can be seen that the product has obvious pore structure, is granular and is convenient for clinical use.
FIG. 2 is a scanning electron microscope image of the calcined bone meal prepared in example 1 of the present invention. As can be seen from fig. 2: the calcined bone powder prepared by the invention has a natural three-dimensional hole structure. The pore size of the product is 150-500 mu m, wherein the pore size close to 150 mu m is suitable for the inward migration of osteoblasts, and the growth of bone tissues is induced; macropores larger than 300 mu m have high permeability and angiogenesis potential, and are beneficial to promoting bone growth and angiogenesis.
FIG. 3 is an elemental mapping of the calcined bone meal prepared in example 1 of the present invention. As can be seen from fig. 3: the calcined bone powder has uniform distribution of each element and compact structure.
FIG. 4 is an EDS spectrum analysis of the calcined bone powder prepared in example 1 of the present invention. As can be seen from fig. 4: the Ca of the bone powder is P=2.08 >1.667, which is far greater than national standard GB23101.3-2010/ISO13779-3:2008, meets the national standard.
FIG. 5 is a XRD contrast pattern of Bei Aolu bioceramic-artificial bone, straumann XenoGraft, geistlich Bio-Os and calcined bone powder prepared in example 1 of the present invention, wherein β is Bei Aolu bioceramic-artificial bone, SX is Straumann XenoGraft, BIO is Geistlich Bio-Os, and BP is calcined bone powder prepared in example 1. As can be seen from fig. 5: the bone powder prepared by the invention mainly comprises hydroxyapatite, and contains a small amount of beta-tricalcium phosphate and calcium oxide, wherein the crystallinity of the hydroxyapatite is smaller in crystal granularity and higher in crystal phase content compared with a commercial product, and is closer to the national standard.
Fig. 6 is a fourier infrared spectrum comparison chart of the calcined bone powder prepared in example 1 of the present invention and the bone powder prepared in comparative examples 1 to 2, as can be seen from fig. 6: the bone powder prepared by the invention is respectively 3575cm -1 、1046 cm -1 、570 cm -1 Has characteristic peak of-OH stretch and PO 4 3- The main component of the calcined bone powder is hydroxyapatite, which is known as the characteristic peak and the O-P-O band. And there was no significant difference from the products obtained in comparative examples 1 to 2.
FIG. 7 is a graph showing the IR spectra of calcined bone meal prepared in example 1 of the present invention, bei Aolu bioceramic-artificial bone, straumann XenoGraft and Geistlich Bio-Oss, wherein β is Bei Aolu bioceramic-artificial bone, SX is Straumann XenoGraft, BIO is Geistlich Bio-Oss, BP is calcined bone meal prepared in example 1. As can be seen from fig. 7: compared with the products sold in foreign countries, the bone powder provided by the invention has similar characteristic peak values.
FIG. 8 is a graph showing comparison of the results of cytotoxicity test of CCK-8 on calcined bone powder, bei Aolu bioceramic-artificial bone, straumann XenoGraft and Geistlich Bio-Os prepared in example 1 of the present invention, as can be seen from FIG. 8: the bone powder prepared by the invention and the rat bone marrow mesenchymal stem cells are mixed and cultured for 24-72 h, so that the bone powder does not influence the growth of the cells, has no cytotoxicity, and has better growth promotion and differentiation effects on the cells compared with the existing products at 72 h.
The steps of CCK-8 cytotoxicity detection are as follows:
the bone powder is sterilized by high-pressure steam for standby. Then, the bone meal was taken and added to DMEM medium (containing fetal calf serum) at a concentration of 0.01 g/mL. At 37℃with 100% relative humidity, 5 v/v% CO 2 Is taken out after 24 hours in the incubator, and is filtered and sterilized by a 0.22 mu m filter membrane to be used as leaching liquor.
The bone marrow mesenchymal stem cells were resuscitated and passaged using DMEM medium containing 10% Fetal Bovine Serum (FBS) and diabodies (containing 100 μg/mL penicillin and 100 μg/mL streptomycin); placing the culture bottle containing cells in a cell culture box for feedingLine culture (temperature 37 ℃,5% CO) 2 ) Changing the culture medium according to the growth condition of the cells; cells in the logarithmic phase are digested with 0.25% trypsin to suspend the cells, a certain amount of fresh culture medium is added after the cells are counted, and the cells are blown and centrifuged to prepare a cell suspension containing a certain number of cells for standby.
The cell suspension to be used was 1X 10 4 cells/mL, 100. Mu.L per well, at 100% relative humidity, 5 v/v% CO, were inoculated into 96-well plates using a pipette 2 Culturing in an incubator at 37 ℃ for 24 hours, removing the original culture solution by a liquid-transferring gun after the cells are grown on the wall completely, replacing the original culture medium, adding 100 mu L of sterilized leaching solution as an experimental group, and culturing for 24 hours. The control group was added with an equal amount of DMEM medium containing 10% fetal bovine serum (fetal bovine serum, FBS).
After the completion of the incubation, 10. Mu.L of CCK-8 reagent was added to each well, the incubation was continued in an incubator for 3 hours, the original liquid was carefully aspirated and discarded, 100. Mu.L of dimethyl sulfoxide solution (dimethyl sulfoxide, DMSO) was added to each well, and the mixture was shaken at a low speed to measure the absorbance (OD) of the sample using an ELISA (lambda=450 nm). Cell viability was calculated according to the formula and evaluated according to the numerical control criteria.
Relative cell viability=
Wherein RCV-relative cell viability (%); OD (optical density) 1 Absorbance, OD of the sample group 2 Absorbance, OD for blank (medium formulated alone, cell free) 3 Absorbance of negative control group (medium and cells formulated only, no sample), experiment was repeated three times.
FIG. 9 is a graph showing the results of the experiment of the living and dead cells of the bone marrow mesenchymal stem cells of rat, which is prepared in example 1 of the present invention. As can be seen from fig. 9: the calcined bone powder prepared by the invention is basically nontoxic to the mesenchymal stem cells of rats.
Fig. 10 is a CT image of the calcined bone powder prepared in example 1 of the present invention for a rabbit tooth extraction experiment, in which the experimental group and the blank control group are sequentially arranged from left to right. The experimental group fills the patent product and sews the wound after tooth extraction, and the blank group is subjected to simulated operation but not tooth extraction, and is all fed for 4 months to be killed. As can be seen from fig. 10: the bone powder prepared by the invention has good osteogenesis effect.
The rabbit tooth extraction experiment comprises the following steps:
and (3) establishing an animal model: the method comprises the steps of carrying out intramuscular injection on a healthy New Zealand white rabbit by atropine sulfate half an hour before operation, carrying out general anesthesia by using sodium pentobarbital through an ear vein, and taking a supine position to fix limbs on an operating table after the muscles of limbs of an experimental animal are completely relaxed, cornea reflection disappears and breathing is stable. Atecan and epinephrine are locally infiltrated and anesthetized. On one hand, the skin around the oral cavity is disinfected and then the gingiva is gently separated by a probe, on the other hand, the pain response of an animal is closely observed while the gingiva is separated, a minimally invasive tooth extraction collar is embedded into the left middle incisor, periodontal ligament tissues and gaps are cut off and separated, after the teeth are touched, loose teeth are completely extracted by using a tooth extraction forceps, and the shape and the structure of corresponding tissues of dental alveolar ridges are kept at the moment in the tooth extraction process. Cleaning tissue fragments in the tooth socket, and implanting 0.1g of bone powder prepared in example 1 into an experimental group; animals in the blank group did not extract teeth. After the local wound is cleaned, the gum is tightly sutured by silk threads, and amoxicillin injection is injected.
Post-operative treatment: after the animals wake up, buprenorphine hydrochloride is administered for about 3-4 hours to relieve pain. The experimental animals were simultaneously given intramuscular injection of penicillin injection to prevent infection of tooth sockets for 3 consecutive days, 1 time per day. During the period, the experimental object is closely concerned, and whether the abnormal phenomenon or symptoms exist or not is observed. The chin specimen was obtained at the end of the 4 th month after the operation, and was fixed in 4% paraformaldehyde for 48 hours (if not handled in time, the mixture was transferred into 75% alcohol after 48 hours).
The above-described embodiments of the present invention do not limit the scope of the present invention. Any of various other corresponding changes and modifications made according to the technical idea of the present invention should be included in the scope of the claims of the present invention.
Claims (10)
1. The preparation method of the calcined bone powder without adding the chemical reagent is characterized by comprising the following steps:
(1) Firstly, cutting the pretreated milk cow bone blocks into small blocks, putting the small blocks into a pressure cooker, carrying out high-pressure treatment, repeating the high-pressure treatment for a plurality of times, separating liquid in the pressure cooker after each high-pressure treatment, carrying out next high-pressure treatment, removing softened cortical bone when the heat is still hot after the last high-pressure treatment is finished, and washing to be neutral after ultrasonic cleaning;
(2) Calcining the washed sample in the step (1) at 700-900 ℃ for 6-10 hours, naturally cooling, ball milling, sieving, ultrasonic cleaning and drying to obtain the calcined bone powder without adding the chemical reagent.
2. The method for preparing calcined bone powder without adding chemical reagent according to claim 1, wherein the pressure of the high-pressure treatment in the step (1) is 1.5-2 times of standard atmospheric pressure.
3. The method for preparing the calcined bone powder without adding the chemical reagent according to claim 2, wherein the time of the high-pressure treatment in the step (1) is 1-5 h each time.
4. A method for preparing calcined bone powder without adding chemical agent according to any one of claims 1 to 3, wherein the pretreatment in step (1) is as follows: taking femur and tibia of fresh dairy cows, freezing and cutting into bone blocks, then placing into a container, adding a clear water cover which is not used for boiling, removing soft tissues and cartilage tissues attached to the bone, cutting into proper sizes, and freezing for later use.
5. The method of claim 4, wherein the washing to neutrality in step (1) is performed by: washing with distilled water.
6. The method for preparing the calcined bone meal without adding any chemical reagent according to any one of claims 1 to 3, wherein the temperature rising rate of the calcination in the step (2) is 2 to 4 ℃/min.
7. The method for preparing calcined bone powder without adding chemical reagent according to claim 6, wherein the particle diameter selected by sieving in the step (2) is 0.335-8 mm.
8. The method for preparing calcined bone powder without adding chemical reagent according to claim 7, wherein the ultrasonic cleaning in the step (2) is as follows: adopting absolute ethyl alcohol and deionized water to alternatively ultrasonically clean for a plurality of times.
9. The calcined bone powder prepared by the method for preparing calcined bone powder without adding chemical reagents according to any one of claims 1 to 8.
10. Use of the calcined bone powder of claim 9 as dental bone powder.
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