CN114209884B

CN114209884B - Fish decalcified bone matrix and preparation method thereof

Info

Publication number: CN114209884B
Application number: CN202111623263.4A
Authority: CN
Inventors: 秦松; 王蕾; 李文军
Original assignee: Yantai Institute of Coastal Zone Research of CAS
Current assignee: Yantai Institute of Coastal Zone Research of CAS
Priority date: 2021-12-28
Filing date: 2021-12-28
Publication date: 2022-07-05
Anticipated expiration: 2041-12-28
Also published as: CN114209884A

Abstract

The invention discloses a fish decalcification bone matrix and a preparation method thereof, wherein the preparation method comprises the following steps: placing fresh fishbone into a reaction kettle, and sequentially carrying out the following treatment in the reaction kettle: stirring sodium chloride solution for 8h at 4 ℃, stirring SDS solution for 8h at 4 ℃, washing with pure water, stirring glycerol for 8h at 4 ℃, washing with pure water, stirring hydrogen peroxide solution for 8h at 37 ℃, washing with pure water, stirring anhydrous ether for 8h at 4 ℃, stirring protease solution for 0.5h at 4 ℃, washing with pure water, dehydrating with anhydrous ethanol, taking out the dehydrated fishbone from the reaction kettle, and freeze-drying to obtain the decalcified fishbone matrix. The fish decalcification bone matrix prepared by the invention well maintains the space structure of the fish bone, has good biocompatibility, biodegradability and bone repair capability, has no risk of diseases of people and livestock, has good mechanical strength after being crushed and remolded, and can be used for various non-bearing bone defect repairs and auxiliary repair of bearing bone defects.

Description

Fish decalcified bone matrix and preparation method thereof

Technical Field

The invention relates to a decalcified bone matrix and a preparation method thereof, in particular to a fish decalcified bone matrix and a preparation method thereof, belonging to the technical field of bone defect repair materials.

Background

The number of patients with various types of bone defects caused by wounds, diseases, infections and the like is always high every year, and a lot of inconvenience is brought to the patients and the families. How to repair bone defects and recover corresponding functions in a short time is the greatest appeal for patients and the direction of efforts of the majority of medical workers.

Currently, the autologous bone graft is considered as the "gold standard" for bone repair in clinic, but the problems of secondary trauma, insufficient bone mass and the like caused by bone harvesting affect the wide application of the autologous bone graft. As a result, a number of different materials, different types of bone repair products have been produced, which mainly include: the bone repair material is an auxiliary fixing, supporting and replacing product made of metal materials such as titanium and tantalum and polymer materials such as polytetrafluoroethylene, a bone repair product made of inorganic materials and organic materials and the like. The bone repair products have the capability of inducing osteogenesis, are degradable, have high biological safety, are widely used in clinic and solve the practical problems of a plurality of patients. Among these bone repair products, collagen-based bone repair products are favored, but the range of application of animal collagen-based bone repair products is limited due to religious beliefs and the risk of zoonosis and the like.

The fish living in the sea and the controlled water area has clean and pollution-free growing environment, so far, no co-morbidity between the fish and human is found, and if the fish is prepared into a collagen-based bone repair product, the fish is not limited by religious belief and is far lower than a terrestrial mammal in risk grade. In addition, the research finds that the collagen derived from fish has similar components and structures with the collagen of terrestrial mammals, is similar in physical and chemical properties, biological properties and the like, and is considered as an important collagen substitute source.

China is a big aquatic product country, various aquatic product processing enterprises have a large number, fish bones left in the aquatic product processing of each enterprise are generally discarded as solid wastes or sold to feed production enterprises at low price for feed production, and the added value is very low. If the characteristics of fish bones can be fully utilized, the fish bones are processed into the decalcified bone matrix with a certain space structure for repairing bone defects, so that not only can an ideal medical bone repairing material raw material be obtained, but also the additional value of the fish bones can be obviously improved.

Disclosure of Invention

The invention aims to: the decalcified bone matrix is prepared with fish bone as material and through impurity eliminating, cell eliminating, decalcification and other treatment.

In order to achieve the above object, the present invention adopts the following technical solutions:

a preparation method of a decalcified bone matrix for fish is characterized by comprising the following steps:

(1) adding the cut fishbone and a sodium chloride solution with the mass concentration of 9% into a reaction kettle, stirring for 8 hours at 4 ℃, and removing the sodium chloride solution;

(2) adding an SDS solution with the mass concentration of 1% into a reaction kettle, stirring for 8 hours at 4 ℃, removing the SDS solution, and repeatedly washing fishbones with pure water for three times;

(3) adding glycerol into a reaction kettle, stirring for 8 hours at 4 ℃, filtering and recovering the glycerol, and washing the fishbone once by using pure water;

(4) adding a hydrogen peroxide solution with the mass concentration of 30% into the reaction kettle, stirring for 8 hours at 37 ℃, removing the hydrogen peroxide solution, and washing the fishbone once by using pure water;

(5) adding anhydrous ether into the reaction kettle, stirring for 8 hours at 4 ℃, and filtering to recover ether;

(6) adding a protease solution with the mass concentration of 5% into the reaction kettle, stirring at 4 ℃ for 0.5h, quickly removing the protease solution, and washing the fishbone once by using pure water;

(7) adding absolute ethyl alcohol into the reaction kettle, dehydrating the fishbone, and then removing the absolute ethyl alcohol;

(8) and taking out the dehydrated fish bones from the reaction kettle, and freeze-drying to obtain the fish decalcified bone matrix.

Preferably, in the step (1), the cut fishbone is in a square shape with the side length of 0.1 cm-0.5 cm, and is rinsed with pure water for three times before being put into the reaction kettle.

Preferably, in step (1), the sodium chloride solution is used in an amount of: 100L of sodium chloride solution is added to 1kg of fishbone.

Preferably, in step (2), the amount of the SDS solution is: 100L of SDS solution was added to 1kg of fishbone.

Preferably, in the step (3), the glycerol is used in an amount of: 100L of glycerol is added into 1kg of fishbone.

Preferably, in the step (4), the amount of the hydrogen peroxide solution is: every 1kg of fishbone is added with 100L of hydrogen peroxide solution.

Preferably, in step (5), the anhydrous diethyl ether is used in an amount of: 100L of anhydrous diethyl ether was added to 1kg of fishbone.

Preferably, in the step (6), the protease solution is used in an amount of: 100L protease solution is added to 1kg fishbone.

Preferably, in the step (7), the absolute ethyl alcohol is used in an amount of: 20L of absolute ethanol is added into each 1kg of fishbone.

The decalcified bone matrix for fish is characterized by being prepared by the preparation method.

The invention has the advantages that:

1. according to the method for preparing the fish decalcification bone matrix, the whole reaction process is completed in the same reaction kettle, so that time and labor are saved, and the prepared decalcification bone matrix is prevented from being polluted by other external impurities;

2. the fish decalcification bone matrix prepared by the invention well maintains the space structure of the fish bone, has good biocompatibility, biodegradability and bone repair capability, and the product obtained after crushing and remodeling has good mechanical strength and can be used for various non-bearing bone defect repairs and auxiliary bearing bone defect repairs;

3. the fish decalcification bone matrix prepared by the method can effectively avoid the risk of people and livestock suffering from diseases;

4. the fish decalcified bone matrix prepared by the method is low in price and has good market application prospect.

Drawings

FIG. 1 is a demineralized bone matrix of fish made from the vertebra of salmon;

FIG. 2 is a powder of a demineralized bone matrix of fish obtained by pulverizing the demineralized bone matrix of fish of FIG. 1;

FIG. 3 is a bone repair product remodeled from the powder of FIG. 2 that may be used in a clinical setting;

FIG. 4 is a 50-fold scanning electron micrograph of the demineralized bone matrix of the fish of FIG. 1;

FIG. 5 is a 500-fold scanning electron micrograph of the demineralized bone matrix of the fish of FIG. 1;

FIG. 6 is a 1000-fold scanning electron micrograph of the demineralized bone matrix of the fish of FIG. 1;

FIG. 7 is a 5000-fold scanning electron micrograph of the demineralized bone matrix of the fish of FIG. 1;

FIG. 8 is an infrared scan of the demineralized bone matrix of fish of FIG. 1;

FIG. 9 is a thermogravimetric analysis of the decalcified bone matrix of the fish of FIG. 1;

FIG. 10 is a graph showing the results of cytotoxicity test of the decalcified bone matrix of the fish of FIG. 1;

FIG. 11 is a graph showing the results of a hemolysis test of the decalcified bone matrix of the fish of FIG. 1;

FIG. 12 is a graph of the results of a pyrogen test of the decalcified bone matrix of the fish of FIG. 1;

FIG. 13 is a graph showing the results of measuring the mechanical strength of the product obtained by pulverizing and remodeling the demineralized bone matrix of fish of FIG. 1;

FIG. 14 is a graph showing HE staining patterns obtained after the fish decalcified bone matrix and the bovine decalcified bone matrix of FIG. 1 are implanted under the skin for 7d, 14d and 21d, respectively.

Detailed Description

The fish bone mainly comprises type III collagen, fish tissue is arranged around the fish bone, cells, calcium and the like are arranged in the fish bone, the spatial structure of the fish bone is well reserved, the fish tissue, the cells, the calcium and the like are removed, the immunogenicity is removed, and the decalcified bone matrix similar to a market-sold terrestrial animal can be prepared.

The invention is described in detail below with reference to the figures and the embodiments.

Preparation of decalcified bone matrix for fish

Fresh salmon vertebra is used as a raw material, a cutting machine is used for cutting the fresh fishbone into blocks with the side length of 0.1 cm-0.5 cm, the blocks are rinsed for three times by using proper amount of pure water, water is drained, 1kg of cut fishbone is weighed and placed in a reaction kettle, and the following process flows are sequentially carried out in the reaction kettle:

1. adding 100L of 9% sodium chloride solution into the reaction kettle, stirring for 8h at 4 ℃, and removing the sodium chloride solution;

2. adding 100L of SDS solution with the mass concentration of 1% into a reaction kettle, stirring for 8 hours at 4 ℃, removing the SDS solution, and repeatedly washing the fishbone with pure water for three times;

3. adding 100L of glycerol into a reaction kettle, stirring for 8h at 4 ℃, filtering and recovering the glycerol, and washing the fishbone once by using pure water;

4. adding 100L of hydrogen peroxide solution with the mass concentration of 30% into a reaction kettle, stirring for 8 hours at 37 ℃, removing the hydrogen peroxide solution, and cleaning the fishbone once by using pure water;

5. adding 100L of anhydrous ether into the reaction kettle, stirring for 8 hours at 4 ℃, and filtering to recover ether;

6. adding 100L of protease solution with the mass concentration of 0.5% into a reaction kettle, stirring at 4 ℃ for 0.5h, quickly removing the protease solution, and washing the fishbone once by using pure water;

7. adding 20L of absolute ethyl alcohol into the reaction kettle, dehydrating the fishbone, and then removing the absolute ethyl alcohol;

8. and taking out the dehydrated fish bones from the reaction kettle, and freeze-drying to obtain the fish decalcified bone matrix.

Secondly, observing the structure of the decalcified bone matrix of the fish

1. Direct viewing

The appearance of the fish decalcified bone matrix prepared by the invention is shown in figure 1, and the bone matrix is milky white, maintains the complete structure of fish bones, and has no impurities.

The obtained bone matrix is pulverized to obtain powder shown in figure 2, which is milky white and free of impurities.

After the powder shown in fig. 2 is remolded, the bone repair product which is shown in fig. 3 and can be directly applied to clinic is obtained, and the bone repair product is milk white, has fine and smooth surface and has no impurities.

2. Scanning electron microscope

Scanning electron micrographs at 50 times, 500 times, 1000 times and 5000 times of the fish decalcified bone matrix prepared by the invention are respectively shown in figure 4, figure 5, figure 6 and figure 7.

As can be seen from fig. 4, 5, 6 and 7: the fish decalcification bone matrix prepared by the invention reserves the space structure of the fishbone skeleton.

3. Infrared spectroscopy scanning

The fish decalcification bone matrix prepared by the method is subjected to infrared spectrum scanning, and the scanning result is shown in figure 8.

As can be seen from fig. 8: the fish decalcified bone matrix prepared by the method has all characteristic absorption peaks of collagen and is high in purity.

4. Porosity of the material

The porosity of the fish decalcified bone matrix prepared by the method is calculated by a swelling method.

The porosity of the decalcified bone matrix of the fish is 46.65 +/-2.00 percent through calculation, so that the decalcified bone matrix of the fish has a good space structure and can provide a scaffold for cell crawling and cell growth.

Thirdly, detecting the thermal stability of the decalcified bone matrix of the fish

Thermogravimetric analysis is carried out on the fish decalcified bone matrix prepared by the invention, and the analysis result is shown in figure 9.

As can be seen from fig. 9: the fish decalcified bone matrix prepared by the method disclosed by the invention is good in thermal stability and suitable for being implanted into a body for use.

Fourth, cytotoxicity test

The experimental procedure is as follows:

(1) soaking the fish decalcified bone matrix in a cell culture medium for 24 hours at 37 ℃ to obtain fish decalcified bone matrix leaching liquor;

(2) culturing the L929 cells to a logarithmic phase, then adding the L929 cells into a 96-well plate, and normally culturing for 2 d;

(3) replacing the cell culture medium (100% leaching solution) with the fish decalcified bone matrix leaching solution or partially replacing the cell culture medium (25% leaching solution, 50% leaching solution, 75% leaching solution);

(4) after culturing for 7d, detecting the cell proliferation rate by using a cell proliferation rate detection kit, and further judging the cytotoxicity.

The results of the experiment are shown in FIG. 10.

As can be seen from fig. 10: the cell proliferation rate of 100% leaching solution group is more than 95%, and the cytotoxicity grade is 0 according to cytotoxicity evaluation standard, namely no cytotoxicity exists.

Experiment on hemolysis

The experimental procedure is as follows:

(1) soaking the fish decalcified bone matrix in normal saline water at 37 deg.C for 24h to obtain fish decalcified bone matrix leaching liquor;

(2) taking 3mL of fresh blood of a rat, adding an anticoagulant, and subpackaging the fresh blood into three 10mL centrifuge tubes in equal amount;

(3) respectively adding 5mL of distilled water, 5mL of physiological saline and 5mL of fish decalcified bone matrix leaching liquor into three centrifuge tubes;

(4) reacting at 37 ℃ for 30min, centrifuging, observing the color of supernatant, and judging whether the erythrocyte swelling and breaking phenomenon occurs.

The results of the experiment are shown in FIG. 11.

As can be seen from fig. 11: the blood added with the fish decalcified bone matrix leaching liquor and the normal saline does not have erythrocyte swelling rupture, namely, the phenomenon of hemolysis does not exist.

Six, pyrogen experiment

The experimental process is as follows:

(2) the decalcified bone matrix leaching liquor of the fishes is injected into a New Zealand white rabbit body intravenously, and the body temperature of the white rabbit is monitored for 3 hours in real time.

The results of the experiment are shown in FIG. 12.

As can be seen from fig. 12: after the fish decalcification bone matrix leaching liquor is injected, the temperature of the white rabbit body has no obvious change, which shows that the fish decalcification bone matrix prepared by the invention has no pyrogen.

Seventhly, detecting the mechanical strength of the product obtained by crushing and remolding the fish decalcified bone matrix specifically as follows:

the fish decalcified bone matrix prepared by the method is crushed and remolded into a cylinder with the diameter of 0.3cm and the height of 0.3cm, and then a universal testing machine is used for testing the compressive strength at the speed of 0.5 mm/s.

The results are shown in FIG. 13.

As can be seen from fig. 13: the product obtained by crushing and remolding the fish decalcification bone matrix has good mechanical strength, which lays a foundation for the remolded product to be used for various non-bearing bone defect repairs and auxiliary repair of bearing bone defects.

Experiment of subcutaneous implantation

The experimental process is as follows:

(1) a 0.5cm long wound was incised on the back of the rat;

(2) respectively implanting the fish decalcified bone matrix prepared by the method and a cattle decalcified bone matrix purchased from the market between the skin and a muscle fascia layer of a rat;

(3) after 7d, 14d and 21d, the rats are sacrificed and subjected to conventional HE staining to observe whether the implantation part has immune reactions such as infection, red swelling and the like and the degradation conditions of the decalcified bone matrix of fish and cattle.

The results of the experiment are shown in FIG. 14.

As can be seen from fig. 14: at 7d, rats implanted with fish decalcified bone matrix and rats implanted with bovine decalcified bone matrix showed many inflammatory cells at the implantation sites; at 14d, inflammatory cells at the implanted parts of the rats implanted with the fish decalcified bone matrix and the rats implanted with the cattle decalcified bone matrix disappear, and the implanted fish decalcified bone matrix and the cattle decalcified bone matrix are wrapped by fibrous tissues and start to degrade; at the 21d, more than 60% of the implanted decalcified bone matrix of the fish is degraded, and more than 60% of the implanted decalcified bone matrix of the cattle is not degraded; in the whole process, rats implanted with the fish decalcified bone matrix and rats implanted with the cattle decalcified bone matrix do not have any infection symptoms.

Therefore, the implanted decalcified bone matrix for fish and the implanted decalcified bone matrix for cattle have good biocompatibility and certain biodegradability.

It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the protection scope of the present invention.

Claims

1. A preparation method of a decalcified bone matrix for fish is characterized by comprising the following steps:

(6) adding a protease solution with the mass concentration of 5% into a reaction kettle, stirring at 4 ℃ for 0.5h, quickly removing the protease solution, and washing the fishbone once by using pure water;

2. The method according to claim 1, wherein in the step (1), the cut fish bone is in a square shape with a side of 0.1cm to 0.5cm, and is rinsed three times with pure water before being put into the reaction vessel.

3. The method according to claim 1, wherein in step (1), the sodium chloride solution is used in an amount of: 100L of sodium chloride solution is added to 1kg of fishbone.

4. The method according to claim 1, wherein in the step (2), the SDS solution is used in an amount of: 100L of SDS solution was added to 1kg of fishbone.

5. The method according to claim 1, wherein in the step (3), the glycerol is used in an amount of: 100L of glycerol is added into 1kg of fishbone.

6. The production method according to claim 1, wherein in the step (4), the hydrogen peroxide solution is used in an amount of: every 1kg of fishbone is added with 100L of hydrogen peroxide solution.

7. The method according to claim 1, wherein in the step (5), the anhydrous diethyl ether is used in an amount of: 100L of anhydrous ether was added to 1kg of fishbone.

8. The method according to claim 1, wherein in the step (6), the protease solution is used in an amount of: 100L protease solution is added to 1kg fishbone.

9. The method according to claim 1, wherein in the step (7), the absolute ethanol is used in an amount of: 20L of absolute ethanol is added into each 1kg of fishbone.

10. A fish decalcified bone matrix prepared by the process according to any one of claims 1 to 9.