CN112642001A

CN112642001A - Method for deproteinizing animal bone particles

Info

Publication number: CN112642001A
Application number: CN202011547925.XA
Authority: CN
Inventors: 马金霞
Original assignee: Shaanxi Bio Regeneration Medicine Co ltd
Current assignee: Shaanxi Bio Regeneration Medicine Co ltd
Priority date: 2020-12-24
Filing date: 2020-12-24
Publication date: 2021-04-13

Abstract

The invention provides a method for deproteinizing animal bone particles, which adopts hydrogen peroxide and ethylenediamine solution to jointly deproteinize, and repeatedly vibrates and cleans through purified water, and repeatedly turns and cleans through water for injection, thereby effectively removing antigenic substances, and simultaneously avoiding the problems of poorer compression resistance and reduced inductivity of the bone particles caused by high-temperature calcination.

Description

Method for deproteinizing animal bone particles

Technical Field

The invention belongs to the technical field of medical materials, and particularly relates to a method for deproteinizing animal bone particles.

Background

The bone grafting operation has been applied to orthopedics clinic for many years, and autologous bone grafting is used as a gold standard for bone grafting and has a good healing effect clinically. The autogenous bone is mostly taken from fibula or hip bone of a patient, the bone taking amount is limited, the patient suffers from secondary operation, and the application of autogenous bone transplantation is limited.

In order to solve the problem of bone transplantation, artificial bone, allogeneic bone, xenogeneic bone and other materials are used for clinical treatment instead of autologous bone transplantation. Most of the artificial bone materials are hydroxyapatite crystals calcined at a high temperature of more than 1000 ℃ or beta-tricalcium phosphate or calcium sulfate crystals synthesized through chemical reaction, but the artificial bone materials cannot simulate the complex microstructure of natural bone materials, and a small amount of carbonate existing in the natural bone, especially in a weak crystal form, has important influence on the degradability of the bone. Therefore, the degradability of the artificial bone material is always a bottleneck restricting the clinical application of the artificial bone material.

The allogeneic bone is derived from human cadaver bone, and only fat and partial protein (mainly antigenic substances) need to be removed in the preparation process, and the bone is obtained by disinfection and sterilization. Because the protein is not completely removed, the compression resistance of the allogeneic bone is strong, the material has good histocompatibility with the human body, the immunological rejection is light after implantation, and the bone induction performance is relatively good. However, due to age differences among donors, the bone sources available for bone grafting are very limited, and allogeneic bones have a certain risk of viral infection.

The xenogenic bone is mostly bovine bone or pig bone, and has wide sources and good application prospect due to the similarity with the structure of human bone. The most representative bovine bone product is Bio-Os bone powder, which is the inorganic cancellous bone particles can be obtained by selecting the end of bovine femur, carrying out Soxhlet extraction and degreasing by toluene, carrying out deproteinization treatment by using an ethylenediamine solution, drying at 160 ℃, and then calcining at 350 ℃. As the calcining process is used, the hydroxyapatite in the Bio-Os bone powder is partially crystallized, so that the bone material has poor compressive resistance, is only suitable for bone defects of oral cavities, maxillofacial bones and other parts, and has no ideal clinical application for repairing bone defects with large defect parts and needing certain mechanical strength support.

The material causing immune reaction in fresh bone is mainly non-collagen and glycoprotein and fat on the surface of bone cell membrane. How to effectively destroy cell surface antigens and remove antigenic substances to obtain xenogenic bone materials with low immunogenicity is an important point in the preparation process of xenogenic bones. In the prior art, antigen removal methods mainly include deep low temperature freezing, chemical reagents (such as methanol/chloroform mixed solution), surfactants (SDS, Triton, etc.), protease treatment, high temperature calcination, and the like. The antigen and organic matter components can be completely removed by high-temperature calcination, but because the organic matter is completely removed, the bone material has poor compression resistance, only has osteoconductivity and loses inducibility, and cannot be applied to defect repair of orthopedics; however, the use of chemical reagents or enzymes for treatment of heterogeneous bones with high antigenicity cannot effectively remove antigens, and the risk of immunological rejection exists after the bone is implanted into the body.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a method for deproteinizing animal bone particles, which can effectively remove non-collagen in allogeneic bone particles and glycoprotein on the surface of a bone cell membrane, and can effectively keep the compression resistance and the osteoconductivity of bone materials.

The purpose of the invention is realized by the following technical scheme:

in one aspect, a method for deproteinizing animal bone particles is provided, comprising the steps of:

1) adding degreased animal bone particles into hydrogen peroxide, standing for 12-24h, removing hydrogen peroxide, placing in purified water, shaking and cleaning for 3-6 times, drying at 75-100 deg.C for 5-7h, and sieving with 14 mesh sieve to obtain sieved animal bone particles;

2) adding the sieved animal bone particles into an ethylenediamine solution, heating to slightly boil, keeping at the temperature of 100-125 ℃ for 7-9h, removing waste liquid, adding purified water, oscillating and cleaning for 3 times, and repeating the step twice;

3) cleaning the animal bone particles obtained in the step 2) in a closed container for 15 times by using purified water, cleaning for 2 times by using water for injection, wherein the cleaning process is 3-6 min/time, turning over the container during cleaning, and discarding waste liquid to obtain deproteinized bone particles.

Preferably, in the step 2), the mass ratio of the defatted bone particles to the hydrogen peroxide is 1 (2-4), for example, 1:2,1:3,1: 4; the mass ratio of the degreased bone particles to the purified water is 1 (2-4), for example, 1:2,1:3, and 1: 4.

Preferably, in step 2), the screened animal bone particles are added with ethylenediamine at a concentration of 70% to 100%, for example, 70%,85%, 100%.

Preferably, in the step 2), the mass ratio of the sieved bone particles to the ethylenediamine solution is 1 (2.0-6.0). For example, 1:2.0, 1:3,1:4, 1:5, 1: 6.

Preferably, in the step 3), the mass ratio of the bone particles to the purified water is 1: 20; the mass ratio of the bone particles to the water for injection is 1: 20.

Preferably, the animal bone particles are prepared from bovine femur or porcine bone.

Compared with the prior art, the invention has the following beneficial effects:

the animal bone particle deproteinizing method provided by the invention adopts the combination of hydrogen peroxide and ethylenediamine solution for deproteinization, and repeatedly vibrates and cleans through purified water, and repeatedly turns and cleans through water for injection, thereby effectively removing antigenic substances, and simultaneously avoiding the problems of poorer compression resistance and reduced inductivity of the bone particles caused by high-temperature calcination.

The animal bone particles are prepared by removing organic matters from animal thighbones, have a three-dimensional reticular microscopic pore structure similar to human bones, are beneficial to formation and growth of new bones at an implanted position, and can partially change the structure of inorganic bones by osteoclasts and osteoblasts along with repair of bone tissues. The polysaccharide macromolecular substance which is natural and biodegradable and has good biocompatibility and cell affinity is used as a carrier, so that the adhesion of inorganic bone particles and a defect part is enhanced, the animal bone particles are gradually degraded and absorbed in a human body along with the repair of bone tissues, the animal bone particles are taken out without a secondary operation, and the biocompatibility is good.

Drawings

FIG. 1A is an SEM image of the product at 50 times magnification;

figure 1B is an SEM image of the product at 500 x magnification,

FIG. 2 is a Micro CT three-dimensional reconstruction map of a bone repair material prepared in example 1 of the present invention;

fig. 3A is a 50-fold SEM image of a bone repair material prepared in inventive example 1;

fig. 3B is a 5000-fold SEM image of the bone repair material prepared in inventive example 1;

FIG. 4 is a graph of the effect of different preparation methods on the compressive strength of a bone hemostatic material;

FIG. 5A is a graph showing the effect of the bone repair material at week 6 after the operation of the experimental group;

FIG. 5B is a graph showing the effect of the bone repair material at week 6 after the operation in the experimental group;

FIG. 5C is a graph showing the effect of the post-operative 6 th week bone repair material in the experimental group;

FIG. 5D is a graph showing the effect of the bone repair material at week 24 after the control group operation;

FIG. 6 is a view of Micro CT observation after 8 weeks of SD rat skull defect repair test;

FIG. 7 shows the detection result of the pixel CT value of the bone defect repair area of SD rat;

FIG. 8A is the HE staining observation experiment group bone regeneration status after 8 weeks of SD rat bone defect repair;

FIG. 8B is the HE staining observation control group bone regeneration status after 8 weeks of SD rat bone defect repair;

FIG. 9A is a photograph of an alveolar bone defect;

FIG. 9B is a photograph showing the filling of an alveolar bone defect with the bone repair material according to example 1 of the present invention;

fig. 10A shows the Masson trichrome staining results of the pathological section for canine alveolar bone filling repair in the experimental group;

fig. 10B is the results of Masson trichrome staining of a pathological section of canine alveolar bone filling repair in the control group;

FIG. 11A is a comparison of bone volume counts for experimental and control groups at week 8 post-surgery;

FIG. 11B is a comparison of trabecular bone thickness at week 8 post-surgery in experimental and control groups;

FIG. 12A is a photograph of histological observation of the experimental group;

FIG. 12B is a photograph showing histological observation of the control group;

FIG. 13A is a graph showing the adhesion of mesenchymal cells of SD rat bone marrow to the surface of the product in example 7;

FIG. 13B is a graph comparing the proliferation of mesenchymal cells on the present product and synthetic hydroxyapatite in example 7;

FIG. 14A is a graph of subcutaneous histological HE staining of SD rats 2 weeks after administration of the product;

FIG. 14B is a graph of subcutaneous histological HE staining of SD rats 4 weeks after administration of the product;

FIG. 14C is a graph of subcutaneous histological HE staining of SD rats 6 weeks after administration of the product;

FIG. 14D is a graph of subcutaneous histological HE staining of SD rats 8 weeks after administration of the product;

FIG. 14E is a graph of subcutaneous histological HE staining of SD rats 12 weeks after administration of the product.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

The embodiment provides a preparation method of a bone repair material, which comprises the following steps:

1) taking animal bone particles, and repeatedly and alternately oscillating and cleaning the animal bone particles for 3-6 times by using isopropanol and purified water to obtain degreased bone particles, wherein the mass ratio of the animal bone particles to the isopropanol is 1: 2.5;

2) adding the degreased bone particles into hydrogen peroxide, standing for 12-24h, removing the hydrogen peroxide, placing in purified water, shaking and cleaning for 3-6 times, drying at 75-100 ℃ for 5-7h, and sieving with a 14-mesh sieve to obtain sieved bone particles;

3) adding the sieved bone particles into 70-100% ethylenediamine solution, heating to slightly boil, keeping at the temperature of 100-125 ℃ for 7-9h, discarding waste liquid, adding purified water, oscillating and cleaning for 3 times, and repeating the step 3) twice;

4) cleaning the bone particles obtained in the step 3) with purified water for 15 times, cleaning with water for injection for 2 times, wherein the cleaning process is 3-6 min/time, turning over the container during cleaning, and discarding waste liquid to obtain deproteinized bone particles;

5) and uniformly mixing the sodium hyaluronate gel and the deproteinized bone particles according to the mass ratio of 10:6, sealing, standing for 4-6min, and freeze-drying to obtain the bone repair material.

In some embodiments, the sodium hyaluronate gel concentration is between 3% and 8%.

In some embodiments, the mass ratio of the sodium hyaluronate gel to the deproteinized bone particles is 10:5 or 10: 7.

In some embodiments, in step 1), the mass ratio of the animal bone particles to the isopropanol is 1:2 or 1:3, and the mass ratio of the animal bone particles to the purified water is 1: 3.

in some embodiments, in step 2), the mass ratio of the defatted bone particles to the hydrogen peroxide is 1 (2-4), for example, 1:2,1:3,1: 4; the mass ratio of the degreased bone particles to the purified water is 1 (2-4), for example, 1:2,1:3, and 1: 4.

In some embodiments, in step 3), the mass ratio of the sieved bone particles to the ethylenediamine solution is 1 (2-6), for example, 1:2,1:3,1:4, 1:5, and 1: 6.

In some embodiments, in step 4), the mass ratio of the bone particles to the purified water is 1: 20; the mass ratio of the bone particles to the water for injection is 1: 20.

In some embodiments, in step 5), the sodium hyaluronate gel is formulated by: weighing 5-8 g of sodium hyaluronate, adding water for injection to 100g, stirring until the sodium hyaluronate is uniformly dispersed, sealing and standing at 2-8 ℃ for 3-16 h, and continuously stirring until the gel is transparent and has no visible particles.

Example 2

The present example provides a method for deproteinizing animal bone particles, comprising the steps of:

2) adding the sieved animal bone particles into 70-100% ethylenediamine solution, heating to slightly boil, keeping at 125 ℃ for 7-9h, discarding waste liquid, adding purified water, oscillating and cleaning for 3 times, and repeating the step twice;

In some embodiments, the mass ratio of the defatted bone particles to hydrogen peroxide in step 2) is 1 (2-4), for example, 1:2,1:3,1: 4; the mass ratio of the degreased bone particles to the purified water is 1 (2-4), for example, 1:2,1:3, and 1: 4.

In some embodiments, the mass ratio of the sieved bone particles of step 3) to the ethylenediamine solution is 1 (2-6), for example, 1:2,1:3,1:4, 1:5, and 1: 6.

In some embodiments, the mass ratio of the bone particles to the purified water of step 4) is 1: 20; the mass ratio of the bone particles to the water for injection is 1: 20.

In some embodiments, the sodium hyaluronate gel of step 5) is configured by: weighing 5-8 g of sodium hyaluronate, adding water for injection to 100g, stirring until the sodium hyaluronate is uniformly dispersed, sealing and standing at 2-8 ℃ for 3-16 h, and continuously stirring until the gel is transparent and has no visible particles.

Example 3

The pore structure of the bone repair material (hereinafter referred to as "the product") prepared in example 1 was analyzed as follows: as shown in fig. 1A-1B, fig. 1A is an SEM image of the product magnified 50 times, fig. 1B is an SEM image of the product magnified 500 times, and it can be seen from fig. 1A-1B that sodium hyaluronate with a porous network structure is uniformly distributed on the surface of the bone repair material, which is beneficial to prolong the retention time of the bone repair material at the operation site and is beneficial to the proliferation, differentiation and migration of osteoblasts.

Meanwhile, the sodium hyaluronate has good hydrophilicity, so that the bone repair material has good plasticity. After three-dimensional reconstruction by Micro CT, see FIG. 2, bone morphological structural analysis was performed to obtain the bone volume fraction (BV/TV), the porosity being equal to 1-BV/TV × 100%. The porosity of the product was about 76.42 ± 8.52% as determined by analysis of 6 samples of about 5mm square. The porosity of the spongy bone is generally 70-80%, and the Micro CT analysis result of the porosity of the product meets the porosity requirement of the spongy bone. 3A-3B, the product maintains the trabecular bone and the through multistage pore space structure of natural bone, including macropores around the trabecular bone and mesopores on the trabecular bone, and is an interconnected pore structure. The size of the macropores is about 200-600 μm, and the size of the mesopores is about 50-100 μm. It is also seen that hydroxyapatite crystals exist finely in a weakly crystalline form, and that there are fine intergranular micropores, the micropore size being <1 μm. The weakly crystallized hydroxyapatite and the porous structure enable the product to have a high specific surface area, increase the contact area of the material with cells and body fluid, be more beneficial to the generation of new bones, and also enable new bone tissues to be fully contacted with the material.

Example 4

The total protein content of example 2 was determined: taking a proper amount of sample, grinding, precisely weighing 1.0g, placing in a 10ml volumetric flask, wetting with 1ml of distilled water, gradually adding 25% phosphoric acid solution while shaking, assisting ultrasonic to accelerate sample dissolution, adding about 6ml of phosphoric acid solution to dissolve the sample, fixing the volume to a scale mark with distilled water, sucking 0.1ml, and placing in a test tube with a plug. The total protein content was < 0.1% (m/m) as determined by the Coomassie Brilliant blue method according to pharmacopoeia of the people's republic of China 2015 edition (0731).

Example 5

The influence of different forms of preparation methods on the compressive strength of the bone repair material is analyzed by taking a product A prepared from free sodium hyaluronate gel and inorganic bone particles, a product B prepared from cross-linked sodium hyaluronate gel and inorganic bone particles, a product C prepared from non-cross-linked sodium hyaluronate gel and inorganic bone particles after electrostatic spinning, and four groups of products, and the method is shown in figure 4. As can be seen from fig. 4, the compressive strength is lower after the pure unmodified sodium hyaluronate solution is mixed with the calcined inorganic bone particles; after the cross-linked sodium hyaluronate is mixed with the calcined inorganic bone particles, the compressive strength is improved, but the effect is limited; the unmodified sodium hyaluronate gel is subjected to electrostatic spinning treatment and then is mixed with calcined inorganic bone particles, and the microscopic nano structure is beneficial to improving the mechanical property of the material, but the material is in a gel state, so that the reinforcing effect is still poor; the product is prepared by the preparation method in the embodiment 1, has a three-dimensional reticular microscopic pore structure similar to a human bone, is beneficial to the adhesion of sodium hyaluronate gel, enhances the combination between an inorganic phase and an organic phase, and greatly improves the compressive strength.

Example 6

In the embodiment, a new zealand white rabbit skull limit defect model, an SD rat skull penetrability limit defect model and a canine alveolar bone defect repair model are used for observing the conditions of bone tissues, vascular ingrowth and bone density recovery of a defect part.

1. Test method

Taking 6 male New Zealand white rabbits with the weight of 2-2.5 kg; 6 SD rats with the weight of about 340-360 g; 6 adult dogs of 2-3 years old weigh 15.0-17.5 kg and are healthy. Are purchased from the laboratory animal center of the fourth military medical university of western and ann.

1) Rabbit skull penetrability defect model making and implantation experiment

24 male New Zealand white rabbits (2-2.5 kg) were anesthetized with 1% sodium pentobarbital at 35mg/kg, depilated at the top of the head with 8% sodium sulfide, washed, fixed on an operating table, and spread with a conventional disinfectant. At the parietal head, the skin was incised arcuately to the subperiosteal, exposing the frontal and parietal bones. Removing bones by using a small osteotome and a dental electric drill to form a circular full-layer bone defect area with the diameter of 15mm, reserving dura mater, and using a bone repair material which is implanted into the product after being washed by normal saline as an experimental group; the blank control group was not filled with any material and the periosteum and skin were sutured layer by layer. Penicillin is injected into muscle for 30min before operation and 20 ten thousand U immediately after operation. Animals were sacrificed at weeks 6, 12, and 24 after surgery, skull specimens were taken, decalcified, HE stained, and histologic microscopic observation was performed, and the material remaining rate and the new bone formation rate were analyzed by calculating the area using Image analysis software Image-Pro plus6.0, and the material remaining rate and the new bone formation rate were the area of the material or the area of the new bone divided by the total area of the analysis chart. The observation groups at 6 weeks, 12 weeks and 24 weeks of the material implantation group and the blank control 24 weeks of the observation group are 6 cases each.

2) SD rat skull penetrability defect model making and implantation experiment

6 SD rats with the weight of 340-360 g are prepared by preparing skins at the tops of the rats, anesthetizing animals by using 1% sodium pentobarbital according to the volume of 30mg/kg, making arc-shaped incisions on the skins at the tops of the heads under aseptic conditions, peeling off and exposing the skull, removing bones by using a small osteotome and a dental electric drill to form 2 left and right adjacent round full-layer bone defect areas with the diameter of 3mm, reserving dura mater, washing by using normal saline, selecting the right defect area to implant the middle bone repair material in the product, making blank control on the left bone defect area, and suturing the incisions. At week 8 after surgery, the animals were sacrificed and skull specimens were taken for Micro CT bone density detection and histological observation.

3) Canine alveolar bone defect repair model and implantation experiment

The right side of the mandible of 6 adult dogs is taken as an experimental side, the left side of the mandible is taken as a control side, and alveolar bone defect models are respectively established:

grinding with a high-speed turbine dental drill to obtain bone defect regions with diameter of 8mm and depth of 6mm, wherein 4 dog bone defect regions are located at the interval of 4 th premolar and 1 st molar alveolus of lower jaw, and 2 dog bone defect regions are located at the root bifurcation of 1 st molar of lower jaw. The bone repair material is implanted into the experimental side, is in the form of scattered particles, is not pressurized, is parallel to the bone surface, is covered with a biological membrane and is sutured; the control side was not implanted with any material. Animals were sacrificed 8 weeks after the postoperative routine feeding. The healing conditions of the alveolar bone at the experimental side and the control side are observed and recorded through a general specimen, and the pathological specimen is taken for optical microscopy.

2. Results of the experiment

1) Rabbit skull defect repair

Experimental groups: at week 6 post-surgery, a large amount of new bone was formed around the bone repair material particles. At 12 weeks after surgery, new bone in the bone defect area has reached the central area, partially connected into pieces, and reformed into lamellar bone, and the bone repair material particles are mostly surrounded by new bone. The maturity of new bone is further improved at 24 weeks after surgery, as shown in fig. 5A, 5B, 5C. Blank control group: at 24 weeks post-surgery, most of the defect area was still fibrous connective tissue, see fig. 5D. At 6 weeks after operation, the new bone formation rate is about 13.27%, and the material residual rate is about 27.45%; at 12 weeks after operation, the new bone generation rate is about 26.73%, and the material residual rate is about 24.62%; at 24 weeks after surgery, the new bone formation rate was about 35.54%, and the material remaining rate was about 19.83%, as shown in Table 1. By calculating the area of the new bone and the area of the residual material, it can be seen that the new bone generation is gradually increased along with the extension of the repair time, and the bone grafting material is slowly degraded.

Table 1 new osteogenesis rate and material remaining rate (%, n = 6) of experimental group

Time	Rate of new bone formation	Residual rate of material
			6 weeks	13.27±3.68	27.45±5.39
For 12 weeks	26.73±5.22	24.62±4.34
			24 weeks	35.54±4.85	19.83±5.17

2) SD rat skull defect repair

SD rats with skull defect repair test for 8 weeks, Micro CT observation shows that the bone defect area has healed, while the control group (unrepaired group) has no bone healing, as shown in figure 6. After 8 weeks, the bone density of the bone repair area and the surrounding normal bone is analyzed by pixel CT value by using Micro CT, the difference between the two is compared, and the detection result is shown in figure 7. Bone density in the 8-week post-operative repair area was nearly close to that of normal bone, with no significant difference between the two groups (F-test, P > 0.05). The bone regeneration state of the experimental group and the control group was observed by HE staining for 8 weeks after the operation, and the results showed that, as shown in fig. 8A-8B, the new bone mass of the experimental group was significantly greater than that of the control group, and due to individual differences, the new bone mass between the experimental groups was also significantly different, and no significant new bone formation occurred in the control group.

3) Canine alveolar bone defect repair

The soft tissues of 6 dogs are well healed, the gum color, the shape and the quality are normal, and the inflammatory reactions such as congestion, swelling, erosion and the like are not seen.

Alveolar bone healing, see figures 9A-9B. There was no significant difference in healing of alveolar bone defects at various sites. The alveolar bone surface of the bone repair material filling set in the product is flat, and the bone regeneration is good. Through histological section observation, the bone wall of the filling group of the product is equivalent to the normal side, the bone density is higher, the bone tissue of the unrepaired group is regenerated, but the height of the bone wall is lower than that of the normal side, the bone gaps are more, and the bone density is low. Meanwhile, at 8 weeks, biofilm residues were still found on the defect tops of the experimental and control groups, and there was no significant fibrous connective tissue ingrowth into the alveolar bone defect area in the experimental group, as shown in fig. 10A. In contrast, the control group had significant fibrous tissue growth into the defect area, as shown in FIG. 10B.

Example 7

This example is a study of the healing effect of bone repair materials on tooth extraction wounds. The absorption of local alveolar bone often takes place among the in-process of the healing of tooth extraction wound rebuild after the tooth extraction operation, leads to alveolar bone height to reduce, and the thickness attenuation to influence later stage artificial tooth and repair, be more unfavorable for the planting body and implant. To solve such problems, a vestibular deepening operation or an alveolar bone heightening operation is often required to obtain a necessary alveolar bone profile. However, these surgical methods are very traumatic, increase the pain of the patient, and are all remedial measures taken after alveolar bone atrophy, and the effect is often unsatisfactory. Therefore, how to promote the healing of the tooth extraction wound and reduce the alveolar bone resorption after tooth loss is one of the important problems in the current oral and maxillofacial surgery research. Some methods for preventing alveolar bone resorption appear at home and abroad in recent years, but the methods are difficult to popularize and apply clinically due to the respective defects. The ideal healing state of the tooth extraction socket is that the bone defect formed after tooth extraction is completely filled with new bone, and the surface of the wound is completely sealed by soft tissue mucosa. The bone graft material filled after tooth extraction is beneficial to the reconstruction of alveolar bone and has important significance for the stability of later-stage implants. The experiment detects the effect of the product on alveolar bone retention by filling the dental extraction socket with single-wall bone defects after tooth extraction with the product.

1. Test method

6 adult domestic dogs of 2-3 years old weigh 15.0-17.5 kg and are healthy. Purchased from the laboratory animal center of western-style medical university, fourth military. The right side of the mandible of 6 adult dogs is taken as an experimental side, the left side of the mandible is taken as a control side, and the bilateral symmetry molar is respectively pulled out. Implanting the bone repairing material into the product at experimental side, and covering with biological membrane without pressurizing to make the bone repairing material flush with bone surface; the control side was not implanted with any material and was covered with a biofilm and sutured. Animals were sacrificed 8 weeks after the postoperative routine feeding. The healing conditions of alveolar bones on the experimental side and the control side are observed and recorded through a general specimen, a pathological specimen is taken for optical microscopy, the difference of the bone mass and the thickness of a trabecula bone between two experimental groups is compared through Micro CT, and the tissue morphology is observed through histological sections. And simultaneously, blood is drawn at 2 weeks, 4 weeks and 8 weeks after the operation, and the calcium and blood phosphorus detection is carried out to investigate whether the material can cause the change of the calcium and phosphorus metabolism level of the animals.

2. Results

At 8 weeks after operation, the soft tissues at the tooth extraction positions of the two groups are well covered, and no inflammatory reaction such as congestion, swelling, erosion and the like is seen. Experimental groups: BV/TV% =81.99 +/-3.12, trabecular bone thickness is 0.492mm +/-0.033 mm. Blank control group: BV/TV% =63.44 ± 3.34, trabecular bone thickness 0.251mm ± 0.061mm, see fig. 11A-11B. The histological observation results show that the formation amount of new bones at the blank group defect is small, soft tissues are taken as main materials, the bone repair degree of the experimental group is very good, and the new bones are continuously fused together, which is shown in fig. 12A and 12B.

EXAMPLE 8 study of cellular compatibility

In the process of repairing bone defects by the product, normal attachment, growth and proliferation of cells with osteogenesis capacity on the surface of the product are key steps for successfully repairing the bone defects, so whether the product can provide a proper space structure for the cells to grow for seed cells is an important index for evaluating whether the material can be used as a bone repair material, and therefore, the biocompatibility of the material at a cell level is evaluated by observing the attachment and proliferation conditions of bone marrow mesenchymal cells on the surface of the product in vitro and the cell state of fibroblasts and the product in co-culture.

1. Test method

1) Adhesion research of SD rat bone marrow mesenchymal cells on surface of product

Aseptically taking down double lower limbs from a super clean bench, shearing off fur, placing in a dish containing 75% ethanol, replacing instruments and the super clean bench, removing muscle tissues around thighbone and shinbone, and repeatedly washing with phosphate buffer solution. Cutting off two ends of the backbone, exposing the marrow cavity, puncturing the bone ends at two sides with a No. 7 needle, and extracting 4ml of DMEM medium containing 10% fetal calf serum by volume fraction with a 5ml syringe to wash out the marrow. Centrifuge at 800 rpm for 5 minutes, resuspend the pellet using α -MEM medium, centrifuge again at 800 rpm for 5 minutes. Blowing, sieving, centrifuging, and re-suspending and precipitating in alpha-MEM culture solution. Culturing at 37 deg.C in 5% CO2 incubator, changing liquid once for 4 hr, and washing off non-adherent cells. Standing at 37 deg.C and 5% CO₂Culturing in incubator, and changing the culture solution once in 1-2 days. Inoculating the 3 rd generation mesenchymal cells on the surface of the product, adding alpha-MEM culture solution, standing at 37 deg.C and 5% CO₂Culturing in an incubator. After 24 hours, the product inoculated with cells was fixed with 3% glutaraldehyde, and after lyophilization, the growth of the cells was observed by scanning electron microscopy.

2) Research on proliferation capacity of bone marrow mesenchymal cells on product

Aseptically taking down double lower limbs from a super clean bench, shearing off coat, placing in a dish containing 75% ethanol, replacing instruments and the super clean bench, removing muscle tissues around thighbone and shinbone, and repeatedly washing with phosphate buffer solution. Cutting off two ends of the backbone, exposing the marrow cavity, puncturing the bone ends at two sides with a No. 7 needle, and extracting 4ml of DMEM medium containing 10% fetal calf serum by volume fraction with a 5ml syringe to wash out the marrow. Centrifuge at 800 rpm for 5 minutes, resuspend the pellet using α -MEM medium, centrifuge again at 800 rpm for 5 minutes. Blowing, sieving, centrifuging, and re-suspending and precipitating in alpha-MEM culture solution. Standing at 37 deg.C and 5% CO₂Culturing in incubator, changing liquid once in 4 hr, and washing to eliminate non-adherent cells. Standing at 37 deg.C and 5% CO₂Culturing in incubator, and changing the culture solution once in 1-2 days.

Inoculating the 3 rd generation mesenchymal cells on the surface of the product and the synthetic hydroxyapatite bioceramic, adding alpha-MEM culture solution, standing at 37 deg.C and 5% CO₂Culturing in an incubator. The number of cells was measured every 24 hours on days 1 to 5 using MTT, and a cell growth curve was plotted.

3) Research on co-culture of fibroblasts and the product

Inoculating human fibroblast to 6-well plate, and putting 3-5 granules of the product into the well plate. The cell state and extracellular matrix formation of the contact surface between the product particles and the surrounding cells were observed at 0, 24, 48, and 96 hours, respectively.

Results

Bone marrow mesenchymal cells were inoculated to the product, and after 24 hours, scanning electron microscopy showed that the cells were all adhered to the scaffold material, indicating that the bone marrow mesenchymal cells were in good extension state, as shown in fig. 13A. The proliferation of bone marrow mesenchymal cells on the product and synthetic hydroxyapatite, the MTT dye method detects the change of the cell number of the bone marrow mesenchymal cells in 1-5 days, reflects the proliferation of the cells on the surfaces of the two materials, and refers to FIG. 13B.

In the experiment, in order to observe the affinity of the product to the mesenchymal cells, the mesenchymal cells are directly inoculated on the surface of the product for in vitro culture, and the scanning electron microscope result shows that the cells can be adhered and grow on the surface of the material and have good growth state. The proliferation conditions of the mesenchymal cells on the surface of the product and the surface of the synthesized hydroxyapatite are compared through measuring the cell number for 1 to 5 days, and the result shows that the adhesion efficiency and the proliferation efficiency of the cells on the surface of the bone induction calcined bone are higher than those of the synthesized hydroxyapatite material. The product has no influence on the normal metabolic process of cells and has good cell compatibility.

The experimental result shows that the prepared product has excellent cell compatibility, can provide a substrate for mesenchymal growth of bone marrow, is beneficial to the adhesion and growth of osteoblasts, and has good cell state and sufficient extracellular matrix secretion in co-culture with human fibroblasts. The product has good cell compatibility.

Example 9 histocompatibility study

1) Experimental methods

SD rats 10 (5 weeks old, male and female unlimited) were anesthetized by intramuscular injection into the legs with 1% sodium pentobarbital 35mg/kg body weight, and were fixed on the operating table in the prone position. Preparing skin on both sides of back, sterilizing with iodophor, and spreading towel. Two sides of the back, chest and waist spine are respectively provided with 2 incisions, subcutaneous tissues are separated in a blunt manner, and 4 subcutaneous lacunae are formed, wherein the transverse spacing is 2cm, and the longitudinal spacing is 2 cm. The product material is implanted and the subcutaneous and cutaneous sutures are layered. The method comprises the steps of wrapping without pressure, feeding, and injecting the penicillin 40 ten thousand units/day after 3 days. Taking 4 pieces of material respectively 2 weeks, 4 weeks, 6 weeks, 8 weeks and 12 weeks after operation, fixing with formalin, decalcifying for 48h, washing with running water for 24h, embedding the section with normal paraffin, and performing HE staining observation.

2) Results

General observation: the materials of the 2-week group are accessible in vitro, the outline is clear, the materials are wrapped by surrounding tissues, the fibrous membrane is thin, the tissue blocks obtained are loose in structure and are easy to separate from the materials. The material can still be touched in vitro after 4 weeks, the fibrous membrane formed by surrounding tissue is thicker, and fibrous connective tissue can grow into the pores of the material after stripping. 6. The material is tightly combined with the surrounding tissue in 8 weeks, the material is in a block shape and has higher hardness, and the interface of the material and the surrounding tissue begins to have fine depressions. A large number of microvessels are formed around the 12-week material, and the material is tightly attached to the surrounding tissue. The tissues around each group of materials have no necrosis or abscess. Histological examination: as shown in fig. 14A-14E, the tissue mass formed by the 2 week group material and the surrounding tissue was loose with fibroblast proliferation and inflammatory cell infiltration dominated by neutrophils, lymphocytes and plasma cells. The 4-week group implanted material has loose connective tissue growing in, and the tissue block formed by the material and the surrounding tissue is more compact than the 2-week group, and still mainly infiltrates with neutrophils and lymphocytes. The number of inflammatory cells is obviously reduced at 6 weeks, the materials in the 8-week group have good biocompatibility with surrounding tissues, no obvious inflammatory cells exist, and blood vessels are formed around the materials. The material in the 12-week group has a degradation tendency, a plurality of blood vessels are formed around the material, fibroblasts grow around the material, and inflammatory cells are not found. Tissue degeneration and necrosis were not observed in any of the groups.

3) Conclusion

In the experiment, the product is buried under the skin of an SD rat, the histocompatibility of the SD rat is detected, and the observation period is up to 12 weeks. The product implanted under the skin has no foreign body rejection reaction. In the early stage of implantation, there was neutrophil and lymphocyte infiltration due to acute inflammatory response of the wound, but with time the number of inflammatory cells decreased significantly and good biocompatibility was achieved at 6 weeks. The product has better tissue compatibility.

Therefore, the product has no immunogenicity, good histocompatibility and can be integrated by organism tissues, and the loose porous structure of the material can guide the formation of new blood vessels and sufficiently support the regeneration and reconstruction of new bone tissues.

Claims

1. A method for deproteinizing animal bone particles, comprising the steps of:

adding degreased animal bone particles into hydrogen peroxide, standing for 12-24h, removing hydrogen peroxide, placing in purified water, shaking and cleaning for 3-6 times, drying at 75-100 deg.C for 5-7h, and sieving with 14 mesh sieve to obtain sieved animal bone particles;

adding the sieved animal bone particles into an ethylenediamine solution, heating to slightly boil, keeping at the temperature of 100-125 ℃ for 7-9h, removing waste liquid, adding purified water, oscillating and cleaning for 3 times, and repeating the step twice;

cleaning the animal bone particles obtained in the step 2) in a closed container for 15 times by using purified water, cleaning for 2 times by using water for injection, wherein the cleaning process is 3-6 min/time, turning over the container during cleaning, and discarding waste liquid to obtain deproteinized bone particles.

2. The preparation method according to claim 1, wherein in the step 1), the mass ratio of the degreased animal bone particles to the hydrogen peroxide is 1 (2-4); the mass ratio of the degreased animal bone particles to the purified water is 1 (2-4).

3. The method according to claim 1, wherein the concentration of ethylenediamine added to the sieved animal bone particles in the step 2) is 70 to 100%.

4. The preparation method according to claim 1, wherein in the step 2), the mass ratio of the sieved animal bone particles to the ethylenediamine solution is 1 (2.0-6.0).

5. The method according to claim 1, wherein in step 3), the mass ratio of the animal bone particles to the purified water is 1: 20; the mass ratio of the animal bone particles to the water for injection is 1: 20.