CN113546216B

CN113546216B - Collagen membrane micro-bone powder composite material and preparation method thereof

Info

Publication number: CN113546216B
Application number: CN202110797340.1A
Authority: CN
Inventors: 范代娣; 康华平; 段志广; 史静静; 殷诗玉; 严建亚
Original assignee: Shaanxi Giant Biogene Technology Co ltd
Current assignee: Shaanxi Giant Biogene Technology Co ltd
Priority date: 2021-07-14
Filing date: 2021-07-14
Publication date: 2023-04-25
Anticipated expiration: 2041-07-14
Also published as: CN113546216A

Abstract

The invention discloses a collagen membrane micro-bone-coated powder composite material and a preparation method thereof, wherein the composite material is prepared by a method comprising the following steps: dissolving collagen, a stabilizer and an osteogenic drug, and then adding a cross-linking agent to obtain a solution A; adding bone powder into the solution A to obtain a mixture; and (3) centrifuging the mixture to obtain a precipitate, and drying the precipitate. According to the composite material disclosed by the invention, the osteogenic medicine is effectively fixed in the collagen molecular structure, and collagen is wrapped on the outer surface of bone powder in a collagen membrane mode, so that the risk of medicine falling is reduced. Meanwhile, the collagen membrane generates larger viscosity after encountering liquid, so that the bone powder is fixed at the bone defect part, and the loss risk is reduced.

Description

Collagen membrane micro-bone powder composite material and preparation method thereof

Technical Field

The invention belongs to the technical field of medical appliances, and particularly relates to a collagen membrane micro-bone powder composite material and a preparation method thereof.

Background

The technology of dental implant restoration is one of important innovations in the development of dental medicine in the last century, and greatly improves the life quality and the mental state of patients with dental defects due to good retention and vivid beautiful effects. The long-term success rate of dental implants depends on a number of factors including the choice of implant site, the state of the morphology of the hard and soft tissues, whether the bone surrounding the implant is intact, the amount of bone meal to be planted, etc. However, it has been found clinically that many patients have bone destruction, bone damage, loss of functional stimulation, and the like of bone tissue during tooth extraction due to destruction of inflammatory substances such as alveolar bone disuse, bone growth, and the like caused by long-term tooth loss. In addition, the bone tissue regeneration capability of patients suffering from systemic diseases such as severe chronic periodontitis, osteoporosis, diabetes and the like is also a difficult problem in oral implantation repair. For this reason, many stomatologists have studied many methods, and although some progress has been made, many disadvantages still remain.

Currently, techniques commonly used clinically are guided bone regeneration, osteoinduction and bone conduction, and the common techniques for repairing bone injuries are: bone grafting techniques, guided bone tissue regeneration techniques, bone compression techniques, bone cleavage techniques, distraction osteogenesis techniques, and the like. Among them, bio-oss bone powder is one of the main means for clinically promoting alveolar bone regeneration, and commercialization has been achieved. Bone powder has a porous structure, but lacks active ingredients such as osteoblasts and bone growth factors, and thus lacks osteogenesis and osteoinduction. Therefore, attempts have been made to combine osteogenic drugs with bone powder in an attempt to solve the drawbacks of using bone powder alone, and the commonly used combination drugs include parathyroid hormone, platelet fibrin, growth factors, etc., but because of technical limitations, the combination of these substances with bone powder still has certain drawbacks.

Disclosure of Invention

In order to solve the problems, the invention provides a collagen membrane micro-bone-coated powder composite material and a preparation method thereof. The collagen membrane micro-bone-coated powder composite material has stronger bone induction activity, allows bone forming cells to migrate, proliferate and differentiate, can be combined with non-collagenous mechanism proteins such as growth factors, effectively controls and forms mineralization reaction, and accelerates bone formation. And simultaneously, the risk of osteoporosis, peri-operative inflammation, bleeding in the operation and the like can be reduced.

The specific technical scheme of the invention is as follows:

1. a collagen membrane micro-bone powder composite material, wherein the composite material is prepared by a method comprising the following steps:

dissolving collagen, a stabilizer and an osteogenic drug, and then adding a cross-linking agent to obtain a solution A;

adding bone powder into the solution A to obtain a mixture;

and (3) centrifuging the mixture to obtain a precipitate, and drying the precipitate.

2. The composite material according to item 1, wherein the collagen is animal derived collagen or recombinant collagen, preferably recombinant collagen.

3. The composite material according to item 1 or 2, wherein the stabilizer is glycerol, epoxidized soybean oil, polysorbate or a glycerol derivative, preferably glycerol.

4. A composite material according to any one of claims 1-3, wherein the osteogenic drug is an estrogen, fluoride, diphosphate, statin or parathyroid hormone, preferably a parathyroid hormone.

5. The composite material according to any one of claims 1-4, wherein the cross-linking agent is maleimide, N-hydroxysuccinimide or a transglutaminase, preferably a transglutaminase.

6. The composite material according to any one of claims 1-5, wherein the bone meal is Bio-oss bone meal, preferably the bone meal has a diameter of 0.1-2mm.

7. The composite material according to any one of claims 1 to 6, wherein the collagen is 1 to 10% (w/v), preferably 2 to 5% (w/v), based on the total volume of the solution a; the stabilizer is 1-5% (w/v), preferably 1-2% (w/v); the osteogenic drug is 0.01-0.1% (w/v), preferably 0.02-0.05% (w/v).

8. The composite material according to any one of claims 1 to 7, wherein the concentration of the crosslinking agent is 0.001 to 0.1% (w/v), preferably 0.005 to 0.01% (w/v), based on the total volume of the solution a.

9. The composite material according to any one of claims 1-8, wherein the bone meal is added in an amount of 0.5-5g/10ml, preferably 1-2.5g/10ml, based on solution a.

10. A method for preparing collagen membrane micro-bone powder composite material, which comprises the following steps:

adding bone powder into the solution A to obtain a mixture;

11. The method according to item 10, wherein the collagen is animal derived collagen or recombinant collagen, preferably recombinant collagen.

12. The method according to item 10 or 11, wherein the stabilizer is glycerol, epoxidized soybean oil, polysorbate or a glycerol derivative, preferably glycerol.

13. The method according to any one of claims 10-12, wherein the osteogenic drug is an estrogen, fluoride, diphosphate, statin or parathyroid hormone, preferably a parathyroid hormone.

14. The method according to any one of claims 10-13, wherein the cross-linking agent is maleimide, N-hydroxysuccinimide or a transglutaminase, preferably a transglutaminase.

15. The method according to any one of claims 10 to 14, wherein the bone meal is Bio-oss bone meal, preferably the bone meal has a diameter of 0.1-2mm.

16. The method according to any one of claims 10-15, wherein the collagen is 1-10% (w/v), preferably 2-5% (w/v), based on the total volume of the solution a; the stabilizer is 1-5% (w/v), preferably 1-2% (w/v); the osteogenic drug is 0.01-0.1% (w/v), preferably 0.02-0.05% (w/v).

17. The process according to any one of claims 10 to 16, wherein the concentration of the crosslinking agent is 0.001 to 0.1% (w/v), preferably 0.005 to 0.01% (w/v), based on the total volume of the solution a.

18. The method according to any one of claims 10 to 17, wherein the bone meal is added in an amount of 0.5-5g/10ml, preferably 1-2.5g/10ml, based on solution a.

ADVANTAGEOUS EFFECTS OF INVENTION

The collagen membrane micro-bone-coated powder composite material provided by the invention effectively fixes the bone-forming medicine in the collagen molecular structure, and wraps the collagen on the outer surface of the bone powder in the form of a collagen membrane, so that the risk of medicine falling is reduced.

Meanwhile, the collagen membrane generates larger viscosity after encountering liquid, so that the bone powder is fixed at the bone defect part, and the loss risk is reduced. The collagen membrane can also be used as a physical barrier to form a space which is favorable for the growth of osteoblasts and promote the regeneration and repair of bone tissues in a defect area, thereby increasing the bone mass.

The adopted collagen membrane is preferably a recombinant collagen membrane, is considered to be a good bone tissue regeneration guiding material, can be completely degraded in vivo, and has good tissue compatibility and biological safety. The synergistic effect of the raw collagen film, the osteogenic medicine and the bio-oss bone powder can effectively promote the repairing process of bone defects and increase the bone regeneration effect of the artificial bone powder.

Detailed Description

The present invention will be described in detail below. While specific embodiments of the invention are shown, it should be understood that the invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

It should be noted that certain terms are used throughout the description and claims to refer to particular components. Those of skill in the art will understand that a person may refer to the same component by different names. The specification and claims do not identify differences in terms of components, but rather differences in terms of the functionality of the components. As referred to throughout the specification and claims, the terms "include" or "comprising" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description hereinafter sets forth a preferred embodiment for practicing the invention, but is not intended to limit the scope of the invention, as the description proceeds with reference to the general principles of the description. The scope of the invention is defined by the appended claims.

The invention provides a collagen membrane micro-bone-coated powder composite material, which is prepared by a method comprising the following steps:

adding bone powder into the solution A to obtain a mixture;

The collagen has strong bone induction activity and can induce bone tissues to form proteins. The triple helix structure of collagen has the function of inducing mineral deposition, the surface of the triple helix structure is rich in a large number of mineral deposition sites, mineralization reaction can be effectively controlled and formed in the process of combining non-collagenous mechanism proteins, especially combining growth factors, bone formation can be accelerated, and the triple helix structure can be implanted into a human body to replace bones.

The micro-wrapping refers to a technology of embedding medicinal powder particles or medicinal liquid microdrops into micro-capsules by using a high polymer material (commonly called capsule wall), and the product has the advantages of prolonged curative effect, improved stability and the like after micro-wrapping.

The osteogenic medicine refers to a medicine capable of promoting bone formation of osteoblasts and accelerating bone cell growth.

The bone powder is a novel bone repair material which is synthesized artificially and has a plurality of excellent physical and chemical properties (self-curing molding, high mechanical strength, convenient use and the like) and biological properties (no toxic or side effect, absorption and degradation, good biocompatibility, capability of inducing bone cells, vascular growth and the like).

In one embodiment, the collagen is animal derived collagen or recombinant collagen, preferably recombinant collagen.

The animal-derived collagen is obtained from tissues such as animal bones, skin, tendons, amniotic membrane and the like through extraction processing, is used as a main component of extracellular matrix, has outstanding biomedical functions, and can be widely used in clinical treatments such as tissue regeneration, hemostasis and the like. The source of the animal-derived collagen is not limited in the present invention, and may be selected as desired by those skilled in the art.

For recombinant collagen, mRNA of human collagen is reverse transcribed into cDNA, and a section of gene after enzyme digestion is recombined in E.coli (Escherichia coli), and the high-molecular biological protein is produced through high-density fermentation, separation, renaturation and purification processes. The source of the recombinant Collagen is not limited in the present invention, and for example, fan Dai of northern west China, which is taught by the university of China, is also called Van's recombinant Collagen or Van's Human-like Collagen (FHLC).

The recombinant collagen is recombinant collagen according to claim 1 of chinese patent application publication CN1371919a, which has a triple-chain, triple-helix structure, and can be prepared by using, for example, the genetic engineering expression method disclosed in the chinese patent application publication CN1371919 a.

In one embodiment, the stabilizer is glycerol, epoxidized soybean oil, polysorbate, or a glycerol derivative, preferably glycerol.

The Epoxidized Soybean Oil (ESO) is an organic compound of the formula (RC) ₂ H ₂ OR'COO) ₃ C ₃ H ₅ Is a pale yellow viscous oily liquid at normal temperature, is nontoxic, is soluble in most organic solvents and hydrocarbons, and is insoluble in water. Has excellent heat resistance, light resistance and compatibility, is commonly used as a plasticizer for polyvinyl chloride products, and is particularly suitable for polyvinyl chloride transparent products, food packaging products and other nontoxic products.

The polysorbate is a light yellow to orange yellow viscous liquid; slightly odorous, slightly bitter and slightly astringent, and warm. The material is mainly used as solubilizer or emulsifier of injection and oral liquid, dispersant for capsule, emulsifier and matrix for ointment, matrix for suppository, etc.

The glycerol derivative refers to a more complex product derived by substituting hydrogen atoms or atomic groups in glycerol with other atoms or atomic groups

In one embodiment, the osteogenic drug is an estrogen, fluoride, diphosphate, statin or parathyroid hormone, preferably parathyroid hormone.

The estrogen refers to substances which promote the development of secondary sex characteristics and the maturation of sexual organs of female animals and are secreted by ovaries and placenta of female animals.

The fluoride refers to an organic or inorganic compound containing fluorine.

The diphosphate refers to a new medicine for various bone diseases and calcium metabolic diseases. Can be specifically combined with hydroxyapatite in bone, and inhibit the activity of osteoclast, thereby inhibiting bone absorption.

The statins refer to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which can not only effectively reduce Total Cholesterol (TC) and Low Density Lipoprotein (LDL), but also reduce Triacylglycerol (TG) to a certain extent and raise High Density Lipoprotein (HDL).

The parathyroid hormone is an alkaline single-chain polypeptide hormone secreted by parathyroid gland main cells. Its main function is to regulate the metabolism of calcium and phosphorus in vertebrate body, promote the rise of blood calcium level and the decrease of blood phosphorus level.

In one embodiment, the cross-linking agent is maleimide, N-hydroxysuccinimide or a transglutaminase, preferably a transglutaminase.

In one embodiment, the bone meal is Bio-oss bone meal, preferably the bone meal has a diameter of 0.1-2mm.

The Bio-oss bone powder is produced by the regeneration medical treatment of the advent, and has the advantages of improving the survival rate of the implant and keeping the osteogenesis environment stable for a long time.

The bone meal may have a diameter of 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1mm, 2mm, etc.

In one embodiment, the collagen is 1-10% (w/v), preferably 2-5% (w/v), based on the total volume of the solution A; the stabilizer is 1-5% (w/v), preferably 1-2% (w/v); the osteogenic drug is 0.01-0.1% (w/v), preferably 0.02-0.05% (w/v).

For example, the collagen may be 1% (w/v), 2% (w/v), 3% (w/v), 4% (w/v), 5% (w/v), 6% (w/v), 7% (w/v), 8% (w/v), 9% (w/v), 10% (w/v), etc. based on the total volume of the solution A;

the stabilizer may be 1% (w/v), 2% (w/v), 3% (w/v), 4% (w/v), 5% (w/v), etc.;

the osteogenic drug may be 0.01% (w/v), 0.02% (w/v), 0.03% (w/v), 0.04% (w/v), 0.05% (w/v), 0.06% (w/v), 0.07% (w/v), 0.08% (w/v), 0.09% (w/v), 0.1% (w/v), etc.

In one embodiment, the concentration of the crosslinking agent is 0.001 to 0.1% (w/v), preferably 0.005 to 0.01% (w/v), based on the total volume of the solution A.

For example, the concentration of the crosslinking agent may be 0.001% (w/v), 0.002% (w/v), 0.003% (w/v), 0.004% (w/v), 0.005% (w/v), 0.006% (w/v), 0.007% (w/v), 0.008% (w/v), 0.009% (w/v), 0.01% (w/v), 0.02% (w/v), 0.03% (w/v), 0.04% (w/v), 0.05% (w/v), 0.06% (w/v), 0.07% (w/v), 0.08% (w/v), 0.09% (w/v), 0.1% (w/v), etc., based on the total volume of the solution A.

In one embodiment, the bone meal is added in an amount of 0.5-5g/10ml, preferably 1-2.5g/10ml, based on solution A.

For example, the bone powder may be added in an amount of 0.5g/10ml, 1g/10ml, 1.5g/10ml, 2g/10ml, 2.5g/10ml, 3g/10ml, 3.5g/10ml, 4g/10ml, 4.5g/10ml, 5g/10ml, etc., based on the solution A.

In one embodiment, after adding bone meal to the solution A, stirring is continued at 2-8deg.C for 12-48 hours, preferably at a rate of 100-500 revolutions per minute.

For example, the stirring and dispersing are continued at 2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃ and the like for 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours;

the stirring speed may be, for example, 100 revolutions per minute, 200 revolutions per minute, 300 revolutions per minute, 400 revolutions per minute, 500 revolutions per minute, etc.

In one embodiment, the mixture is centrifuged at 50-300rpm/min to obtain a precipitate, which is preferably allowed to stand at 2-8deg.C for 24-72 hours.

In one embodiment, the precipitate after standing is put into a freeze dryer to be dried for 24 to 100 hours to obtain the collagen membrane micro-bone powder composite material.

The collagen membrane micro-bone-coated powder composite material effectively fixes the osteogenic drugs in the collagen molecular structure, and wraps the collagen on the outer surface of the bone powder in the form of collagen membrane, thereby reducing the risk of drug shedding. Meanwhile, the collagen membrane generates larger viscosity after encountering liquid, so that the bone powder is fixed at the bone defect part, and the loss risk is reduced. In addition, the collagen membrane can also be used as a physical barrier to form a space which is favorable for the growth of osteoblasts and promote the regeneration and repair of bone tissues in a defect area, thereby increasing the bone mass.

The invention provides a collagen membrane micro-bone-coated powder composite material, which comprises the following steps:

adding bone powder into the solution A to obtain a mixture;

The concentration of the cross-linking agent refers to the mass volume percentage of the solution A.

The addition amount of the bone powder is calculated by the solution A, and 0.5-5g of bone powder is added into each 10ml of the solution A.

The collagen film micro-bone powder composite material prepared by the method has good uniformity, good wrapping effect, better guiding regeneration of bone tissues and good application prospect.

The guiding tissue regeneration technology is the most commonly used method in the current oral implantation, and along with the development and the gradual maturity of the technology, the membrane is used as a physical barrier to help the bone cells to grow preferentially, prevent heterogeneous cells such as non-bone connective tissue from invading, provide a favorable environment for the growth of the bone cells, increase the bone mass and promote the regeneration and repair of bone tissue in a defect area, and is the main direction of the development of the GBR technology. According to the invention, through strict control of key technological parameters, collagen is crosslinked into a layer of film on the surface of bio-oss bone powder by using a related technology, a physical barrier and a favorable environment are provided for growth of bone cells at a defect part, and an osteogenic medicament is firmly locked in a collagen film and is released to the bone defect part along with degradation of the collagen film after being implanted into a body, so that growth of the osteoblasts is induced. Experimental results show that although the effect of new bone formation can be achieved by applying the bio-oss bone powder, compared with the invention, the collagen membrane has obvious advantages in effective combination with the bio-oss bone powder because the bone formation effect and the growth speed are obviously different. The invention organically combines the guiding bone regeneration theory, the bone induction theory and the bone conduction theory to obtain the collagen membrane micro-bone-coated powder composite material with stable quality and obvious effect.

Examples

The materials used in the test and the test methods are described generally and/or specifically in the examples which follow,% represents wt%, i.e. weight percent, unless otherwise specified. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge, wherein Table 1 is a raw material source table for the examples.

Table 1 table of sources of raw materials used in examples

	Purity of	Manufacturing factories
			Recombinant collagen	95％	Siam giant biological Gene technologies Co
Parathyroid hormone	/	BEIJING J&K SCIENTIFIC Ltd.
			Glutamine transferase	/	SHANGHAI YUANYE BIOTECHNOLOGY Co.,Ltd.
bio-oss bone powder	/	Gai's pharmaceutical

Example 1

(1) Weighing 2g of recombinant collagen, 2g of glycerol and 0.05g of parathyroid hormone, dissolving in 100ml of water for injection, and then adding 0.005g of transglutaminase to form a solution A;

(2) Weighing 10g of bio-oss bone powder with the diameter of 1mm, adding the bio-oss bone powder into 100ml of A solution to obtain a mixed solution, placing the mixed solution at the temperature of 4 ℃ and continuously stirring and dispersing the mixed solution for 20 hours by using a magnetic stirrer, wherein the stirring speed is 300 revolutions per minute to obtain a uniformly dispersed mixture;

(3) Centrifuging the above mixture in a centrifuge at 100rpm/min for 5min to obtain precipitate, filtering the precipitate with 50 mesh standard sieve, dispersing, standing at 2-8deg.C for 24 hr, and drying in a freeze dryer (LG-02, shanghai Dongfulong technologies Co., ltd.) for 60 hr to obtain the final product.

Determining the thickness of the collagen membrane in the collagen membrane micro-bone-coated powder composite material by using a microscopic image method: 1g of the final product is taken, 6 particles are randomly selected for slicing, and 8 positions of each slice particle are taken along the circumference to obtain microscopic images. Then, image information is obtained by using Photoshop, the thickness of the collagen film is calculated, and the experimental results are shown in Table 2:

TABLE 2 results of collagen film thickness from microscopic images

As can be seen from Table 2, the collagen membrane of the collagen membrane micro-bone powder composite material prepared in the first embodiment has a thickness distribution of 20.3-21.3 μm, and has good uniformity and good wrapping effect.

Example 2

(1) 1g of recombinant collagen, 1g of polysorbate and 0.01g of estrogen are weighed and dissolved in 100ml of water for injection, and then 0.001g of maleimide is added to form solution A.

(2) 6g of bio-oss bone powder with the diameter of 0.5mm is weighed and added into the 100ml of A solution, and the mixture is placed under the condition of 4 ℃ and continuously stirred and dispersed for 28 hours by a magnetic stirrer, and the stirring speed is 350 revolutions per minute, so as to obtain a uniformly dispersed mixture.

(3) Centrifuging the mixture in a centrifuge at 100rpm/min for 10min to obtain precipitate, filtering the precipitate with 24 mesh sieve, dispersing, standing at 2-8deg.C for 24 hr, and drying in a freeze dryer for 48 hr to obtain the final product.

The thickness of the collagen film was measured by the method described in example 1, and 0.5g of the above product was measured, and the results are shown in Table 3.

TABLE 3 results of collagen film thickness from microscopic images

From the above table, the collagen film of the collagen film micro-bone powder coating composite material has the thickness distribution of 14.3-16.5 μm, good uniformity and good coating effect.

Example 3

(1) Weighing 1g of recombinant collagen, 1g of polysorbate and 0.01g of estrogen, dissolving in 100ml of water for injection, and adding 0.001g of transglutaminase to form a solution A;

(2) Weighing 5g of bio-oss bone powder with the diameter of 0.5mm, adding the bio-oss bone powder into the 100ml of A solution, placing the mixed solution at the temperature of 4 ℃ and continuously stirring and dispersing for 24 hours by using a magnetic stirrer, wherein the stirring speed is 350 revolutions per minute, so as to obtain a uniformly dispersed mixture;

(3) Centrifuging the mixture in a centrifuge at 100rpm/min for 5min to obtain precipitate, filtering the precipitate with 24 mesh sieve, dispersing, standing at 2-8deg.C for 24 hr, and drying in a freeze dryer for 48 hr to obtain the final product.

The thickness of the collagen film was measured by the method described in example 1, and 0.5g of the above product was measured, and the results are shown in Table 4.

TABLE 4 results of collagen film thickness from microscopic images

From the above table, the collagen film of the collagen film micro-bone powder coating composite material has the thickness distribution of 14.0-16.8 μm, good uniformity and good coating effect.

Example 4

(1) Weighing 10g of recombinant collagen, 5g of polysorbate and 0.1g of estrogen, dissolving in 100ml of water for injection, and adding 0.1g of transglutaminase to form solution A;

(2) Weighing 10g of bio-oss bone powder with the diameter of 0.5mm, adding the bio-oss bone powder into the 100ml of A solution, placing the mixed solution at the temperature of 4 ℃ and continuously stirring and dispersing the mixed solution for 40 hours by using a magnetic stirrer, wherein the stirring speed is 350 revolutions per minute, so as to obtain a uniformly dispersed mixture;

(3) Centrifuging the mixture in a centrifuge at 100rpm/min for 15min to obtain precipitate, filtering the precipitate with 24 mesh sieve, dispersing, standing at 2-8deg.C for 50 hr, and drying in a freeze dryer for 48 hr to obtain the final product.

The thickness of the collagen film was measured by the method described in example 1, and 0.5g of the above product was measured, and the results are shown in Table 5.

TABLE 5 results of collagen film thickness from microscopic images

From the above table, the collagen film of the collagen film micro-bone powder coating composite material has the thickness distribution of 14.7-17.3 μm, good uniformity and good coating effect.

Example 5

(2) Weighing 6g of bio-oss bone powder with diameter of 0.5mm, adding into the 100ml of A solution, placing the mixture at 4deg.C, and continuously stirring with magnetic stirrer for 40 hr at stirring speed of 400 rpm to obtain uniformly dispersed mixture

(3) Centrifuging the mixture in a centrifuge at 100rpm/min for 10min to obtain precipitate, filtering the precipitate with a 10 mesh sieve, dispersing the precipitate, standing at 2-8deg.C for 24 hr, and drying in a freeze dryer for 30 hr to obtain the final product.

2g of the above product was measured as described in example 1, and the thickness of the collagen film was calculated, and the results are shown in Table 6.

TABLE 6 results of collagen film thickness from microscopic images

From the above table, the collagen film of the collagen film micro-bone powder coating composite material has the thickness distribution of 15.2-16.8 μm, good uniformity and good coating effect.

Example 6

(2) Weighing 10g of bio-oss bone powder with diameter of 0.1mm, adding into the 100ml of A solution, placing the mixture at 4deg.C, and continuously stirring with magnetic stirrer for 30 hr at stirring speed of 350 rpm to obtain uniformly dispersed mixture

(3) Centrifuging the mixture in a centrifuge at 100rpm/min for 15min to obtain precipitate, filtering the precipitate with 120 mesh sieve, dispersing, standing at 2-8deg.C for 40 hr, and drying in a freeze dryer for 35 hr to obtain the final product.

The thickness of the collagen film was measured by the method described in example 1, and the result was shown in Table 7.

TABLE 7 results of collagen film thickness from microscopic images

From the above table, the collagen film of the collagen film micro-bone powder coating composite material has the thickness distribution of 13.5-17.0 μm, good uniformity and good coating effect.

Example 7

(1) Weighing 5g of recombinant collagen, 1g of glycerol and 0.02g of parathyroid hormone, dissolving in 100ml of water for injection, and then adding 0.01g of transglutaminase to form a solution A;

(2) Weighing 25g of bio-oss bone powder with the diameter of 1mm, adding the bio-oss bone powder into 100ml of A solution to obtain a mixed solution, placing the mixed solution at the temperature of 4 ℃ and continuously stirring and dispersing the mixed solution for 20 hours by using a magnetic stirrer, wherein the stirring speed is 300 revolutions per minute to obtain a uniformly dispersed mixture;

(3) Centrifuging the mixture in a centrifuge at 100rpm/min for 5min to obtain precipitate, filtering the precipitate with 50 mesh sieve, dispersing, standing at 2-8deg.C for 24 hr, and drying in a freeze dryer for 60 hr to obtain the final product.

The thickness of the collagen film was measured by the method described in example 1, and the result was shown in Table 8.

TABLE 8 results of collagen film thickness from microscopic images

Example 8

(1) Weighing 10g of recombinant collagen, 5g of glycerol and 0.1g of parathyroid hormone, dissolving in 100ml of water for injection, and then adding 0.1g of transglutaminase to form a solution A;

(2) Weighing 50g of bio-oss bone powder with the diameter of 0.5mm, adding the bio-oss bone powder into 100ml of A solution to obtain a mixed solution, placing the mixed solution at the temperature of 4 ℃ and continuously stirring and dispersing the mixed solution for 20 hours by using a magnetic stirrer, wherein the stirring speed is 300 revolutions per minute to obtain a uniformly dispersed mixture;

The thickness of the collagen film was measured by the method described in example 1, and the result was shown in Table 9.

TABLE 9 results of collagen film thickness from microscopic images

Example 9

(1) Weighing 5g of recombinant collagen, 2g of glycerol and 0.05g of parathyroid hormone, dissolving in 100ml of water for injection, and adding 0.005g of transglutaminase to form a solution A;

1g of the above product was measured as described in example 1, and the thickness of the collagen film was calculated, and the results are shown in Table 10.

Table 10 results of microscopic image measurement of collagen film thickness

Example 10

(1) Weighing 1g of recombinant collagen, 2g of glycerol and 0.05g of parathyroid hormone, dissolving in 100ml of water for injection, and then adding 0.005g of transglutaminase to form a solution A;

1g of the above product was measured as described in example 1, and the thickness of the collagen film was calculated, and the results are shown in Table 11.

TABLE 11 results of collagen film thickness from microscopic images

Example 11

(1) Weighing 10g of recombinant collagen, 2g of glycerol and 0.05g of parathyroid hormone, dissolving in 100ml of water for injection, and adding 0.005g of transglutaminase to form a solution A;

1g of the above product was measured as described in example 1, and the thickness of the collagen film was calculated, and the results are shown in Table 12.

Table 12 results of microscopic image measurement of collagen film thickness

The raw material consumption amounts of the examples are shown in Table 13:

table 13 raw material consumption table of examples

Comparative example 1

(2) Weighing 50g of sub-nano hydroxyapatite with the diameter of 1mm, adding the sub-nano hydroxyapatite into 100ml of solution A to obtain a mixed solution, placing the mixed solution at the temperature of 4 ℃ and continuously stirring and dispersing the mixed solution for 20 hours by using a magnetic stirrer, wherein the stirring speed is 300 revolutions per minute to obtain a uniformly dispersed mixture;

(3) Centrifuging the above mixture in a centrifuge at 100rpm/min for 5min to obtain precipitate, filtering the precipitate with 50 mesh sieve, dispersing, standing at 2-8deg.C for 24 hr, and drying in a freeze dryer for 60 hr to obtain the final product, wherein the preparation method of sub-nanometer hydroxyapatite is carried out by the method disclosed in CN109908405A example 1

The thickness of the collagen film was measured by the method described in example 1, and the result was shown in Table 14.

TABLE 14 results of collagen film thickness from microscopic images

Test examples

The collagen membrane micro-encapsulated bone powder composite materials obtained in examples 1 to 11 and comparative example 1 were used as experimental groups 1 to 12, respectively, and the non-mixed bio-oss bone powder was used as a control group for animal experiments without any treatment.

15 healthy adult male hybrid dogs are taken, 1-2 years old and weight is 16-20kg. The materials after operation are divided into two groups according to the degradation characteristics of the materials, wherein 3 groups are respectively three month groups and six month groups. Animals are fasted for 12 hours before anesthesia, water is forbidden for 4 hours, muscles at gum are fixed on cheek sides of mandibles at two sides are respectively cut through the mouth, the length of the cut is about 5cm, and in order to avoid influence of periosteum on bone regeneration, periosteum of an experimental area is peeled off and removed, and bone surfaces are fully exposed. The experimental area of about 1cm x 1cm was randomly formed on both the exposed buccal bone surfaces of the left and right mandibular bones using a low-speed handpiece and a split drill, where small holes of about 2mm in diameter were punched to a depth of cancellous bone to obtain good blood supply. And placing a proper amount of bone powder on the bone surface of the experimental area of the experimental group and the control group respectively. Finally, the mandibular gingival mucosa and muscle cut at the two sides of the dog are tightly sutured without tension, 80 ten thousand units of penicillin is injected immediately after the operation, and routine nursing is carried out. The experimental results are shown in tables 15 and 16, wherein the area percentage of new bone growth of the experimental group and the control group at three or six months after operation is calculated by the following method: randomly observing 4 visual fields/sheet after the bone surface subjected to conventional nursing is dyed by HE, carrying out bone histomorphometric measurement by adopting image morphological analysis software, respectively measuring the areas of the newly-formed bones and the non-newly-formed bones, and calculating the average newly-formed bone area percentage according to the following formula:

new bone area percentage (%) =new bone area/(new bone area+area of non-new bone area) ×100%.

TABLE 15 general observations of post-operative animals

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TABLE 16 comparison of the area percentage of new bone growth between experimental and control groups at three or six months after surgery

Remarks: * Indicating a significant difference in the area percentage of new bone as compared to the control group

As can be seen from table 16, at the third month of the experiment, the percentage of new areas of bones in examples 1 to 8 compared with the comparative example was greater than that in the comparative example, but there was no statistical difference, and the percentage of new areas of bones compared with the comparative group had a statistical difference (P < 0.05); at the sixth month of the experiment, the area percentage of the new bone of the examples 1-8 and the control group also have statistical difference (P < 0.05) and have no statistical difference with the comparative example, which shows that the collagen membrane micro-coated bone powder composite material prepared by the examples 1-8 has the same capability of guiding the regeneration of bone tissue in the oral bone implantation operation as the bio-oss bone powder most commonly used in the market at present, and has good application prospect.

In conclusion, the collagen membrane micro-bone-coated powder composite material can effectively fix the osteogenic drugs in the collagen molecular structure, and wrap collagen on the outer surface of the bone powder in the form of a collagen membrane, so that the risk of drug shedding is reduced, and the composite material has good application prospect compared with the most commonly used bio-oss bone powder in the market at present in the capability of guiding bone tissue regeneration in the oral bone implantation operation.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

adding bone powder into the solution A, and continuously stirring and dispersing for 12-48 hours at the temperature of 2-8 ℃ at the stirring speed of 100-500 revolutions per minute to obtain a mixture;

centrifuging the mixture at 50-300rpm to obtain precipitate, standing at 2-8deg.C for 24-72 hr, and drying to obtain precipitate;

the stabilizer is glycerol, epoxidized soybean oil, polysorbate or a glycerol derivative;

the bone powder is Bio-oss bone powder, and the diameter of the bone powder is 0.1-2mm;

taking the solution A as the total volume, the collagen is 1-10% (w/v); the stabilizer is 1-5% (w/v); the osteogenic medicine is 0.01-0.1% (w/v);

the concentration of the cross-linking agent is 0.001-0.1% (w/v);

the addition amount of the bone powder is 0.5-5g/10mL calculated by the solution A.

2. The composite of claim 1, wherein the collagen is animal-derived collagen or recombinant collagen.

3. The composite of claim 1, wherein the collagen is recombinant collagen.

4. The composite of claim 1, wherein the stabilizer is glycerol.

5. The composite of claim 1, wherein the osteogenic drug is an estrogen, fluoride, diphosphate, statin, or parathyroid hormone.

6. The composite of claim 1, wherein the osteogenic drug is parathyroid hormone.

7. The composite of claim 1, wherein the cross-linking agent is maleimide, N-hydroxysuccinimide, or transglutaminase.

8. The composite of claim 1, wherein the cross-linking agent is a transglutaminase.

9. The composite of any one of claims 1-8, wherein the collagen is 2-5% (w/v) based on the total volume of the a solution; the stabilizer is 1-2% (w/v); the osteogenic medicine is 0.02-0.05% (w/v).

10. The composite material according to any one of claims 1-8, wherein the concentration of the cross-linking agent is 0.005-0.01% (w/v) based on the total volume of the a solution.

11. The composite material of any one of claims 1-8, wherein the bone meal is added in an amount of 1-2.5g/10mL, based on solution a.

12. A method for preparing collagen membrane micro-bone powder composite material, which comprises the following steps:

the concentration of the cross-linking agent is 0.001-0.1% (w/v);

13. The method of claim 12, wherein the collagen is animal-derived collagen or recombinant collagen.

14. The method of claim 12, wherein the collagen is recombinant collagen.

15. The method of claim 12, wherein the stabilizer is glycerol.

16. The method of claim 12, wherein the osteogenic drug is an estrogen, fluoride, diphosphate, statin, or parathyroid hormone.

17. The method of claim 12, wherein the osteogenic drug is parathyroid hormone.

18. The method of claim 12, wherein the cross-linking agent is maleimide, N-hydroxysuccinimide, or transglutaminase.

19. The method of claim 12, wherein the cross-linking agent is a transglutaminase.

20. The method of any one of claims 12-19, wherein the collagen is 2-5% (w/v) based on the total volume of solution a; the stabilizer is 1-2% (w/v); the osteogenic medicine is 0.02-0.05% (w/v).

21. The method of any one of claims 12-19, wherein the concentration of the cross-linking agent is 0.005-0.01% (w/v) based on the total volume of the solution a.

22. The method of any one of claims 12-19, wherein the bone meal is added in an amount of 1-2.5g/10mL, based on solution a.