CN115645617A - Preparation method of bone matrix gel with biological activity - Google Patents
Preparation method of bone matrix gel with biological activity Download PDFInfo
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Abstract
The invention discloses a preparation method of bone matrix gel with bioactivity. The method adopts fresh or deep low temperature frozen cortical bone, cancellous bone and cartilage, and comprises the steps of rough treatment, primary degreasing, secondary degreasing, impurity removal, crushing, sequential decalcification, antigen removal, puffing, primary dialysis, crosslinking, secondary dialysis and centrifugation. The bone matrix gel prepared by the invention has no cytotoxicity, good biocompatibility, strong heat denaturation resistance and moderate viscosity, and improves the use efficiency of bones.
Description
Technical Field
The invention belongs to the technical field of medical materials, and particularly relates to a preparation method of bone matrix gel with biological activity.
Background
At present, in clinical practice, patients with fractures and local bone defects at different parts of the whole body caused by diseases such as trauma and tumor are increasingly used, and bone graft substitute materials are also increasingly used, wherein the commonly used materials comprise autologous bones, allogeneic bones, artificial bones and the like, and the autologous bones are limited in large-scale application due to limited sources and complications. The artificial bone also has the defects of poor biocompatibility, poor osteoconductivity, insufficient mechanical property and the like. The allogeneic bone is used in large clinical scale after being processed by a series of processes, and the performance is good. In allogenic bone materials, the spongy bone series products are mostly used at present, and the popularization of the use of a large amount of cortical bone is limited due to the high calcium content, so that the problem of how to improve the use efficiency of the cortical bone becomes an urgent need to be solved.
Researchers have prepared decalcified bone matrix by decalcifying bone material and further developed different products. Zhanchunli et al prepared the decalcified bone matrix micropowder by dynamic decalcification and secondary acid-changing process, and implanted in nude mice can regenerate new bones (Zhanchunli, zhao waves, white jade dragon, korean Wei, houshu, the influence of an efficient preparation process on the performance of the decalcified bone matrix, J. Chinese bone and joint injury, 2015, no. 30, no. 1: 79-81.), the method effectively improves the utilization rate of bone materials, but in the clinical operation process, the decalcified bone matrix powder has poor formability, and cannot form good defect repair form after operation, the free particles influence the bone conduction and bone induction of the material at the defect broken end repair part, and the decalcified bone matrix powder scattered around the wound also influences the normal healing of the wound. From the perspective of an extraction process, the preparation process is to completely dissolve the decalcified bone matrix, a dissolving solution is an acid-soluble type I collagen solution, and after the pH value is adjusted by NaOH, collagen is precipitated to form a jelly-like substance, and the method is not beneficial to large-scale preparation and application from the perspective of clinical transformation. Also, patent publication No. CN112999420A discloses a method for using cortical bone, which comprises defatting with chloroform and formaldehyde, soaking in water bath at 59-61 deg.C for 10 hr, decalcifying with 0.6M hydrochloric acid to obtain demineralized bone fibers, soaking the bone fibers in 1M hydrochloric acid, neutralizing with NaOH, centrifuging to remove supernatant, placing the residue in a mold, and lyophilizing to obtain spongy substance, wherein the residual chloroform and formaldehyde used in the preparation process can cause toxicity of the product, and the water bath at 37 deg.C, 59-61 deg.C and 1M hydrochloric acid can cause collagen in the demineralized bone matrix to denature, thereby causing the material to lose natural bioactivity of collagen, and not beneficial to the biological efficacy of the material.
Disclosure of Invention
The invention aims to provide a preparation method of a bone matrix gel with bioactivity.
A preparation method of a bone matrix gel with bioactivity comprises the following steps:
(1) Selecting one or more of fresh or deep low temperature frozen cortical bone, cancellous bone and cartilage;
(2) Rough treatment: removing soft tissues outside the bone tissue by using a blade, cleaning the bone tissue by using 0.01-0.03M PBS buffer solution (pH 7.2-7.4, solarbio, product number: P1020), cutting the bone into bone blocks, and flushing the bone blocks by using a high-pressure water gun until bone marrow components do not exist in bone strips;
(3) Primary degreasing: 1g of bone small pieces: soaking the bone small blocks in the starch suspension for 1-5 hours at the ratio of 10-50 mL of the starch suspension, and washing the bone small blocks with 0.01-0.03M PBS buffer solution after the soaking is finished until the washing solution is clear and transparent and has no starch particles;
(4) Secondary degreasing: 1g of bone pieces: adding the bone small blocks into 0.01-0.10M glucopyranoside solution according to the proportion of 10-50 mL glucopyranoside solution, soaking for 1-5 h, and cleaning with 0.01-0.03M PBS buffer solution after soaking is finished until the cleaning solution is clear and transparent;
(5) Impurity removal: 1g of bone pieces: adding the bone small blocks into 0.10-3.00 percent NaOH solution according to the proportion of 10-50 mL of NaOH solution, soaking for 1-5 h to remove impurities, and after soaking, washing with 0.01-0.03M PBS buffer solution until the pH value of the washing solution is neutral;
(6) Crushing: freeze-drying the bone small blocks, adding the bone small blocks into an automatic ball mill, and grinding the bone small blocks into bone powder with the particle size of 1-999 microns;
(7) Sequential decalcification: 1g of bone meal: adding the bone meal into 0.01-1.00M hydrochloric acid, formic acid or acetic acid solution according to the proportion of 10-50 mL of acid solution, vibrating to decalcify, centrifuging to remove the acid solution, replacing with 0.10-1.00M EDTA solution, and continuously decalcifying until complete decalcification;
(8) Antigen removal: 1g of bone meal: adding the bone meal into 0.10-50.00U/mL totipotent nuclease solution and/or 1.00-100.00U/mL alpha-Gal enzyme according to the proportion of 10-50 mL enzyme solution, oscillating, removing DNA and alpha-Gal antigen, and freeze-drying to obtain bone matrix powder;
(9) Puffing: 1g of bone matrix powder: adding the bone matrix powder into 0.01-1.00M hydrochloric acid, acetic acid or citric acid solution according to the proportion of 10-50 mL of solution, adding pepsin, trypsin, papain or ficin accounting for 0.01-10% of the weight of the bone matrix powder into the solution, and vibrating and puffing for 1-7 days;
(10) Primary dialysis: centrifuging the expanded liquid mixture at the speed of 5000-10000 rpm, removing supernatant, transferring the centrifuged precipitate into a dialysis bag, clamping the bag opening, placing the bag opening into a container, adding dialysis external liquid into the container, replacing the dialysis external liquid once every 24 hours, then replacing the dialysis external liquid with 0.01-0.03M PBS buffer solution (pH 7.2-7.4, solarbio, product number: P1020) and replacing the dialysis external liquid once every 24 hours;
(11) And (3) crosslinking: centrifuging the liquid mixture in the dialysis bag at the speed of 5000-10000 rpm, removing supernatant, and crosslinking the centrifugal precipitate in a physical or chemical mode;
(12) And (3) secondary dialysis: centrifuging the crosslinked mixture at 5000-10000 rpm, removing supernatant, transferring the centrifuged precipitate into a dialysis bag, clamping the bag mouth, placing the bag mouth into a container, adding dialysis external liquid into the container, and replacing the dialysis external liquid once every 24 hours, wherein the cut-off molecular weight of the dialysis bag is 10-500 kDa, and the dialysis external liquid is 0.01-0.03M PBS buffer solution (pH 7.2-7.4, solambio, product number: P1020) or 0.9% NaCl solution;
(13) Centrifuging: centrifuging the liquid mixture in the dialysis bag at 3000-5000 rpm, and removing supernatant to obtain jelly-like substance, namely the bone matrix gel with bioactivity.
The cortical bone, the cancellous bone and the cartilage are derived from one or more of human, non-human primate, fish, birds, cattle, pig, horse, sheep, dog, mule, chicken, duck and goose.
The deep low temperature is-80 to-20 ℃.
The starch suspension is prepared from starch from wheat, corn, rice, sorghum, soybean, potato or sweet potato and PBS buffer solution, and the starch content is 0.10-50.00%.
The glucopyranoside is one or more of n-octyl-beta-D glucopyranoside, octyl-beta-D-thioglucopyranoside, nonyl glucopyranoside and dodecyl glucopyranoside.
The cut-off molecular weight of the dialysis bag in the step (10) is 10-500 kDa, and the dialysis external liquid is 0.01-1.00M hydrochloric acid, acetic acid or citric acid solution.
The physical crosslinking mode is ultraviolet irradiation; the chemical crosslinking mode is to add any one or a combination of a plurality of aldehydes, epoxy compounds, plant extracts, EDC/NHS, sulfur-containing compounds, inorganic salts and photosensitive curing agents.
The plant extract is a viola yedoensis makino extract and a fenugreek seed extract according to the mass ratio of 1: 1.
And (7) the acid solution is a mixed acid solution of 0.2M hydrochloric acid and 0.2M malic acid.
The invention has the beneficial effects that: the method removes soft tissue, fat, impure protein and the like from the bone material under the low-temperature condition, and can keep the biological activity of collagen in the bone tissue unaffected. The bone material is degreased in a milder mode, the starch can adsorb grease substances, the cost performance is higher, the glucopyranoside reagent is an environment-friendly and low-toxicity surfactant, and the grease of the bone material can be better removed by sequentially using the glucopyranoside reagent and the low-toxicity surfactant; the bone meal is soaked in NaOH, so that impurities can be removed, and the effect of virus inactivation is achieved. The sequential decalcification adopts a mild decalcification reagent, so that the damage of the decalcification reagent to the structure and activity of the collagen is avoided. The antigen substance is removed by using the combination of the holonuclease and the alpha-Gal enzyme, so that DNA, RNA and alpha-Gal antigen in extracellular matrix can be removed as much as possible, and the immunogenicity of the extracellular matrix is reduced. The bone meal particles are expanded by combining an acid and enzyme mode, the acid can open the collagen fiber space structure in the extracellular matrix, water molecules can enter the collagen fiber space structure to be expanded, and the enzyme can specifically cut terminal peptide sequences of the amino terminal and the carboxyl terminal of the collagen to further reduce the immunogenicity. The bone matrix gel can improve the degradation resistance of the bone matrix gel through crosslinking, and is more beneficial to the returning and the action of materials. Ineffective or foreign components in the bone matrix gel can be removed in a dialysis manner, which is more effective than a simple cleaning manner.
Drawings
Figure 1 is the appearance of the bone matrix gel prepared in example 1.
Fig. 2 is a microstructure of a bone matrix gel after lyophilization.
FIG. 3 shows the growth state of MC3T3 cells in the bone matrix gel prepared in example 1.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following more detailed description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Example 1
A preparation method of a bone matrix gel with bioactivity comprises the following steps:
(1) Fresh cortical bone and cancellous bone from pig are selected, and the mass ratio of the two bones is 1:1;
(2) Rough treatment: removing soft tissues outside the bone tissue by using a blade, cleaning the bone tissue by using 0.02M PBS buffer solution, cutting the bone into bone small blocks, and flushing the bone small blocks by using a high-pressure water gun until bone marrow components do not exist in bone strips;
(3) Primary degreasing: 1g of bone pieces: soaking the bone small blocks in the starch suspension for 2 hours at the ratio of 30mL of the starch suspension, and washing the bone small blocks with 0.02M PBS buffer solution after the soaking is finished until the washing solution is clear and transparent and has no starch particles; the starch suspension is prepared from wheat starch and 0.02M PBS buffer solution, and the starch content is 20%;
(4) Secondary degreasing: 1g of bone pieces: according to the proportion of 30 mLn-octyl-beta-D glucopyranoside solution, small bone blocks are added into n-octyl-beta-D glucopyranoside solution to be soaked for 2 hours, and after soaking is finished, the small bone blocks are washed by 0.02M PBS buffer solution until the washing solution is clear and transparent;
(5) Impurity removal: 1g of bone small pieces: 30mL of NaOH solution, adding the bone small blocks into 0.50 percent of NaOH solution, soaking for 2 hours to remove impurities, and after soaking, washing with 0.02M PBS buffer solution until the pH value of the washing solution is neutral;
(6) Crushing: freeze-drying the bone small blocks, adding the bone small blocks into an automatic ball mill, and grinding into bone powder with the particle size of 50 microns;
(7) Sequential decalcification: 1g of bone meal: adding the bone meal into an acid solution with the proportion of 30mL, oscillating to decalcify, centrifuging to remove the acid solution, replacing the acid solution with 0.50M EDTA solution, and continuously decalcifying until complete decalcification; the acid solution is a mixed acid solution of 0.20M hydrochloric acid and 0.20M malic acid;
(8) Antigen removal: 1g of bone meal: adding bone meal into totipotent nuclease solution (15mL, 40.00U/mL) and alpha-Gal enzyme (15mL, 40.00U/mL) according to the proportion of 30mL of enzyme solution, oscillating, removing DNA and alpha-Gal antigen, and freeze-drying to obtain bone matrix powder;
(9) Puffing: 1g of bone matrix powder: adding bone matrix powder into 0.20M hydrochloric acid solution at a ratio of 30mL, adding pepsin 0.1% of the weight of the bone matrix powder into the solution, and vibrating and puffing for 5 days;
(10) Primary dialysis: centrifuging the expanded liquid mixture at 8000rpm, removing supernatant, transferring the centrifuged precipitate into a dialysis bag, clamping the bag opening, placing the bag opening into a container, adding dialysis external liquid into the container, replacing the dialysis external liquid once every 24 hours, replacing the dialysis external liquid with 0.02M PBS buffer liquid, and replacing the dialysis external liquid once every 24 hours; the cut-off molecular weight of the dialysis bag is 100kDa, and the dialysis external liquid is 0.2M hydrochloric acid solution;
(11) And (3) crosslinking: centrifuging the liquid mixture in the dialysis bag at 8000rpm, removing supernatant, and crosslinking the precipitate by adding plant extract at an amount of 10% of the precipitate; the plant extract is a viola yedoensis makino extract and a fenugreek seed extract according to the mass ratio of 1:1; the extraction method of the viola yedoensis makino extract comprises the following steps: taking roots of violet, drying in the sun, grinding into powder, adding 5 times of water by weight, extracting for 3 times by reflux, combining filtrates, and concentrating to 1/10 of the filtrate volume to prepare the traditional Chinese medicine composition; the extraction method of the fenugreek extract comprises the following steps: taking seeds of common fenugreek seeds, drying in the sun, grinding into powder, adding 5 times of water by weight, refluxing and extracting for 3 times, combining filtrates, and concentrating to 1/10 of the volume of the filtrate to prepare the traditional Chinese medicine composition;
(12) And (3) secondary dialysis: centrifuging the crosslinked mixture at 8000rpm, removing supernatant, transferring the centrifugal precipitate into a dialysis bag, clamping the bag opening, placing into a container, adding dialysis external liquid into the container, and replacing the dialysis external liquid once every 24 hours, wherein the cut-off molecular weight of the dialysis bag is 100kDa, and the dialysis external liquid is 0.9% NaCl solution;
(13) Centrifuging: centrifuging the liquid mixture in the dialysis bag at 4000rpm, and removing the supernatant to obtain jelly-like substance, i.e. bone matrix gel with bioactivity.
The bone matrix gel prepared in this example is clear in appearance, smooth in surface, rich in moisture, and jelly-like, as shown in fig. 1. After the bone matrix gel is frozen and dried, the observation is carried out by a scanning electron microscope, the result is shown in figure 2, as can be seen from figure 2, the freeze-dried product of the bone matrix gel is in a lamellar shape, the distance between the lamellar layers is 20-50 mu m, pores between the lamellar layers can form a channel of a capillary vessel, and a large amount of phase change materials can be loaded, so that the loaded materials can still be kept from losing under the action of external force.
Example 2
A preparation method of a bone matrix gel with bioactivity comprises the following steps:
(1) Selecting bovine cancellous bone and cartilage frozen at a deep low temperature of (-20 ℃), wherein the mass ratio of the two bones is 1:1;
(2) Rough treatment: removing soft tissues outside the bone tissue by using a blade, cleaning the bone tissue by using 0.01M PBS buffer solution, cutting the bone into bone small blocks, and flushing the bone small blocks by using a high-pressure water gun until bone marrow components do not exist in bone strips;
(3) Primary degreasing: 1g of bone small pieces: soaking the bone small blocks in 20mL of starch suspension for 1h, and washing the bone small blocks with 0.01M PBS buffer solution after soaking till the washing solution is clear and transparent and has no starch particles; the starch suspension is prepared from corn starch and 0.01M PBS buffer solution, and the starch content is 10%;
(4) And (3) secondary degreasing: 1g of bone small pieces: adding the small bone blocks into an octyl-beta-D-thiopyranoside solution according to the proportion of 20mL of the octyl-beta-D-thiopyranoside solution, soaking for 1h, and cleaning with 0.01M PBS buffer solution after soaking is finished until the cleaning solution is clear and transparent;
(5) Impurity removal: 1g of bone pieces: 20mL of NaOH solution, adding the bone small blocks into 0.50 percent of NaOH solution, soaking for 1h to remove impurities, and after soaking, washing with 0.01M PBS buffer solution until the pH value of the washing solution is neutral;
(6) Crushing: freeze-drying the bone small blocks, adding the bone small blocks into an automatic ball mill, and grinding into bone powder with the particle size of 100 mu m;
(7) Sequential decalcification: 1g of bone meal: adding the bone meal into 0.40M formic acid solution according to the proportion of 20mL of acid solution, oscillating to decalcify, centrifuging to remove the acid solution, replacing the acid solution with 0.40M EDTA solution, and continuously decalcifying until complete decalcification;
(8) Antigen removal: 1g of bone meal: adding bone meal into totipotent nuclease solution (10mL, 50.00U/mL) and alpha-Gal enzyme (10mL, 50.00U/mL) according to the proportion of 20mL of enzyme solution, oscillating, removing DNA and alpha-Gal antigen, and freeze-drying to obtain bone matrix powder;
(9) Puffing: 1g of bone matrix powder: adding bone matrix powder into 0.30M acetic acid solution according to the proportion of 20mL of solution, adding trypsin accounting for 0.50 percent of the weight of the bone matrix powder into the solution, and vibrating and puffing for 3 days;
(10) Primary dialysis: centrifuging the puffed liquid mixture at 6000rpm, removing supernatant, transferring the centrifuged precipitate into a dialysis bag, clamping the bag opening, placing into a container, adding dialysis external liquid into the container, replacing the dialysis external liquid once every 24 hours, replacing the dialysis external liquid with 0.01M PBS buffer liquid, and replacing the dialysis external liquid once every 24 hours; the cut-off molecular weight of the dialysis bag is 200kDa, and the dialysis external solution is 0.40M acetic acid solution;
(11) And (3) crosslinking: centrifuging the liquid mixture in the dialysis bag at 6000rpm, removing supernatant, and crosslinking the centrifuged precipitate by ultraviolet irradiation;
(12) And (3) secondary dialysis: centrifuging the crosslinked mixture at 6000rpm, removing supernatant, transferring the centrifuged precipitate into a dialysis bag, clamping the bag opening, placing into a container, adding dialysis external liquid into the container, and replacing the dialysis external liquid every 24 hours, wherein the dialysis bag has a molecular weight cutoff of 200kDa, and the dialysis external liquid is 0.02M PBS buffer solution;
(13) Centrifuging: centrifuging the liquid mixture in the dialysis bag at 3000rpm, and removing the supernatant to obtain jelly-like substance, i.e. bone matrix gel with bioactivity.
Example 3
A preparation method of bone matrix gel with bioactivity comprises the following steps:
(1) Selecting the same cortical bone and cancellous bone frozen at a deep low temperature of (-60 ℃), wherein the mass ratio of the two bones is 1:1;
(2) Rough treatment: removing soft tissues outside the bone tissue by using a blade, cleaning the bone tissue by using 0.03M PBS buffer solution, cutting the bone into bone small blocks, and flushing the bone small blocks by using a high-pressure water gun until bone marrow components do not exist in bone strips;
(3) Primary degreasing: 1g of bone small pieces: soaking the bone small blocks in the starch suspension for 4 hours at the ratio of 40mL of the starch suspension, and washing the bone small blocks with 0.03M PBS (phosphate buffer solution) after soaking till the washing solution is clear and transparent and has no starch particles; the starch suspension is prepared from potato starch and 0.03M PBS buffer solution, and the starch content is 20%;
(4) And (3) secondary degreasing: 1g of bone pieces: adding small bone blocks into the nonyl glucopyranoside solution according to the proportion of 40mL, soaking for 4 hours, and cleaning with 0.03M PBS buffer solution after soaking is finished until the cleaning solution is clear and transparent;
(5) Impurity removal: 1g of bone small pieces: adding the bone small blocks into 0.30 percent NaOH solution according to the proportion of 40mL of NaOH solution, soaking for 4 hours to remove impurities, and after soaking is finished, washing with 0.03M PBS buffer solution until the pH value of the washing solution is neutral;
(6) Crushing: freeze-drying the bone small blocks, then adding the bone small blocks into an automatic ball mill, and grinding the bone small blocks into bone powder with the particle size of 200 mu m;
(7) Sequential decalcification: 1g of bone meal: adding the bone meal into 0.30M acetic acid solution according to the proportion of 40mL of acid solution, carrying out vibration decalcification, centrifuging to remove the acid solution, replacing the acid solution with 0.30M EDTA solution, and continuously decalcification until complete decalcification;
(8) Antigen removal: 1g of bone meal: adding the bone meal into totipotent nuclease solution (40mL, 30.00U/mL) according to the ratio of 40mL enzyme solution, oscillating, removing DNA, and freeze-drying to obtain bone matrix powder;
(9) Puffing: 1g of bone matrix powder: adding bone matrix powder into 0.60M citric acid solution at a ratio of 40mL, adding ficin 0.20% of the weight of the bone matrix powder into the solution, and vibrating and puffing for 3 days;
(10) Primary dialysis: centrifuging the expanded liquid mixture at 9000rpm, removing supernatant, transferring the centrifuged precipitate into a dialysis bag, clamping the bag opening, placing into a container, adding dialysis external liquid into the container, replacing the dialysis external liquid once every 24 hours, replacing the dialysis external liquid with 0.03M PBS buffer liquid, and replacing the dialysis external liquid once every 24 hours; the cut-off molecular weight of the dialysis bag is 300kDa, and the dialysis external liquid is 0.50M citric acid solution;
(11) And (3) crosslinking: centrifuging the liquid mixture in the dialysis bag at 9000rpm, removing supernatant, and crosslinking the centrifuged precipitate by adding plant extract, wherein the addition amount is 10% of the mass of the centrifuged precipitate; the plant extract is a viola yedoensis makino extract and a fenugreek seed extract according to the mass ratio of 1:1; the extraction method of the viola yedoensis makino extract comprises the following steps: taking roots of violet, drying in the sun, grinding into powder, adding 6 times of water by weight, extracting for 3 times by reflux, combining filtrates, and concentrating to 1/10 of the filtrate volume to prepare the traditional Chinese medicine composition; the extraction method of the fenugreek extract comprises the following steps: taking seeds of common fenugreek seeds, drying in the sun, grinding into powder, adding 6 times of water by weight, refluxing and extracting for 3 times, combining filtrates, and concentrating to 1/10 of the volume of the filtrate to prepare the traditional Chinese medicine composition;
(12) And (3) secondary dialysis: centrifuging the crosslinked mixture at 9000rpm, removing supernatant, transferring the centrifugal precipitate into a dialysis bag, clamping the bag opening, placing into a container, adding dialysis external liquid into the container, and replacing the dialysis external liquid every 24 hours, wherein the cut-off molecular weight of the dialysis bag is 300kDa, and the dialysis external liquid is 0.03M PBS buffer solution;
(13) Centrifuging: centrifuging the liquid mixture in the dialysis bag at 5000rpm, and removing the supernatant to obtain jelly-like substance, i.e. bone matrix gel with bioactivity.
Comparative example 1
A method for preparing a bioactive bone matrix gel, the other experimental steps being the same as in example 1, except that the operation of step (7) is: sequential decalcification: 1g of bone meal: adding the bone meal into 0.40M hydrochloric acid solution according to the proportion of 30mL acid solution, shaking to decalcify, centrifuging to remove the hydrochloric acid solution, replacing with 0.50M EDTA solution, and continuously decalcifying till complete decalcification.
Comparative example 2
A method for preparing a bioactive bone matrix gel, the other experimental steps being the same as in example 1, except that the operation of step (7) is: sequential decalcification: 1g of bone meal: adding the bone meal into 0.40M malic acid solution according to the proportion of 30mL of acid solution, carrying out vibration decalcification, centrifuging to remove the malic acid solution, replacing the malic acid solution with 0.50M EDTA solution, and continuing to decalcify until the bone meal is completely decalcified.
Comparative example 3
A method for preparing a bioactive bone matrix gel, the other experimental steps being the same as in example 1, except that the operation of step (11) is: and (3) crosslinking: centrifuging the liquid mixture in the dialysis bag at high speed, removing supernatant, and crosslinking the centrifugal precipitate in a manner of adding herba Violae extract, wherein the addition amount is 10% of the centrifugal precipitate; the extraction method of the viola yedoensis makino extract comprises the following steps: taking the root of viola yedoensis makino, drying in the sun, grinding into powder, adding 5 times of water by weight, extracting under reflux for 3 times, combining filtrates, and concentrating to 1/10 of the volume of the filtrate to prepare the viola yedoensis makino.
Comparative example 4
A method for preparing a bioactive bone matrix gel, the other experimental steps being the same as those of example 1, except that the operation of step (11) is: and (3) crosslinking: centrifuging the liquid mixture in the dialysis bag at high speed, removing supernatant, and crosslinking the centrifugal precipitate in a manner of adding fenugreek extract, wherein the addition amount is 10% of the mass of the centrifugal precipitate; the extraction method of the fenugreek extract comprises the following steps: taking seeds of the fenugreek, drying in the sun, grinding into powder, adding 5 times of water by weight, refluxing and extracting for 3 times, combining filtrates, and concentrating to 1/10 of the volume of the filtrate to prepare the fenugreek seed extract.
Comparative example 5
A method for preparing a bioactive bone matrix gel, the other experimental steps being the same as those of example 1, except that the operation of step (8) is: 1g of bone meal: 30mL of enzyme solution, adding the bone meal into totipotent nuclease solution (30mL, 40.00U/mL), oscillating, removing DNA, and freeze-drying to obtain bone matrix powder.
Comparative example 6
A method for preparing a bioactive bone matrix gel, the other experimental steps being the same as in example 1, except that the operation of step (8) is: antigen removal: 1g of bone meal: 30mL of enzyme solution, adding the bone meal into alpha-Gal enzyme (30mL, 40.00U/mL), oscillating, removing alpha-Gal antigen, and freeze-drying to obtain bone matrix powder.
Experimental example 1: viscosity measurement experiment
The bone matrix gels prepared in examples 1 to 3 and comparative examples 3 to 4 were sampled and analyzed for viscosity characteristics using a rotational viscometer. The temperature is set at 150 ℃ for 30min, and then the measurement is carried out, wherein the rotating speed is 12r/min. The results are shown in Table 1:
TABLE 1
Note: * Represents a P <0.05 compared to example 1.
As can be seen from table 1, the combined crosslinking of viola extract and fenugreek extract increases the viscosity of the bone matrix gel and synergizes, and under the action of viola extract and fenugreek extract, proteins in the gel are easily crosslinked with each other to form crosslinked aggregates, and the hydrogen bonding effect in and among molecules of the bone matrix gel is enhanced, resulting in the increase of the viscosity.
Experimental example 2: cytotoxicity
The bone matrix gels prepared in examples 1 to 3 and comparative examples 1 to 6 were collected, subjected to enzymatic digestion to obtain MC3T3 cells, and the cell density was adjusted to 4X 10 5 Perml, 100. Mu.l per well, were plated into 96-well plates. Inoculating cells into 5 holes of each group of the test group, and adding the leaching liquor of each group of samples; 5 wells of each control group were inoculated with cells and complete medium was added; blank 5 wells, no cells were inoculated, only complete medium was added.
After culturing for 72 hours in the cell incubator, the medium was discarded from each well, 100. Mu.l of a 10-percent CCK8-containing whole medium was added to each well, and after incubation for 30min in an incubator at 37 ℃, absorbance (OD value) was measured at a wavelength of 450nm in a microplate reader. The cell proliferation rate is calculated, and the result shows that the cell proliferation rate of each experimental group is slightly higher than that of a control group, the compatibility of the bone matrix gel cells is excellent, and the cell proliferation is vigorous.
Experimental example 3: biocompatibility
Taking the bone matrix gels prepared in examples 1-3 and comparative examples 1-6, obtaining MC3T3 cells by enzymatic digestion, adjusting the cell density to 4X 10 with 1640 medium containing 20% FBS 5 Per ml, cell suspensions were gelled with bone matrix of varying concentrationsMixing, adding the mixture into 24-well plate, and adding 5% CO at 37 deg.C 2 Culturing in an incubator for 48h. After the culture was completed, the cytoskeleton and nucleus of MC3T3 cells in the gel were stained with α -Tubulin (green) and DAPI (blue), respectively.
The experimental results of example 1 are shown in fig. 3, and it can be seen that the cell growth state is good and the cell survival rate is above 85%, indicating that the bone matrix gel has good biocompatibility. The experimental results of examples 2-3 and comparative examples 1-4 are similar to example 1, and have no significant difference. The cell viability of comparative example 5 was about 60%, the cell viability of comparative example 6 was about 65%, and the biocompatibility was significantly lower than that of example 1.
Experimental example 4: thermal denaturation
The bone matrix gel prepared in example 1 was divided into 4 portions, of which 1 portion was refrigerated in a refrigerator (4 ℃) for 24 hours, 1 portion was allowed to stand at room temperature (22 ℃) for 24 hours, 1 portion was incubated in a water bath at 50 ℃ for 24 hours, and 1 portion was incubated in a water bath at 80 ℃ for 24 hours. The thermal denaturation temperature was determined by differential scanning calorimetry after freeze-drying 4 parts of the bone matrix gel. The results are shown in Table 2, and it is understood that the temperature of 50 ℃ or higher can denature the collagen fibers in the bone matrix gel, thereby lowering the overall thermal denaturation resistance temperature of the material.
TABLE 2 Heat denaturation temperature of each group of samples
The bone matrix gels prepared in examples 2 to 3 and comparative examples 1 to 2 were incubated in a water bath at 50 ℃ for 24 hours, and the heat denaturation temperature was measured by differential scanning calorimetry after the bone matrix gels were lyophilized, and the results are shown in table 3:
TABLE 3
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (9)
1. A method for preparing a bone matrix gel with bioactivity is characterized by comprising the following steps:
(1) Selecting one or more of fresh or deep low temperature frozen cortical bone, cancellous bone and cartilage;
(2) Rough treatment: removing soft tissues outside the bone tissue by using a blade, cleaning the bone tissue by using 0.01-0.03M PBS buffer solution, cutting the bone into bone small blocks, and flushing the bone small blocks by using a high-pressure water gun until bone marrow components do not exist in bone strips;
(3) Primary degreasing: 1g of bone pieces: soaking the bone small blocks in the starch suspension for 1-5 h at the ratio of 10-50 mL of the starch suspension, and washing the bone small blocks with 0.01-0.03M PBS (phosphate buffer solution) after soaking till the washing solution is clear and transparent and has no starch particles;
(4) And (3) secondary degreasing: 1g of bone pieces: adding the bone small blocks into 0.01-0.10M glucopyranoside solution according to the proportion of 10-50 mL glucopyranoside solution, soaking for 1-5 h, and cleaning with 0.01-0.03M PBS buffer solution after soaking is finished until the cleaning solution is clear and transparent;
(5) Impurity removal: 1g of bone pieces: adding the bone small blocks into 0.10-3.00 percent NaOH solution according to the proportion of 10-50 mL of NaOH solution, soaking for 1-5 h to remove impurities, and cleaning with 0.01-0.03 MPBS buffer solution after soaking till the pH value of the cleaning solution is neutral;
(6) Crushing: freeze-drying the bone small blocks, then adding the bone small blocks into an automatic ball mill, and grinding the bone small blocks into bone powder with the particle size of 1-999 microns;
(7) Sequential decalcification: 1g of bone meal: adding the bone meal into 0.01-1.00M hydrochloric acid, formic acid or acetic acid solution according to the proportion of 10-50 mL acid solution, oscillating to decalcify, centrifugally removing the acid solution, replacing with 0.10-1.00M EDTA solution, and continuously decalcifying until complete decalcification;
(8) Antigen removal: 1g of bone meal: adding the bone meal into 0.10-50.00U/mL totipotent nuclease solution and/or 1.00-100.00U/mL alpha-Gal enzyme according to the proportion of 10-50 mL enzyme solution, oscillating, removing DNA and alpha-Gal antigen, and freeze-drying to obtain bone matrix powder;
(9) Puffing: 1g of bone matrix powder: 10-50 mL of solution, adding the bone matrix powder into 0.01-1.00M of hydrochloric acid, acetic acid or citric acid solution, adding pepsin, trypsin, papain or ficin accounting for 0.01-10% of the weight of the bone matrix powder into the solution, and vibrating and puffing for 1-7 days;
(10) Primary dialysis: centrifuging the expanded liquid mixture at the speed of 5000-10000 rpm, removing supernatant, transferring the centrifugal precipitate into a dialysis bag, clamping the bag opening, placing the bag opening into a container, adding dialysis external liquid into the container, replacing the dialysis external liquid once every 24 hours, replacing the dialysis external liquid with 0.01-0.03M PBS buffer solution, and replacing the dialysis external liquid once every 24 hours;
(11) And (3) crosslinking: centrifuging the liquid mixture in the dialysis bag at the speed of 5000-10000 rpm, removing supernatant, and crosslinking the centrifugal precipitate in a physical or chemical way;
(12) And (3) secondary dialysis: centrifuging the crosslinked mixture at 5000-10000 rpm, removing supernatant, transferring the centrifuged precipitate into a dialysis bag, clamping the bag mouth, placing the bag mouth into a container, adding dialysis external liquid into the container, and replacing the dialysis external liquid once every 24 hours, wherein the cut-off molecular weight of the dialysis bag is 10-500 kDa, and the dialysis external liquid is 0.01-0.03M PBS buffer solution or 0.9 percent NaCl solution;
(13) Centrifuging: centrifuging the liquid mixture in the dialysis bag, and removing supernatant to obtain jelly-like substance, i.e. bone matrix gel with bioactivity.
2. The method for preparing the bone matrix gel with bioactivity according to claim 1, wherein the cortical bone, the cancellous bone and the cartilage are derived from one or more of human, non-human primate, fish, birds and birds, cattle, pig, horse, sheep, dog, mule, chicken, duck and goose.
3. The method for preparing a bioactive bone matrix gel according to claim 1, wherein the deep cryogenic temperature is-80 to-20 ℃.
4. The method of claim 1, wherein the starch suspension is prepared from a starch derived from wheat, corn, rice, sorghum, soy, potato, or sweet potato and PBS buffer, and the starch content is 0.10-50.00%.
5. The method of preparing a bioactive bone matrix gel as claimed in claim 1, wherein the glucopyranoside is one or more of n-octyl- β -D glucopyranoside, octyl- β -D-thioglucopyranoside, nonylpyranoside, dodecylglucopyranoside.
6. The method for preparing a bioactive bone matrix gel according to claim 1, wherein the cut-off molecular weight of the dialysis bag in the step (10) is 10-500 kDa, and the dialysate is 0.01-1.00M hydrochloric acid, acetic acid or citric acid solution.
7. The method of preparing a bioactive bone matrix gel as claimed in claim 1, wherein the physical crosslinking is ultraviolet irradiation; the chemical crosslinking mode is to add any one or a combination of a plurality of aldehydes, epoxides, plant extracts, EDC/NHS, sulfur-containing compounds, inorganic salts and photosensitive curing agents.
8. The method for preparing a bioactive bone matrix gel according to claim 7, wherein the plant extract is viola yedoensis makino extract and fenugreek extract in a mass ratio of 1: 1.
9. The method for preparing a bone matrix gel having biological activity according to claim 1, wherein the acid solution of step (7) is a mixed acid solution of 0.2M hydrochloric acid and 0.2M malic acid.
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