CN114392394A - Preparation method of composite bone morphogenetic protein bionic bone repair material - Google Patents
Preparation method of composite bone morphogenetic protein bionic bone repair material Download PDFInfo
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Abstract
The invention discloses a preparation method of a composite bone morphogenetic protein bionic bone repair material, which comprises the steps of sampling, degreasing and decalcifying, washing and freeze-drying, digesting and centrifuging, neutralizing and compounding, gelling and shaping, crosslinking, sterilizing and packaging. The invention has the beneficial effects that: BMP and a gel-like biological bone-derived material are directly compounded in the gelation process to prepare a product, so that the BMP is uniformly distributed inside and outside the structure of the gel-like material, the BMP compounded on the surface of the material can play a role after being implanted into a human body, and the BMP inside the material structure is slowly released along with the degradation of the material to continuously play a bone induction role, thereby reducing the using amount of the BMP and lowering the cost.
Description
Technical Field
The invention relates to a preparation method of a bionic bone repair material, in particular to a preparation method of a composite bone morphogenetic protein biogenic gel bionic bone repair material, and belongs to the technical field of medical treatment.
Background
Although BMP has been found to be a positive and most powerful bone growth factor so far and has great clinical application value, because BMP has the defects of short half-life and easy removal of the contents of a single implant, the BMP needs to be used in combination with a carrier, and thus, researchers have conducted a great deal of research on BMP. The BMP carrier material can be mainly prepared from the following components: natural polymer materials typified by collagen, hyaluronic acid, chitosan, and the like; synthetic polymer materials typified by polycaprolactone and polylactic acid-polyglycolic acid, and inorganic materials typified by hydroxyapatite and calcium phosphate. Although there are many related studies, there are two major categories currently in true clinical use: one is collagen/decalcified bone matrix composite BMPs such as the inflase products and recombinant xenogeneic bones produced by Meidunli, USA, and one is hydroxyapatite artificial bone composite BMPs such as Ribangtai.
Although these two types of products are widely used, there are some problems in their production and preparation:
1. the Infuse product, the recombined allogenic bone and the rapangbon bone Tai are all prepared by a surface adsorption method through carrier and factor compounding, and most BMP is lost and degraded due to extrusion or early exposure to a microenvironment of a human body after the product is implanted into a bone injury part of the human body, so that the biggest problem of the product is that the bone growth factor BMP has general slow release performance, the required concentration is high during compounding, and the product cost is high;
2. the infuse product is inconvenient to use because the collagen sponge and the BMP are independently packaged and adsorbed and compounded when in use;
3. when the heterogeneous bone is recombined, a BMP needs to be subjected to a 'degeneration-renaturation' process, and the process can also cause partial BMP activity loss and reduce the titer.
Disclosure of Invention
The present invention has been made to solve the above problems, and an object of the present invention is to provide a method for producing a composite bone morphogenetic protein-mimetic bone repair material.
The invention realizes the purpose through the following technical scheme: a preparation method of a composite bone morphogenetic protein bionic bone repair material comprises the following steps:
step A: sampling, namely taking fresh calf limb cortical bone, removing soft tissues such as muscles and the like, freezing in liquid nitrogen, respectively carrying out primary crushing, low-temperature fine crushing and superfine crushing by using a superfine crusher, and sieving to obtain bone particles with the size of 100-;
and B: defatting and decalcifying, defatting bone grains with mixed defatting liquid of chloroform and methanol in the ratio of 1 to 1 in the ratio of 30ml to bone powder for 4 hr, and decalcifying with 0.6M hydrochloric acid in the ratio of 30ml to bone powder for 24 hr;
and C: washing and freeze-drying, washing with deionized water 10 times the weight of bone powder for 3-5 times until the pH value is equal to 7.4, pre-freezing in-40 deg.C refrigerator for 4 hr, freeze-drying in freeze dryer, and sealing for storage;
step D: digesting and centrifuging, weighing a certain amount of freeze-dried bone powder, adding 100ml of 0.01M hydrochloric acid and 100mg of pepsin into 1g of bone powder, digesting the bone powder at the temperature of 4 ℃, and continuously stirring for 96 hours by using a magnetic stirrer;
step E: neutralizing and compounding, adding 1/10 volume, 0.1M sodium hydroxide solution and 1/9 volume 10XPBS (pH7.4) into the digestion solution at 4 ℃, fully and uniformly stirring, then adding 10% PVA200 microliter/ml, 0.25 mg/ml BMP and 0.2 mg/ml calcium phosphate powder, and fully and uniformly stirring;
step F: performing gelation and shaping, namely subpackaging the gel solution into a special mould for shaping, and transferring the special mould and the special mould into a 37 ℃ incubator until a gel compound is formed;
step G: crosslinking, namely freeze-drying the gel-like compound, and placing the lyophilized BMP gel compound in a 70 ℃ incubator for 2 hours;
step H: and (3) sterilizing and packaging, namely sterilizing the tool loaded with the BMP gel compound, and packaging the BMP gel compound.
As a still further scheme of the invention: in the step A, the cortex bones of the fresh calf limbs are removed, the soft tissues such as muscles and the like are removed, the fresh calf limbs are frozen in liquid nitrogen, and the fresh calf limbs are sieved for multiple times after being crushed, and the freezing is carried out at room temperature when a sample is taken out.
As a still further scheme of the invention: in step D, the digestion solution is centrifuged at 1000rpm4 ℃ for 5 minutes to remove residues.
As a still further scheme of the invention: in the step E, stirring is performed while adding 200. mu.l/ml of 10% PVA, 0.25 mg/ml of BMP and 0.2 mg/ml of calcium phosphate powder, and the stirring time is prolonged.
As a still further scheme of the invention: in the step H, the sterilization and packaging operation is carried out in an aseptic environment, and the final gel compound is packaged integrally and independently.
The invention has the beneficial effects that:
1. compared with the products sold on the market at present, the BMP is compounded in the gelation process of the carrier material and is formed once through the freeze drying process, compared with the infuse product, the use is convenient, compared with the recombination of the allogeneic bone, the BMP does not need to be denatured and renatured, compared with the Ribangtai, the preparation process is coherent;
2. compared with the current products sold on the market, the method obviously prolongs the slow release time of the BMP, reduces the consumption of the BMP and obviously reduces the cost;
3. the BMP carrier material belongs to biological bone source, main collagen and endogenous growth factors of the BMP carrier material are reserved, a proper amount of calcium phosphate powder is added, and compared with single type I collagen of an infuse product, a carrier bracket after severe antigen removal treatment of recombinant allogeneic bone and pure calcium phosphate powder of Ribangtai, the components of the product produced by the method are closer to the components of actual bone, so the BMP carrier material has more bionic property.
Drawings
FIG. 1 is a schematic view of the structure of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
Referring to fig. 1, a method for preparing a composite bone morphogenetic protein bionic bone repair material includes the following steps:
step A: sampling, namely taking fresh calf limb cortical bone, removing soft tissues such as muscles and the like, freezing in liquid nitrogen, respectively carrying out primary crushing, low-temperature fine crushing and superfine crushing by using a superfine crusher, and sieving to obtain bone particles with the size of 100 um;
and B: defatting and decalcifying, defatting bone grains with mixed defatting liquid of chloroform and methanol in the ratio of 1 to 1 in the ratio of 30ml to bone powder for 4 hr, and decalcifying with 0.6M hydrochloric acid in the ratio of 30ml to bone powder for 24 hr;
and C: washing and freeze-drying, washing with deionized water 10 times the weight of bone powder for 3 times until the pH value is 7.4, pre-freezing in-40 deg.C refrigerator for 4 hr, freeze-drying in freeze-drying machine, and sealing for storage;
step D: digesting and centrifuging, weighing a certain amount of freeze-dried bone powder, adding 100ml of 0.01M hydrochloric acid and 100mg of pepsin into 1g of bone powder, digesting the bone powder at the temperature of 4 ℃, and continuously stirring for 96 hours by using a magnetic stirrer;
step E: neutralizing and compounding, adding 1/10 volume, 0.1M sodium hydroxide solution and 1/9 volume 10XPBS (pH7.4) into the digestion solution at 4 ℃, fully and uniformly stirring, then adding 10% PVA200 microliter/ml, 0.25 mg/ml BMP and 0.2 mg/ml calcium phosphate powder, and fully and uniformly stirring;
step F: performing gelation and shaping, namely subpackaging the gel solution into a special mould for shaping, and transferring the special mould and the special mould into a 37 ℃ incubator until a gel compound is formed;
step G: crosslinking, namely freeze-drying the gel-like compound, and placing the lyophilized BMP gel compound in a 70 ℃ incubator for 2 hours;
step H: and (3) sterilizing and packaging, namely sterilizing the tool loaded with the BMP gel compound, and packaging the BMP gel compound.
In the embodiment of the invention, in the step A, the cortex bones of the fresh calf limbs, the soft tissues such as muscles and the like are removed, the fresh calf limbs are frozen in liquid nitrogen, and the crushed calf limbs are sieved for multiple times, and the sample is taken out at room temperature, so that the safety of the sample taking out is ensured, the rubber parts of the storage equipment are prevented from being damaged, the service life of the device is prolonged, and the quality of the sample used in the subsequent operation is ensured by sieving for multiple times, thereby ensuring the quality of the final product.
In the embodiment of the invention, in the step D, the digestive juice is centrifuged at 1000rpm4 ℃ for 5 minutes to remove residues, so that the solid-liquid separation is effectively carried out, and the residues are safely stored for convenient centralized treatment.
Example two
Referring to fig. 1, a method for preparing a composite bone morphogenetic protein bionic bone repair material includes the following steps:
step A: sampling, namely taking fresh calf limb cortical bone, removing soft tissues such as muscles and the like, freezing in liquid nitrogen, respectively carrying out primary crushing, low-temperature fine crushing and superfine crushing by using a superfine crusher, and sieving to obtain 150-micron bone particles;
and B: defatting and decalcifying, defatting bone grains with mixed defatting liquid of chloroform and methanol in the ratio of 1 to 1 in the ratio of 30ml to bone powder for 4 hr, and decalcifying with 0.6M hydrochloric acid in the ratio of 30ml to bone powder for 24 hr;
and C: washing and freeze-drying, washing with deionized water 10 times the volume of the bone meal for 4 times until the pH value is equal to 7.4, pre-freezing in a refrigerator at-40 deg.C for 4h in air-dry water, freeze-drying in a freeze-drying machine, and sealing for storage;
step D: digesting and centrifuging, weighing a certain amount of freeze-dried bone powder, adding 100ml of 0.01M hydrochloric acid and 100mg of pepsin into 1g of bone powder, digesting the bone powder at the temperature of 4 ℃, and continuously stirring for 96 hours by using a magnetic stirrer;
step E: neutralizing and compounding, adding 1/10 volume, 0.1M sodium hydroxide solution and 1/9 volume 10XPBS (pH7.4) into the digestion solution at 4 ℃, fully and uniformly stirring, then adding 10% PVA200 microliter/ml, 0.25 mg/ml BMP and 0.2 mg/ml calcium phosphate powder, and fully and uniformly stirring;
step F: performing gelation and shaping, namely subpackaging the gel solution into a special mould for shaping, and transferring the special mould and the special mould into a 37 ℃ incubator until a gel compound is formed;
step G: crosslinking, namely freeze-drying the gel-like compound, and placing the lyophilized BMP gel compound in a 70 ℃ incubator for 2 hours;
step H: and (3) sterilizing and packaging, namely sterilizing the tool loaded with the BMP gel compound, and packaging the BMP gel compound.
In the embodiment of the invention, in the step E, 200 μ l/ml of 10% PVA, 0.25 mg/ml of BMP and 0.2 mg/ml of calcium phosphate powder are added while stirring, and the stirring time is prolonged, so that the mixing efficiency of the calcium phosphate powder is improved, and the stirring time of BMP is prolonged, thereby allowing full expression of BMP, further reducing the amount of BMP, and reducing the cost.
EXAMPLE III
Referring to fig. 1, a method for preparing a composite bone morphogenetic protein bionic bone repair material includes the following steps:
step A: sampling, namely taking fresh calf limb cortical bone, removing soft tissues such as muscles and the like, freezing in liquid nitrogen, respectively carrying out primary crushing, low-temperature fine crushing and superfine crushing by using a superfine crusher, and sieving to obtain bone particles with the size of 200 um;
and B: defatting and decalcifying, defatting bone grains with mixed defatting liquid of chloroform and methanol in the ratio of 1 to 1 in the ratio of 30ml to bone powder for 4 hr, and decalcifying with 0.6M hydrochloric acid in the ratio of 30ml to bone powder for 24 hr;
and C: washing and freeze-drying, washing with deionized water 10 times the weight of bone powder for 5 times until the pH value is 7.4, pre-freezing in-40 deg.C refrigerator for 4 hr, freeze-drying in freeze-drying machine, and sealing for storage;
step D: digesting and centrifuging, weighing a certain amount of freeze-dried bone powder, adding 100ml of 0.01M hydrochloric acid and 100mg of pepsin into 1g of bone powder, digesting the bone powder at the temperature of 4 ℃, and continuously stirring for 96 hours by using a magnetic stirrer;
step E: neutralizing and compounding, adding 1/10 volume, 0.1M sodium hydroxide solution and 1/9 volume 10XPBS (pH7.4) into the digestion solution at 4 ℃, fully and uniformly stirring, then adding 10% PVA200 microliter/ml, 0.25 mg/ml BMP and 0.2 mg/ml calcium phosphate powder, and fully and uniformly stirring;
step F: performing gelation and shaping, namely subpackaging the gel solution into a special mould for shaping, and transferring the special mould and the special mould into a 37 ℃ incubator until a gel compound is formed;
step G: crosslinking, namely freeze-drying the gel-like compound, and placing the lyophilized BMP gel compound in a 70 ℃ incubator for 2 hours;
step H: and (3) sterilizing and packaging, namely sterilizing the tool loaded with the BMP gel compound, and packaging the BMP gel compound.
In the embodiment of the invention, in the step H, the sterilization and packaging operation is performed in an aseptic environment, the final gel compound is integrally and independently packaged, the use of the finally prepared product is convenient, the external packaging amount is reduced compared with the traditional infuse product, the packaging cost is reduced, the aseptic packaging of the product is ensured, the whole product process is simpler, the use is convenient and fast, the sustained release performance is outstanding, the sustained action time of the growth factor BMP is prolonged, the use amount is reduced, the product cost is lower, and meanwhile, the safety risk caused by excessive use of the BMP is eliminated.
The working principle is as follows: BMP and a gel-like biological bone-derived material are directly compounded in the gelation process to prepare a product, so that the BMP is uniformly distributed inside and outside the structure of the gel-like material, the BMP compounded on the surface of the material can play a role after being implanted into a human body, and the BMP inside the material structure is slowly released along with the degradation of the material to continuously play a role in inducing bones of the material, thereby reducing the using amount of the BMP and lowering the cost
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (5)
1. A preparation method of a composite bone morphogenetic protein bionic bone repair material is characterized by comprising the following steps: the method comprises the following steps:
step A: sampling, namely taking fresh calf limb cortical bone, removing soft tissues such as muscles and the like, freezing in liquid nitrogen, respectively carrying out primary crushing, low-temperature fine crushing and superfine crushing by using a superfine crusher, and sieving to obtain bone particles with the size of 100-;
and B: defatting and decalcifying, defatting bone grains with mixed defatting liquid of chloroform and methanol in the ratio of 1 to 1 in the ratio of 30ml to bone powder for 4 hr, and decalcifying with 0.6M hydrochloric acid in the ratio of 30ml to bone powder for 24 hr;
and C: washing and freeze-drying, washing with deionized water 10 times the weight of bone powder for 3-5 times until the pH value is equal to 7.4, pre-freezing in-40 deg.C refrigerator for 4 hr, freeze-drying in freeze dryer, and sealing for storage;
step D: digesting and centrifuging, weighing a certain amount of freeze-dried bone powder, adding 100ml of 0.01M hydrochloric acid and 100mg of pepsin into 1g of bone powder, digesting the bone powder at the temperature of 4 ℃, and continuously stirring for 96 hours by using a magnetic stirrer;
step E: neutralizing and compounding, adding 1/10 volume, 0.1M sodium hydroxide solution and 1/9 volume of 10XPBS into the digestion solution at 4 ℃, fully and uniformly stirring, adding 10% PVA200 microliter/milliliter, 0.25 milligram/milliliter BMP and 0.2 milligram/milliliter calcium phosphate powder, and fully and uniformly stirring;
step F: performing gelation and shaping, namely subpackaging the gel solution into a special mould for shaping, and transferring the special mould and the special mould into a 37 ℃ incubator until a gel compound is formed;
step G: crosslinking, namely freeze-drying the gel-like compound, and placing the lyophilized BMP gel compound in a 70 ℃ incubator for 2 hours;
step H: and (3) sterilizing and packaging, namely sterilizing the tool loaded with the BMP gel compound, and packaging the BMP gel compound.
2. The method for preparing a composite bone morphogenetic protein bionic bone repair material according to claim 1, which is characterized in that: in the step A, the cortex bones of the fresh calf limbs are removed, the soft tissues such as muscles and the like are removed, the fresh calf limbs are frozen in liquid nitrogen, and the fresh calf limbs are sieved for multiple times after being crushed, and the freezing is carried out at room temperature when a sample is taken out.
3. The method for preparing a composite bone morphogenetic protein bionic bone repair material according to claim 1, which is characterized in that: in step D, the digestion solution is centrifuged at 1000rpm4 ℃ for 5 minutes to remove residues.
4. The method for preparing a composite bone morphogenetic protein bionic bone repair material according to claim 1, which is characterized in that: in the step E, stirring is performed while adding 200. mu.l/ml of 10% PVA, 0.25 mg/ml of BMP and 0.2 mg/ml of calcium phosphate powder, and the stirring time is prolonged.
5. The method for preparing a composite bone morphogenetic protein bionic bone repair material according to claim 1, which is characterized in that: in the step H, the sterilization and packaging operation is carried out in an aseptic environment, and the final gel compound is packaged integrally and independently.
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