CN115920135B - Bone repair material and preparation method thereof - Google Patents
Bone repair material and preparation method thereof Download PDFInfo
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- CN115920135B CN115920135B CN202211663072.5A CN202211663072A CN115920135B CN 115920135 B CN115920135 B CN 115920135B CN 202211663072 A CN202211663072 A CN 202211663072A CN 115920135 B CN115920135 B CN 115920135B
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Materials For Medical Uses (AREA)
Abstract
The invention belongs to the field of biomedical materials, and provides a bone repair material and a preparation method thereof, wherein the bone repair material comprises the following components: bovine cancellous bone inorganic salt particles; the surfaces of the pores of the bovine spongy bone inorganic salt particles are loaded with a polytrimethylene carbonate coating; the inside of the pores of the bovine spongy bone inorganic salt particles is filled with a mixture of sodium carboxymethyl starch and gelatin in a spongy state. Solves the problems of poor strength, fragility, no hemostasis and induced bone activity of the heterogeneous bone material, can be filled in the defect of the oral bone, and plays roles of supporting tooth extraction sockets, absorbing blood, stabilizing blood clots and promoting bone formation.
Description
Technical Field
The invention belongs to the field of biomedical materials, and in particular relates to a bone repair material and a preparation method thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
The healing process of alveolar bone after tooth loss can be divided into 3 phases: ① inflammatory phase. This process refers to blood clot formation followed by massive inflammatory cell infiltration, ultimately forming granulation tissue. ② proliferation stage. This process is mainly the replacement of granulation tissue by fibrous connective tissue, and the formation of woven bone. ③ New bone formation and bone remodeling stage. In the process, the mature bone replaces the woven bone and bone reconstruction occurs, so that the form of the alveolar socket, the height and width of the alveolar ridge and the like are changed. One system evaluation result indicated that the average bone width of the tooth socket was reduced to 3.79.+ -. 0.23mm, the average bone height was reduced to 1.24.+ -. 0.11mm, the horizontal bone resorption rate was 29 to 63% and the vertical bone resorption rate was 11 to 22% within 6 months after tooth extraction.
With the rapid development of the oral medical technology, the oral implantation clinical technology is rapidly developed and matured. A number of important factors affecting the long-term effect of the implant have been demonstrated, one of which is the requirement that there is sufficient bone mass at the implant site to achieve good implant osseointegration. In the course of implant repair, if the bone augmentation technique cannot be applied to improve the bone defect at the implant site, the selection of implant surgery scheme, the effect of aesthetic repair and the long-term implant efficacy can be affected. Therefore, in the case of insufficient bone mass in the implant area, good initial stability is provided to the implant, and guiding bone regeneration becomes necessary. In order to obtain a good clinical effect of guiding bone regeneration, bone repair materials play an important role.
The bone repair materials commonly used in clinic at present can be classified into autologous bone, allogeneic bone, xenogeneic bone and synthetic bone according to the sources thereof. Autologous bone is considered as a gold standard for bone grafting materials because of its excellent bone conduction, bone induction and bone regeneration capabilities, but autologous bone grafting has the disadvantages of secondary operation area, limited bone extraction amount, more complications and the like, and thus is often limited in clinical application. The allogeneic bone is generally used in the forms of fresh frozen bone, allogeneic freeze-dried bone or demineralized allogeneic freeze-dried bone, and the like, and the allogeneic bone has strong compression resistance because protein is not completely removed, the material has good compatibility with human tissues, the immune rejection after implantation is light, the bone induction performance is relatively good, but due to the age difference of donors, the bone source for bone grafting is very limited, and the allogeneic bone has a certain risk of virus infection. The heterologous bones are mostly pig bones or bovine bones, the structure of the heterologous bones is similar to that of human bones, the source is wide, the application prospect is good, the representative product is deproteinized bovine bone particles (Bio-Oss bone powder) of Geistlich company in Switzerland, and the deproteinized bovine bone particles become the first choice of the oral implant filling material due to good physicochemical property and biocompatibility. However, the disadvantages are also apparent, such as lack of bioactivity, poor compression resistance, slow degradation, etc. The artificial bone is generally hydroxyapatite crystal after high temperature calcination or beta-tricalcium phosphate or calcium sulfate crystal synthesized by reaction, has low porosity, nonuniform pores and the like, cannot simulate the complex microstructure of the natural bone material, and a small amount of carbonate existing in a weak crystal form in the natural bone has an important influence on the degradability of the bone. Thus, the degradability of the artificial bone material has been a bottleneck limiting its clinical application.
Chinese patent publication No. CN 101020082a discloses a bone repair material for guiding regeneration of bone tissue, which is a composition of calcined bone powder with different particle sizes and bioabsorbable polymer material, wherein the particle size of the calcined bone powder ranges from 0.05 mm to 2.5mm; the bioabsorbable polymeric material is one or the derivatives of collagen, chitin, chitosan, hyaluronic acid, chondroitin sulfate, calcium alginate, fibrin and elastin, or the mixture of two or more of them, which overcomes the problem that the hardness, strength and plasticity of the existing synthetic materials are poor, but the inventor finds that: the bio-absorbable polymer material selected by the patent has poor mechanical strength, and the polymer material of the composition prepared by the method is attached to the surface of the calcined bone powder, has poor liquid absorbability, and does not have the effect of stabilizing blood clots and promoting bone formation.
The poly (trimethylene carbonate) (PTMC) is prepared by ring opening of trimethylene carbonate monomer, is a biomedical material with good biocompatibility and degradability, and the degradation product is neutral, and can not cause acid inflammatory reaction after being implanted into human body, and has certain elasticity and good mechanical processing property at body temperature, thus being widely used in the fields of drug controlled release, in vivo implantation materials and the like.
Sodium carboxymethyl starch (CMS-Na) is a modified starch etherified with carboxymethyl, which is odorless, non-toxic, and readily soluble in water. CMS-Na has a very wide application in medicine and can be used as a raw material for making capsules, tablets and sugar-coats. CMS-Na has strong water absorption and expansibility and can be degraded into glucose of small molecules.
Gelatin is a hot water-soluble polypeptide mixture obtained by converting a collagen triple helix structure into an irregular chain, and has wide application in the medical field due to good affinity with biological tissues and no antigen reaction. The gelatin sponge can stop bleeding, has no toxicity, no immune rejection and local verification reaction, can improve the activity and reproduction of cells, and has degradation products of amino acid and water which can be absorbed by human bodies, but the gelatin sponge has poor hydrophilicity and small water absorption.
Disclosure of Invention
In order to solve the problems, the invention provides a bone repair material and a preparation method thereof, solves the problems of poor strength, fragility, no hemostasis and induced bone activity of heterogeneous bone materials, can be filled in the defect of oral bone, and plays roles of supporting tooth extraction sockets, absorbing blood, stabilizing blood clots and promoting osteogenesis.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a bone repair material comprising:
Bovine cancellous bone inorganic salt particles;
The surfaces of the pores of the bovine spongy bone inorganic salt particles are loaded with a polytrimethylene carbonate coating;
The inside of the pores of the bovine spongy bone inorganic salt particles is filled with a mixture of sodium carboxymethyl starch and gelatin in a sponge state;
The particle diameter of the bovine spongy bone inorganic salt in the bone repair material is 0.25-5 mm, the fat content is less than 1%, and the protein content is less than 0.1%;
the thickness of the polytrimethylene carbonate coating in the bone repair material is 10-100 mu m, and the viscosity average molecular weight is 20-50 w;
The ratio of the carboxymethyl starch sodium to the gelatin in the bone repair material is 1:1-5.
In a second aspect of the present invention, there is provided a method for preparing a bone repair material, comprising:
removing organic components in bovine cancellous bone, and pulverizing to obtain inorganic salt particles of bovine cancellous bone;
immersing the bovine spongy bone inorganic salt particles in methylene dichloride solution of the polytrimethylene carbonate for 2-6 hours, and then taking out and drying to obtain the bovine spongy bone inorganic salt particles with the surfaces loaded with the polytrimethylene carbonate coating;
Immersing the bovine spongy bone inorganic salt particles with the surface loaded with the polytrimethylene carbonate coating in a mixed aqueous solution of carboxymethyl starch sodium and gelatin, carrying out vacuum defoaming to ensure that the solution completely enters the pores of the bovine spongy bone inorganic salt particles, then pre-freezing for more than 6-8 hours at the temperature of minus 20-minus 18 ℃, and freeze-drying to obtain the bovine spongy bone inorganic salt.
In a third aspect of the present invention, there is provided a bone repair material prepared by the method described above.
In a fourth aspect of the present invention there is provided the use of a bone repair material as described above in the field of dentistry.
The beneficial effects of the invention are that
(1) The bone repair material has the macroscopic and microscopic structures of natural inorganic cancellous bone, and is endowed with good strength and toughness through the polytrimethylene carbonate coating, so that the bone repair material is not easy to break after being filled, can play a good supporting role, and slows down bone absorption;
(2) The pores of the bone repair material are sponge structures composed of sodium carboxymethyl starch and gelatin, so that the bone repair material has good hemostatic and blood seepage absorption effects, blood clots formed by blood seepage can be stabilized between the pores of the bone repair material, good environment and support are provided for blood mechanized bone formation, and the bone repair material is beneficial to blood bone formation;
(3) The poly (trimethylene carbonate) degradation product is neutral, does not cause inflammatory reaction, and is beneficial to the proliferation of osteoblasts;
(4) The degradation products of gelatin are amino acids, can be absorbed by osteoblasts, and play a role in promoting osteoblast proliferation.
(5) The preparation method is simple, has strong practicability and is easy to popularize.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
Fig. 1: photographs of the bone repair material prepared in example 1 of the present invention;
fig. 2: SEM image of bone repair material prepared in example 1 of the present invention;
fig. 3: the embodiment 2 of the invention is a surgical chart for a canine alveolar restoration effect test of a bone restoration material;
fig. 4: the canine alveolar socket repair effect test of the embodiment 2 of the invention is a CBCT chart of an experimental group at 12 weeks;
Fig. 5: the canine alveolar socket repair effect test of the embodiment 2 of the invention is a CBCT image of a control group at 12 weeks;
Fig. 6: the canine alveolar prosthetic effect test of the embodiment 2 of the invention is based on a tissue section chart (Masson staining) of an experimental group at 12 weeks;
fig. 7: example 2 canine alveolar repair effect of the present invention control tissue section (Masson staining) at 12 weeks.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In one aspect, the present invention provides a bone repair material prepared from bovine spongy bone inorganic salt particles, polytrimethylene carbonate, sodium carboxymethyl starch and gelatin, wherein the polytrimethylene carbonate is coated on the surfaces of the pores of the bovine spongy bone inorganic salt particles, and the sodium carboxymethyl starch and the gelatin are filled in the inside of the pores of the bovine spongy bone inorganic salt particles in a spongy state.
In some embodiments, the particle diameter of the bovine cancellous bone inorganic salt in the bone repair material is 0.25 mm-5 mm, the fat content is less than 1%, the protein content is less than 0.1%;
In some embodiments, the thickness of the polytrimethylene carbonate coating in the bone repair material is 10 μm to 100 μm and the viscosity average molecular weight is 20w to 50w.
In some embodiments, the ratio of sodium carboxymethyl starch to gelatin in the bone repair material is 1 (1-5).
In some embodiments, the sodium carboxymethyl starch has a degree of substitution D.S of 0.3 to 0.6.
In another aspect, the present invention provides a method for preparing a bone repair material, the method comprising the steps of:
step 1: taking bovine cancellous bone as a raw material, completely removing organic matters and crushing to obtain bovine cancellous bone inorganic salt particles;
Step 2: completely immersing bovine spongy bone inorganic salt particles in methylene dichloride solution of the polytrimethylene carbonate for 2-6 hours, and then taking out and drying to obtain the bovine spongy bone inorganic salt particles with the surface of the polytrimethylene carbonate coating;
Step 3: placing bovine spongy bone inorganic salt particles with the surface poly (trimethylene carbonate) coating in a mixed aqueous solution of carboxymethyl starch sodium and gelatin until the mixed aqueous solution is completely immersed, carrying out vacuum defoaming, enabling the solution to completely enter pores of the bovine spongy bone inorganic salt particles, then pre-freezing for more than 6 hours at the temperature below minus 20 ℃, and obtaining the bone repair material after freeze drying.
In some embodiments, the step 1 of obtaining bovine cancellous bone inorganic salt particles comprises: cutting bovine cancellous bone into slices with the thickness of less than 2cm, soaking purified water to remove blood, drying, reflux-extracting with alkane organic reagent to remove fat components, reflux-extracting with amine organic reagent to remove protein components, washing with purified water to be neutral, drying, pulverizing and sieving to obtain bovine cancellous bone inorganic salt particles;
In some embodiments, the alkane organic solvent used for degreasing in the step 1 is petroleum ether or n-hexane, the reflux extraction temperature is 80-120 ℃, and the reflux extraction cycle times are not less than 24 times;
in some embodiments, the amine-based organic reagent for removing protein components in the step 1 is propylamine, ethylenediamine or propylenediamine, and the deproteinization time is 24h to 72h;
In some embodiments, the methylene carbonate in step 2 has a methylene chloride solution concentration of 0.02g/mL to 0.2g/mL; the drying conditions of the bovine spongy bone inorganic salt particles are as follows: drying at normal pressure and at a drying temperature of 50-80 ℃;
In some embodiments, the vacuum degassing conditions in step 3 are: vacuum degree is not less than 0.1MPa, and defoaming time is not less than 30min; the concentration of the mixed aqueous solution of sodium carboxymethyl starch and gelatin is 1-5%.
The invention will now be described in further detail with reference to the following specific examples, which should be construed as illustrative rather than limiting.
Example 1:
Cutting bovine cancellous bone into slices with thickness of about 1cm, repeatedly soaking and cleaning with purified water to remove blood, drying at 100deg.C for 12 hr, placing completely dried cancellous bone in Soxhlet extractor, reflux extracting fat component with petroleum ether at 110deg.C, and circularly extracting for 24 times to obtain defatted bone. The defatted bone is placed in a propylene diamine solution for deproteinization treatment for 24 hours, bone tissues are taken out after deproteinization is finished, the bone tissues are repeatedly washed to be neutral by purified water, then the dried deproteinized bone is obtained by drying for 12 hours at 150 ℃, and the dried bone tissues are crushed and screened to obtain 0.5-5 mm bovine spongy bone inorganic salt particles. The fat content and the protein content of the obtained bovine cancellous bone inorganic salt particles are detected: taking 2g of bovine cancellous bone inorganic salt particles, and detecting according to a first method for detecting fat in national food safety standard of GB5009.6-2016, wherein the detection result of fat content is 0.85%; in addition, 2g of bovine cancellous bone inorganic salt particles are taken and detected according to a second method of Lin Fenfa (Lowry method) of protein content determination method of four parts 0731 of the pharmacopoeia of the people's republic of China, and the protein content detection result is 0.06%.
20G of polytrimethylene carbonate having a viscosity average molecular weight of 20w was weighed out and dissolved in 100mL of methylene chloride solution to obtain a polytrimethylene carbonate solution having a concentration of 0.2 g/mL. 20g of bovine spongy bone inorganic salt particles are taken and placed in a poly (trimethylene carbonate) solution to be fully immersed for 2 hours, and then the bovine spongy bone inorganic salt particles are taken out and dried for 6 hours under normal pressure, wherein the drying temperature is 60 ℃, so as to obtain the bovine spongy bone inorganic salt particles with the poly (trimethylene carbonate) coating. The thickness of the polytrimethylene carbonate coating was observed by scanning electron microscopy, and 5 fields of view were randomly selected, and the average thickness of the polytrimethylene carbonate coating was 50.2 μm.
1G of carboxymethyl starch sodium with the substitution degree D.S of 0.3 is weighed, 4g of gelatin is weighed, 250mL of purified water is added, and the mixture is heated and dissolved to obtain a mixed solution of carboxymethyl starch sodium with the concentration of 2 percent and gelatin. And (3) putting the bovine spongy bone inorganic salt particles with the polytrimethylene carbonate coating into a mixed solution of carboxymethyl starch sodium and gelatin for complete immersion, vacuumizing to a vacuum degree of more than 0.1MPa, continuously defoaming for 30min, then putting into a temperature of minus 20 ℃ for prefreezing for 6h, and freeze-drying to obtain the bone repair material.
Example 2
Cutting bovine cancellous bone into slices with thickness of about 1cm, repeatedly soaking and cleaning with purified water to remove blood, drying at 110deg.C for 8 hr, placing completely dried cancellous bone in Soxhlet extractor, reflux extracting fat component with n-hexane at 120deg.C, and circularly extracting for 48 times to obtain defatted bone. The defatted bone is placed in ethylenediamine solution for deproteinization treatment for 72 hours, bone tissues are taken out after deproteinization is completed, the bone tissues are repeatedly washed to be neutral by purified water, then the dried deproteinized bone is obtained by drying for 12 hours at 120 ℃, and the dried bone tissues are crushed and screened to obtain 0.5-5 mm bovine spongy bone inorganic salt particles. The fat content and the protein content of the obtained bovine cancellous bone inorganic salt particles are detected: taking 2g of bovine cancellous bone inorganic salt particles, and detecting according to a first method for detecting fat in GB5009.6-2016 food safety national standard food, wherein the fat content detection result is 0.13%; in addition, 2g of bovine cancellous bone inorganic salt particles are taken and detected according to a second method of Lin Fenfa (Lowry method) of protein content determination method of four parts 0731 of the pharmacopoeia of the people's republic of China, and the protein content detection result is 0.02%.
10G of polytrimethylene carbonate having a viscosity average molecular weight of 50w was weighed out and dissolved in 100mL of methylene chloride solution to obtain a polytrimethylene carbonate solution having a concentration of 0.1 g/mL. 20g of bovine spongy bone inorganic salt particles are taken and placed in a poly (trimethylene carbonate) solution to be fully immersed for 2 hours, and then the bovine spongy bone inorganic salt particles are taken out and dried for 6 hours under normal pressure, wherein the drying temperature is 60 ℃, so as to obtain the bovine spongy bone inorganic salt particles with the poly (trimethylene carbonate) coating. The thickness of the polytrimethylene carbonate coating was observed by scanning electron microscopy, and 5 fields of view were randomly selected, and the average thickness of the polytrimethylene carbonate coating was found to be 23.5 μm.
2G of sodium carboxymethyl starch with the substitution degree D.S of 0.6 is weighed, 2g of gelatin is weighed, 100mL of purified water is added, and the mixture is heated and dissolved to obtain a mixed solution of sodium carboxymethyl starch with the concentration of 4 percent and gelatin. And (3) putting the bovine spongy bone inorganic salt particles with the polytrimethylene carbonate coating into a mixed solution of carboxymethyl starch sodium and gelatin for complete immersion, vacuumizing to a vacuum degree of more than 0.1MPa, continuously defoaming for 2 hours, then putting into a temperature of minus 20 ℃ for prefreezing for 8 hours, and freeze-drying to obtain the bone repair material.
Comparative example 1
Cutting bovine cancellous bone into slices with thickness of about 1cm, repeatedly soaking and cleaning with purified water to remove blood, drying at 110deg.C for 8 hr, placing completely dried cancellous bone in Soxhlet extractor, reflux extracting fat component with n-hexane at 120deg.C, and circularly extracting for 20 times to obtain defatted bone. The defatted bone is placed in ethylenediamine solution for deproteinization treatment for 20 hours, bone tissues are taken out after deproteinization is completed, the bone tissues are repeatedly washed to be neutral by purified water, then the dried deproteinized bone is obtained by drying for 12 hours at 120 ℃, and the dried bone tissues are crushed and screened to obtain 0.5-5 mm bovine spongy bone inorganic salt particles. The fat content and the protein content of the obtained bovine cancellous bone inorganic salt particles are detected: taking 2g of bovine cancellous bone inorganic salt particles, and detecting according to a first method for detecting fat in national food safety standard of GB5009.6-2016, wherein the fat content detection result is 1.2%; in addition, 2g of bovine cancellous bone inorganic salt particles are taken and detected according to a second method of Lin Fenfa (Lowry method) of protein content determination method of four parts 0731 of the pharmacopoeia of the people's republic of China, and the protein content detection result is 0.15%.
5G of polytrimethylene carbonate having a viscosity average molecular weight of 10w was weighed out and dissolved in 100mL of methylene chloride solution to obtain a polytrimethylene carbonate solution having a concentration of 0.1 g/mL. 20g of bovine spongy bone inorganic salt particles are taken and placed in a poly (trimethylene carbonate) solution to be fully immersed for 2 hours, and then the bovine spongy bone inorganic salt particles are taken out and dried for 6 hours under normal pressure, wherein the drying temperature is 60 ℃, so as to obtain the bovine spongy bone inorganic salt particles with the poly (trimethylene carbonate) coating. The thickness of the polytrimethylene carbonate coating was observed by scanning electron microscopy, and 5 fields of view were randomly selected, and the average thickness of the polytrimethylene carbonate coating was found to be 8.5 μm.
1G of carboxymethyl starch sodium with the substitution degree D.S of 0.6 is weighed, 2g of gelatin is weighed, 100mL of purified water is added, and the mixture is heated and dissolved to obtain a mixed solution of carboxymethyl starch sodium with the concentration of 3 percent and gelatin. And (3) putting the bovine spongy bone inorganic salt particles with the polytrimethylene carbonate coating into a mixed solution of carboxymethyl starch sodium and gelatin for complete immersion, vacuumizing to a vacuum degree of more than 0.1MPa, continuously defoaming for 2 hours, then putting into a temperature of minus 20 ℃ for prefreezing for 8 hours, and freeze-drying to obtain the bone repair material.
Comparative example 2
Cutting bovine cancellous bone into slices with thickness of about 1cm, repeatedly soaking and cleaning with purified water to remove blood, drying at 110deg.C for 8 hr, placing completely dried cancellous bone in Soxhlet extractor, reflux extracting fat component with n-hexane at 105deg.C, and circularly extracting for 36 times to obtain defatted bone. The defatted bone is placed in propylamine solution for deproteinization treatment for 60 hours, bone tissues are taken out after deproteinization is finished, purified water is used for repeatedly cleaning to neutrality, then the dried deproteinized bone is obtained after drying for 12 hours at 110 ℃, and the dried bone tissues are crushed and screened to obtain 0.5 mm-5 mm bovine spongy bone inorganic salt particles. The fat content and the protein content of the obtained bovine cancellous bone inorganic salt particles are detected: taking 2g of bovine cancellous bone inorganic salt particles, and detecting according to a first method for detecting fat in national food safety standard of GB5009.6-2016, wherein the detection result of fat content is 0.09%; in addition, 2g of bovine cancellous bone inorganic salt particles are taken and detected according to a second method of Lin Fenfa (Lowry method) of protein content determination method of four parts 0731 of the pharmacopoeia of the people's republic of China, and the protein content detection result is 0.03%.
1G of carboxymethyl starch sodium with the substitution degree D.S of 0.5 is weighed, 2g of gelatin is weighed, 100mL of purified water is added, and the mixture is heated and dissolved to obtain a mixed solution of carboxymethyl starch sodium with the concentration of 4 percent and gelatin. And (3) putting the bovine spongy bone inorganic salt particles of the polytrimethylene carbonate coating into a mixed solution of carboxymethyl starch sodium and gelatin for complete immersion, vacuumizing to a vacuum degree of more than 0.1MPa, continuously defoaming for 1h, then putting into a temperature of minus 20 ℃ for prefreezing for 8h, and freeze-drying to obtain the bone repair material without the polytrimethylene carbonate coating.
Effect verification 1 particle crush strength test
(1) Test grouping: the experiments were divided into 12 groups, respectively, an inorganic bone particle group prepared in example 1 (labeled as group a), an inorganic salt particle group of the polytrimethylene carbonate coating prepared in example 1 (labeled as group B), a final product group prepared in example 1 (labeled as group C), an inorganic bone particle group prepared in example 2 (labeled as group D), an inorganic salt particle group of the polytrimethylene carbonate coating prepared in example 2 (labeled as group E), a final product group prepared in example 2 (labeled as group F), an inorganic bone particle group prepared in comparative example 1 (labeled as group G), an inorganic salt particle group of the polytrimethylene carbonate coating prepared in comparative example 1 (labeled as group H), a final product group prepared in comparative example 1 (labeled as group I), an inorganic bone particle group prepared in comparative example 2 (labeled as group J), a bone repair material group of the polytrimethylene carbonate-free coating prepared in comparative example 2 (labeled as group K), and a commercially available product Bio-s (labeled as group L).
(2) The test method comprises the following steps: accurately weighing 1.0g of each group of samples, namely m 0, placing the samples in a cylinder with the diameter of 2cm, uniformly applying a force of 1kg to each group of samples for 1 minute, sieving the samples, weighing each group of samples with the particle size of more than 0.25mm, namely m 1, and calculating the crushing strength coefficient of the particles.
Particle crush strength coefficient = m 1/m0 x 100%
(3) Test results
The test results show that the crushing strength coefficient of the inorganic salt particles of the polytrimethylene carbonate coating is greatly improved compared with that of inorganic salt particles without the polytrimethylene carbonate coating, is far higher than that of a pure inorganic bone particle material, and is superior to that of a commercial product Bio-Oss group.
Effect validation 2 cytotoxicity test
(1) Test grouping: the test was divided into sample groups [ 9 groups in total, namely, an inorganic bone particle group prepared in example 1 (labeled as group a), a final product group prepared in example 1 (labeled as group B), an inorganic bone particle group prepared in example 2 (labeled as group C), a final product group prepared in example 2 (labeled as group D), an inorganic bone particle group prepared in comparative example 1 (labeled as group E), a final product group prepared in comparative example 1 (labeled as group F), an inorganic bone particle group prepared in comparative example 2 (labeled as group G), a final product group prepared in comparative example 2 (labeled as group H), a commercial product Bio-Oss group (labeled as group I) ], a positive control group (10% dmso solution), a negative control group (high density polyethylene) and a medium control group (cell culture broth containing no test sample).
(2) Test liquid preparation: each group of samples was taken and placed in 10mL of MEM cell culture medium containing 10% fetal bovine serum, and the extract was prepared by leaching (24.+ -. 2) at (37.+ -. 1) DEG C for h.
(3) Test system:
cell lines: mouse fibroblast L929 was purchased from the China academy of sciences typical culture Collection Committee Kunming cell bank. The test adopts cells which grow vigorously after passage for 48-72 hours.
MTT solution: MTT powder is prepared into MTT solution with the mass concentration of 5mg/mL, and the MTT solution is sterilized by a sterile filter (the pore diameter is less than or equal to 0.22 mu m) for standby. Before use, the MTT solution with the mass concentration of 1mg/mL is diluted with serum-free MEM, the solution is prepared immediately before use, and the solution is used on the same day.
Mouse fibroblasts were cultured in a cell culture medium at 37℃in a cell culture incubator at 5% CO 2 (after thawing the stored cells, passage was performed 2 to 3 times for the test). All the test processes are carried out in a sterile environment of the biosafety cabinet, and the solutions, vessels and the like used for the test are sterile.
(4) L929 cells in good growth state were collected and cell suspension was prepared with cell culture medium, the cell concentration was adjusted to 1X10 5 cells/mL, and 100. Mu.L/well cell suspension was added to a 96-well cell culture plate. Incubation was performed in a cell incubator (5% CO 2, 37 ℃) for 24h to form a near confluent monolayer of cells. Each well was examined under a microscope to ensure that the cell growth was relatively equal for each well, the original medium was aspirated, and 100 μl of each column of 6 wells (peripheral wells) was added with each of the negative control, positive control, and medium control, respectively. The cells were incubated in a cell incubator for 24 hours, and the cell morphology of each plate well was observed under a microscope. The medium was carefully removed, 50. Mu.L of MTT solution was added to each well, the liquid in the well was discarded after culturing in a cell culture tank for 2 hours, 100. Mu.L of DMSO was added to each well, the plate was shaken, and the absorbance at 570nm (refer to 650 nm) was measured with a microplate reader, and the survival rate (%) was calculated as follows.
Survival (%) = OD570e/OD570b x 100%
Wherein:
OD570e—absorbance of each test group (sample group, positive control group);
OD570 b-absorbance of medium control.
(5) Evaluation of results
The average value of the medium control OD570b is more than or equal to 0.2, and the average value of the left and right medium control is different from the average value of all the medium control by not more than 15%, so that the test meets the acceptance standard. If the survival rate drops to less than 70% of the vehicle control, there is potential cytotoxicity.
(6) Test results
The mean value of the media control OD570b was 0.856, the mean value of the left and right two columns of media control was 2% different from the mean value of all media controls, and the test met the acceptance criteria.
And observing under a microscope, wherein most cells in each sample group are normal in morphology, and a small number of cells are round, loose and adherent, have no intracellular particles or show morphological changes. The negative control group had a normal morphology of most cells, and a small number of cells were round, loosely adherent, free of cytoplasmic granules or showed morphological changes. The positive control group had almost complete disruption of the cell layer. The 24h MTT assay results are shown in the following table, and each group of samples has no cytotoxicity.
Cytotoxicity test results
Effect verification 3 hemolysis test
(1) Test grouping: the tests were divided into sample groups [ respectively, inorganic bone particle group prepared in example 1 (labeled as group a), final product group prepared in example 1 (labeled as group B), inorganic bone particle group prepared in example 2 (labeled as group C), final product group prepared in example 2 (labeled as group D), inorganic bone particle group prepared in comparative example 1 (labeled as group E), final product group prepared in comparative example 1 (labeled as group F), inorganic bone particle group prepared in comparative example 2 (labeled as group G), final product group prepared in comparative example 2 (labeled as group H), commercial product Bio-Oss group (labeled as group I) ], positive control group (distilled water group) and negative control group (physiological saline group).
(2) Test liquid preparation: the samples of each group are respectively taken and placed in normal saline, and subjected to 60r/min oscillation leaching (72+/-2) h at the temperature of (37+/-1) ℃ to serve as test solutions, so that 3 tubes are prepared in parallel. Negative control and positive control were prepared in the same way as 3 tubes.
(3) The test steps are as follows:
a) 10mL of rabbit heart blood is collected, and 0.5mL of potassium oxalate solution with the mass concentration of 20g/L is added to prepare fresh anticoagulated rabbit blood. Fresh anticoagulated rabbit blood (8 mL) is taken and diluted with 10mL of 0.9% sterile sodium chloride injection.
B) Each tube of the test sample group is added with 2.1g of test sample and 10.5mL of physiological saline;
10mL of physiological saline is added into each tube of the negative control group; distilled water was added 10mL per tube for the positive control group. 3 tubes were prepared in parallel for each group.
C) All test tubes are subjected to oscillation leaching (72+/-2) for 60r/min at the temperature of 37+/-1 ℃ to prepare test solutions, negative control and positive control are prepared under the same conditions respectively, diluted rabbit blood is added into each test tube according to the ratio of 0.2mL diluted rabbit blood to 10mL test solution, and the test solutions are gently mixed and placed in a water bath at the temperature of 37+/-1 ℃ for continuous incubation for 60min. The liquid in the pouring tube was centrifuged at 800g for 5min. The supernatant was pipetted into a cuvette and absorbance was measured with a spectrophotometer at 545nm wavelength.
(4) Result calculation
The absorbance of each of the test sample group and the control group was averaged over 3 tubes. The absorbance of the negative control should be no greater than 0.03, the absorbance of the positive control should be 0.8.+ -. 0.3, otherwise the test should be repeated. The hemolysis rate of the test sample is calculated according to the following formula:
HR＝(A-B)/(C-B)×100％
Wherein:
hr—rate of hemolysis of test sample,%;
a—absorbance of test sample group;
b—negative control absorbance;
c-absorbance of positive control group.
(5) Result judgment
The criterion for qualification is generally defined as a hemolysis ratio of less than 5%.
(6) Test results
The hemolysis rate of each group of samples is less than 5 percent, and no hemolysis reaction exists. The test results are as follows:
Effect verification 4 Water absorbency test
The test was divided into 12 groups, namely, the inorganic bone particle group prepared in example 1 (labeled as group a), the inorganic salt particle group of the polytrimethylene carbonate coating prepared in example 1 (labeled as group B), the final product group prepared in example 1 (labeled as group C), the inorganic bone particle group prepared in example 2 (labeled as group D), the inorganic salt particle group of the polytrimethylene carbonate coating prepared in example 2 (labeled as group E), the final product group prepared in example 2 (labeled as group F), the inorganic bone particle group prepared in comparative example 1 (labeled as group G), the inorganic salt particle group of the polytrimethylene carbonate coating prepared in comparative example 1 (labeled as group H), the final product group prepared in comparative example 1 (labeled as group I), the inorganic bone particle group prepared in comparative example 2 (labeled as group J), the bone repair material group without the polytrimethylene carbonate coating prepared in comparative example 2 (labeled as group K), and the commercially available products (labeled as group Bio-L). About 1.5g of each group of samples are taken respectively, the samples are immersed in water, taken out and weighed after 1 hour, and the water absorption multiple of the product is calculated. The water absorption multiple is calculated according to the following formula:
Water absorption multiple= (m) 1-m0)/m0
Wherein:
m 0, the mass before water absorption, g;
m 1 -the mass after water absorption, g.
The test results are as follows:
the test results show that the water absorption performance of the bone repair material filled with sodium carboxymethyl starch and gelatin sponge is greatly improved, and the bone repair material is favorable for absorbing blood and playing a role in stopping bleeding and stabilizing blood clots.
Verification effect 5 canine alveolar socket repair effect verification
(1) Test group
8 Adult beagle dogs were selected and tested with the third premolars on the left side of the mandible and the third premolars on the right side of the mandible as controls. Experimental group the bone repair material prepared in example 2 of the present invention was implanted in the extraction socket and covered with Bio-Gide absorbable biofilm; the control side was implanted with the marketed product Bio-Os bone filler material at the extraction socket and covered with Bio-Gide resorbable biofilm.
(2) Surgical method
After anesthesia, cutting gums in third premolars on two sides of a mandible, exposing a bone surface, removing third premolars on two sides to form tooth extraction sockets, filling the tooth extraction sockets of an experimental group with the bone repair material prepared by the invention after scratching to enable the tooth extraction sockets to be level with the bone surface, cutting a Bio-Gide absorbable biological film into a proper size, covering the tooth extraction sockets, and finally suturing gums; the control group was operated in the same manner, filling the site of tooth extraction with the marketed product Bio-Oss bone filling material, covering with Bio-Gide absorbable biofilm, and finally suturing the gingiva.
(3) Evaluation of results
Animals were sacrificed after 12 weeks of normal post-operative feeding, CBCT examination was performed on the bone filling sites, bone volume fraction and bone trabecular thickness of each group of bone defect regions were measured, and quality and strength of the new bone were compared. After CBCT detection, the defect site and surrounding tissues were removed, 10% formaldehyde was fixed, and then the structure and morphology of the new bone tissue in each group of bone defect regions were observed under a microscope after treatment with histological methods (decalcification, embedding, slicing, masson staining).
(4) Test results
12 Weeks after the operation, the new bones of the experimental group are filled with tooth sockets, the bones are small Liang Miji, the situation of the bones of the control group is slightly worse, the data analysis shows that the average level of the bone volume fraction of the experimental group is 92.58+/-5.20%, the control group is 81.22+/-4.92%, and the two groups have statistical difference (P is less than 0.05). The thickness of the trabecula bone of the test group is 0.589 +/-0.036 mm, that of the control group is 0.352+/-0.056 mm, and the results of the two groups are statistically different (P is less than 0.05).
12 Weeks after the operation, histological observation results show that the periphery of the implant material of the experimental group is tightly surrounded by new bone tissue, a large amount of new bone is formed, part of the implant material is connected into a piece, and a large amount of new bone is formed in the implant material; the control group implant material was tightly surrounded by new bone, and there was formation of new bone, but there was almost no new bone inside the implant material.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A bone repair material, comprising:
Bovine cancellous bone inorganic salt particles;
The surfaces of the pores of the bovine spongy bone inorganic salt particles are loaded with a polytrimethylene carbonate coating;
The inside of the pores of the bovine spongy bone inorganic salt particles is filled with a mixture of sodium carboxymethyl starch and gelatin in a sponge state;
The particle diameter of the bovine spongy bone inorganic salt in the bone repair material is 0.25-5 mm, the fat content is less than 1%, and the protein content is less than 0.1%;
the thickness of the polytrimethylene carbonate coating in the bone repair material is 10-100 mu m, and the viscosity average molecular weight is 20-50 w;
the ratio of the sodium carboxymethyl starch to the gelatin in the bone repair material is 1:1-5;
The preparation method of the bovine spongy bone inorganic salt particles comprises the steps of removing organic matters in bovine spongy bone and crushing to obtain bovine spongy bone inorganic salt particles;
The steps for removing organic components in bovine cancellous bone include: cutting bovine cancellous bone into pieces with thickness less than 2cm, soaking in purified water to remove blood, drying, reflux extracting with alkane organic reagent to remove fat component, reflux extracting with amine organic reagent to remove protein component, washing with purified water to neutrality, drying at 150deg.C for 12h or 120 deg.C for 12h;
The reflux extraction temperature is 80-120 ℃, and the reflux extraction cycle times are not less than 24 times;
the deproteinization time is 24-72 h.
2. The bone repair material of claim 1, wherein the sodium carboxymethyl starch has a degree of substitution D.S of 0.3 to 0.6.
3. A method of preparing a bone repair material according to claim 1, comprising:
removing organic components in bovine cancellous bone, and pulverizing to obtain inorganic salt particles of bovine cancellous bone;
immersing the bovine spongy bone inorganic salt particles in methylene dichloride solution of the polytrimethylene carbonate for 2-6 hours, and then taking out and drying to obtain the bovine spongy bone inorganic salt particles with the surfaces loaded with the polytrimethylene carbonate coating;
Immersing the bovine spongy bone inorganic salt particles with the surface loaded with the polytrimethylene carbonate coating in a mixed aqueous solution of sodium carboxymethyl starch and gelatin, carrying out vacuum defoaming to ensure that the solution completely enters the pores of the bovine spongy bone inorganic salt particles, pre-freezing for more than 6-8 hours at the temperature of minus 20-minus 18 ℃, and freeze-drying to obtain the bovine spongy bone inorganic salt.
4. The method for preparing a bone repair material according to claim 3, wherein the step of removing organic components from bovine cancellous bone comprises: cutting bovine cancellous bone into pieces with thickness less than 2cm, soaking in purified water to remove blood, drying, reflux extracting with alkane organic reagent to remove fat component, reflux removing protein component with amine organic reagent, washing with purified water to neutrality, and drying.
5. The method for preparing a bone repair material according to claim 4, wherein the alkane-based organic solvent is petroleum ether or n-hexane;
the reflux extraction temperature is 80-120 ℃, and the reflux extraction cycle times are not less than 24 times.
6. The method for preparing a bone repair material according to claim 4, wherein the amine-based organic agent is propylamine, ethylenediamine or propylenediamine;
the deproteinization time is 24-72 h.
7. The method for producing a bone repair material according to claim 3, wherein the methylene chloride solution of the polytrimethylene carbonate has a concentration of 0.02g/mL to 0.2g/mL;
The drying temperature is 50-80 ℃.
8. The method for preparing a bone repair material according to claim 3, wherein the vacuum degassing conditions are: vacuum degree is not less than 0.1MPa, and defoaming time is not less than 30min;
the concentration of the mixed aqueous solution of sodium carboxymethyl starch and gelatin is 1-5%.
9. A bone repair material prepared by the method of any one of claims 3-8.
10. Use of the bone repair material of claim 9 in the preparation of an oral medical product.
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