阅读说明：本技术 一种mbmp2蛋白复合水凝胶及其制备方法和应用 (MBMP2 protein composite hydrogel and preparation method and application thereof ) 是由 彭松林 陈欣 谭宝玉 唐榕泽 杨大志 于 2021-08-26 设计创作，主要内容包括：本发明提供了一种MBMP2蛋白复合水凝胶及其制备方法和应用,所述MBMP2蛋白复合水凝胶的制备原料包括明胶、琼脂糖-MBMP2复合微球和TG酶,所述制备方法包括以下步骤：(1)将明胶溶于水中,并调节pH,得到明胶溶液；(2)将步骤(1)得到的明胶溶液与琼脂糖-MBMP2复合微球混合,之后与TG酶溶液混合,孵育得到所述MBMP2蛋白复合水凝胶。本发明提供的MBMP2蛋白复合水凝胶稳定性好,制备工艺简单,生物安全性高,成本低,易于临床操作,可实现MBMP2长期缓释的效果。(The invention provides an MBMP2 protein composite hydrogel and a preparation method and application thereof, wherein the preparation raw materials of the MBMP2 protein composite hydrogel comprise gelatin, agarose-MBMP 2 composite microspheres and TG enzyme, and the preparation method comprises the following steps: (1) dissolving gelatin in water, and adjusting pH to obtain gelatin solution; (2) and (2) mixing the gelatin solution obtained in the step (1) with agarose-MBMP 2 composite microspheres, then mixing with a TG enzyme solution, and incubating to obtain the MBMP2 protein composite hydrogel. The MBMP2 protein composite hydrogel provided by the invention has the advantages of good stability, simple preparation process, high biological safety, low cost and easy clinical operation, and can realize the effect of long-term slow release of MBMP 2.)

1. The MBMP2 protein composite hydrogel is characterized in that raw materials for preparing the MBMP2 protein composite hydrogel comprise gelatin, agarose-MBMP 2 composite microspheres and TG enzyme.

2. The MBMP2 protein composite hydrogel according to claim 1, wherein the agarose-MBMP 2 composite microspheres are prepared by a preparation method comprising the following steps: mixing the agarose microsphere solution with the recombinant protein MBMP2 to obtain the agarose-MBMP 2 composite microsphere;

preferably, the recombinant protein MBMP2 is prepared by a preparation method comprising the following steps:

(1') amplifying the DNA fragment of the T4L-BMP2, and then subcloning the DNA fragment into a vector to obtain a recombinant plasmid;

(2') introducing the recombinant plasmid into a competent cell, and then culturing, breaking the strain, centrifuging, resuspending, hanging a column and purifying to obtain the recombinant protein MBMP 2;

preferably, the vector is a pET28a expression vector;

preferably, the competent cell is a competent cell of BL21(DE 3).

3. The MBMP2 protein composite hydrogel of claim 2, wherein the degree of crosslinking of the agarose microspheres is 3-5%;

preferably, the concentration of the agarose microsphere solution is 10-50 mg/mL;

preferably, the concentration of the recombinant protein MBMP2 in the agarose microsphere solution is 2-15 mg/mL;

preferably, the temperature for mixing the agarose microsphere solution and the recombinant protein MBMP2 is 4-8 ℃ and the time is 4-12 h.

4. A method for preparing the MBMP2 protein composite hydrogel according to any one of claims 1 to 3, comprising the steps of:

(1) dissolving gelatin in water, and adjusting pH to obtain gelatin solution;

(2) and (2) mixing the gelatin solution obtained in the step (1) with agarose-MBMP 2 composite microspheres, then mixing with a TG enzyme solution, and incubating to obtain the MBMP2 protein composite hydrogel.

5. The method for preparing an MBMP2 protein composite hydrogel according to claim 4, wherein the gelatin in step (1) comprises type A gelatin;

preferably, the mass fraction of the gelatin in the gelatin solution is 10-20%;

preferably, the gelatin solution has a pH of 7.5 to 8.5.

6. The method for preparing the MBMP2 protein composite hydrogel according to claim 4 or 5, wherein the gelatin solution obtained in step (1) is further subjected to the following steps (1'): mixing the gelatin solution obtained in the step (1) with tannic acid, and stirring to obtain a tannic acid-doped gelatin solution;

preferably, the mass fraction of tannic acid in the tannic acid-doped gelatin solution of step (1') is 0 to 4%, excluding 0;

preferably, the temperature of the stirring in the step (1') is 45-55 ℃;

preferably, the stirring time in step (1') is 4-10 h.

7. The method for preparing the MBMP2 protein composite hydrogel according to any one of claims 4 to 6, wherein the concentration of the agarose-MBMP 2 composite microspheres in the gelatin solution in the step (2) is 0.5 to 2 mg/mL;

preferably, the volume ratio of the gelatin solution to the TG enzyme solution in the step (2) is 5:1-8: 1;

preferably, the incubation temperature is 35-60 ℃ and the incubation time is 0.5-5 min.

8. The method for preparing an MBMP2 protein composite hydrogel according to any one of claims 4 to 7, wherein the concentration of TG enzyme in the TG enzyme solution of step (2) is 0.5-1 mg/mL.

9. The method for preparing an MBMP2 protein composite hydrogel according to any one of claims 4 to 8, wherein the method for preparing comprises the following steps:

(1) dissolving gelatin in water, and adjusting pH to 7.5-8.5 to obtain gelatin solution with mass fraction of 10-20%;

(2) mixing the gelatin solution obtained in the step (1) with tannic acid, and stirring for 4-10h at 45-55 ℃ to obtain a tannic acid-doped gelatin solution;

(3) and (3) mixing the tannin-doped gelatin solution obtained in the step (2) with agarose-MBMP 2 composite microspheres, then mixing with a TG enzyme solution, and incubating at 35-60 ℃ for 0.5-5min to obtain the MBMP2 protein composite hydrogel.

10. Use of the MBMP2 protein composite hydrogel according to any one of claims 1-3 for the preparation of a tissue engineering material.

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to an MBMP2 protein composite hydrogel and a preparation method and application thereof, in particular to an MBMP2 protein composite hydrogel with high stability and a preparation method and application thereof.

Background

The repair of bone defect and bone regeneration are still important problems in clinical medicine, and currently, for patients with large-section bone defect, the bone defect is repaired and healed mainly by means of autologous bone transplantation, allogeneic bone transplantation, tissue engineering material transplantation and the like. In recent years, tissue engineering materials are receiving attention, so that the problems of immunological rejection and insufficient donor sources can be avoided, but the defects of low biological activity and poor osteogenesis induction capability generally exist. Therefore, effective bone tissue engineering requires effective combination of scaffold materials and bioactive factors to achieve a minimally invasive treatment with high osteogenic activity.

The most used bioactive factor at present is recombinant bone morphogenetic protein (BMP-2), however, BMP-2 has the following problems in clinical application: 1) BMP-2 protein has short half-life and is easy to be enzymolyzed or diluted by body fluid quickly; 2) the carrier biological material is difficult to operate, needs surgical operation when being implanted, can not well fill the defect part, and influences the healing and regeneration of bones; 3) the carrier is expensive, the biocompatibility is limited, and the degradation is too fast, so that the BMP-2 is rapidly lost. Therefore, it is of great clinical significance to find bone repair biomaterials that are economical, convenient, and that efficiently load and release BMP-2.

CN109692344A discloses a bone repair material containing rH-BMP2, which comprises modified gelatin and rH-BMP 2. The material can be used for repairing bone injury. The invention provides a bone repair material containing rH-BMP2, which comprises the following components in percentage by mass: 21-5% of rH-BMP, 60-86% of modified gelatin, 5-20% of hydroxyapatite and 8-15% of calcium chloride. The bone repair material containing rH-BMP2 has improved mechanical property, and can be used as slow release carrier to realize great improvement in degradation/release time and reduce allergy. The effect which is obviously superior to that of chitosan and the prior similar material is obtained in both in-vitro release experiments and animal experiments. The cytotoxicity test also preliminarily proves the safety of the compound. However, the slow release of rH-BMP2 cannot be realized, and the treatment effect is fast to pass.

As the repair of bone defect and bone regeneration are still important problems in clinical medicine at present, the clinical application of BMP-2 also has the problems of high price and low stability at present. Therefore, how to provide a BMP-2 bone repair biomaterial with high stability becomes a problem to be solved urgently.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide an MBMP2 protein composite hydrogel and a preparation method and application thereof, and particularly provides an MBMP2 protein composite hydrogel with high stability and a preparation method and application thereof. The MBMP2 protein composite hydrogel provided by the invention has the advantages of good stability, simple preparation process, high biological safety, low cost and easy clinical operation, and can realize the effect of long-term slow release of MBMP 2.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides an MBMP2 protein composite hydrogel, and raw materials for preparing the MBMP2 protein composite hydrogel comprise gelatin, agarose-MBMP 2 composite microspheres and TG enzyme.

The MBMP2 protein composite hydrogel provided by the invention can realize gelation under physiological conditions, has short gelation time and is easy for clinical operation; the agarose microspheres and hydrogel dual-release carrier are utilized, so that the purpose of long-term slow release of MBMP2 can be realized while the protein activity of the carrier is maintained; the prepared MBMP2 protein composite hydrogel has a controllable three-dimensional net structure, compact and uniform holes, good adhesion performance and strong mechanical strength, can realize adhesion and efficient retention on defective tissues, is suitable for filling various irregular-shaped defects, can efficiently promote the performance of material performance, and realizes the repair of the defective tissues.

Preferably, the agarose-MBMP 2 composite microspheres are prepared by a preparation method comprising the following steps: and mixing the agarose microsphere solution with the recombinant protein MBMP2 to obtain the agarose-MBMP 2 composite microspheres.

Preferably, the recombinant protein MBMP2 is prepared by a preparation method comprising the following steps:

(1') amplifying the DNA fragment of the T4L-BMP2, and then subcloning the DNA fragment into a vector to obtain a recombinant plasmid;

(2') introducing the recombinant plasmid into competent cells, and then culturing, breaking the bacteria, centrifuging, resuspending, hanging a column and purifying to obtain the recombinant protein MBMP 2.

Preferably, the vector is a pET28a expression vector.

Preferably, the competent cell is a competent cell of BL21(DE 3).

Preferably, the subcloning sites are NdeI and BamHI sites.

The protein sequence of the T4L-BMP2 is as follows:

HHHHMNIFEMLRIDEGLRLKIYKDTEGYYTIGIGHLLTKSPSLNAAKSELDKAIGRNTNGVITKDEAEKLFNQDVDAAVRGILRNAKLKPVYDSLDAVRRAALINMVFOMGETGVAGFTNSLRMLQQKRWDEAAVNLAKSRWYNQTPNRAKRVITTFRTGTWDAYGGSGGSGGSGGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVOTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR。

the recombinant protein MBMP2 is designed and constructed through a genetic engineering technology, and T4 lysozyme protein with good solubility and stable property is added to the nitrogen tail end of BMP-2, so that on one hand, the solubility and stability of the BMP-2 protein are enhanced, and on the other hand, the recombinant protein MBMP2 can be covalently connected to a biological material through the T4 lysozyme at the nitrogen tail end to achieve the effect of slowly releasing growth factors for a long time.

Preferably, the crosslinking degree of the agarose microspheres is 3-5%;

preferably, the concentration of the agarose microsphere solution is 10-50 mg/mL;

preferably, the concentration of the recombinant protein MBMP2 in the agarose microsphere solution is 2-15 mg/mL;

preferably, the temperature for mixing the agarose microsphere solution and the recombinant protein MBMP2 is 4-8 ℃ and the time is 4-12 h.

Wherein the degree of crosslinking may be 3%, 3.2%, 3.4%, 3.6%, 3.8%, 4%, 4.2%, 4.4%, 4.6%, 4.8%, or 5%, etc., the concentration of the agarose microsphere solution may be 10mg/mL, 15mg/mL, 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, or 50mg/mL, etc., the concentration of the recombinant protein MBMP2 in the agarose microsphere solution may be 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, 10mg/mL, 11mg/mL, 12mg/mL, 13mg/mL, 14mg/mL, or 15mg/mL, etc., and the temperature at which the agarose microsphere solution and the recombinant protein MBMP2 are mixed may be 4 ℃. (see, The time may be 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours, etc., at 5 ℃, 6 ℃, 7 ℃ or 8 ℃, etc., but is not limited to the above-mentioned values, and other values not listed in the above-mentioned range of values are also applicable.

The specific agarose microspheres have good biocompatibility and uniform particle size distribution, and can be efficiently combined with the recombinant protein BMP2C to realize slow release; meanwhile, the carrier is cooperated with hydrogel to release the recombinant protein BMP2C doubly, so that the purpose of long-term slow release of BMP2C can be realized while the protein activity of the carrier is maintained.

In a second aspect, the present invention provides a preparation method of an MBMP2 protein composite hydrogel, comprising the following steps:

(1) dissolving gelatin in water, and adjusting pH to obtain gelatin solution;

(2) and (2) mixing the gelatin solution obtained in the step (1) with agarose-MBMP 2 composite microspheres, then mixing with a TG enzyme (transglutaminase) solution, and incubating to obtain the MBMP2 protein composite hydrogel.

The preparation method has simple preparation process, does not need to introduce any other chemical cross-linking agent, and has good biological safety and low cost.

Preferably, the gelatin of step (1) comprises type a gelatin.

Preferably, the mass fraction of the gelatin in the gelatin solution is 10-20%.

Preferably, the gelatin solution has a pH of 7.5 to 8.5.

The mass fraction of gelatin may be 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc., and the pH may be 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, etc., but is not limited to the above-mentioned values, and other values not listed in the above-mentioned numerical range are also applicable.

Preferably, the gelatin solution obtained in step (1) further comprises step (1'): and (2) mixing the gelatin solution obtained in the step (1) with tannic acid, and stirring to obtain the tannic acid-doped gelatin solution.

Preferably, the mass fraction of tannic acid in the tannic acid-doped gelatin solution of step (1') is 0 to 4% excluding 0.

Preferably, the temperature of said stirring of step (1') is 45-55 ℃.

Preferably, the stirring time in step (1') is 4-10 h.

The mass fraction of tannic acid may be 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, or 4%, the temperature may be 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃, 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, or 55 ℃, and the time may be 4h, 5h, 6h, 7h, 8h, 9h, or 10h, but the present invention is not limited to the above-mentioned values, and other values not listed in the above-mentioned range of values are also applicable.

Preferably, the concentration of the agarose-MBMP 2 composite microspheres in the gelatin solution in the step (2) is 0.5-2 mg/mL.

Preferably, the volume ratio of the gelatin solution to the TG enzyme solution in the step (2) is 5:1-8: 1.

Preferably, the incubation temperature is 35-60 ℃ and the incubation time is 0.5-5 min.

Wherein, the concentration of the agarose-MBMP 2 composite microspheres in the tannin-doped gelatin solution can be 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL, 1mg/mL, 1.1mg/mL, 1.2mg/mL, 1.3mg/mL, 1.4mg/mL, 1.5mg/mL, 1.6mg/mL, 1.7mg/mL, 1.8mg/mL, 1.9mg/mL or 2mg/mL, etc., the volume ratio of the gelatin solution to the TG enzyme solution can be 5:1, 6:1, 7:1 or 8:1, etc., the incubation temperature can be 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, or 60 ℃, etc., the incubation time can be 0.5min, 1min, 1.5min, 2min, 2.5min, 3min, 3.5min, 4min, 4.5min, or 5min, etc., but not limited to, the above-listed numerical values, and other numerical values not listed in the above numerical range are also applicable.

The preparation process steps are simplified by adopting the TG enzyme to replace a chemical cross-linking agent, and the MBMP2 protein composite hydrogel has good biological safety; meanwhile, the hydrogel prepared under specific conditions can be cooperated with agarose microspheres to doubly release the recombinant protein BMP2C carrier, so that the purpose of long-term sustained release of BMP2C can be realized while the protein activity of the carrier is maintained.

Preferably, the concentration of TG enzyme in the TG enzyme solution in step (2) is 0.5-1mg/mL, such as 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL, or 1mg/mL, but not limited to the above-listed values, and other values not listed within the above-mentioned range of values are equally applicable.

As a preferred technical scheme of the invention, the preparation method comprises the following steps:

(1) dissolving gelatin in water, and adjusting pH to 7.5-8.5 to obtain gelatin solution with mass fraction of 10-20%;

(2) mixing the gelatin solution obtained in the step (1) with tannic acid, and stirring for 4-10h at 45-55 ℃ to obtain a tannic acid-doped gelatin solution;

(3) and (3) mixing the tannin-doped gelatin solution obtained in the step (2) with agarose-MBMP 2 composite microspheres, then mixing with a TG enzyme solution, and incubating at 35-60 ℃ for 0.5-5min to obtain the MBMP2 protein composite hydrogel.

In a third aspect, the invention also provides application of the MBMP2 protein composite hydrogel in preparation of tissue engineering materials.

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

(1) the invention provides the MBMP2 protein composite hydrogel, the preparation method of the MBMP2 protein composite hydrogel is simple in preparation process, no other chemical cross-linking agent is required to be introduced, the MBMP2 protein composite hydrogel has good biological safety and low cost; the prepared MBMP2 protein composite hydrogel can realize gelation under physiological conditions, has short gelation time and is easy for clinical operation; the agarose microspheres and hydrogel dual-release carrier are utilized, so that the purpose of long-term slow release of MBMP2 can be realized while the protein activity of the carrier is maintained; the prepared MBMP2 protein composite hydrogel has a controllable three-dimensional reticular structure, compact and uniform holes, good adhesion performance and strong mechanical strength, can realize adhesion and efficient retention on defective tissues, is suitable for filling various irregular-shaped defects, can efficiently promote the performance of material performance, and realizes the repair of the defective tissues;

(2) the recombinant protein MBMP2 is designed and constructed through a genetic engineering technology, and T4 lysozyme protein with good solubility and stable property is added to the nitrogen tail end of BMP-2, so that on one hand, the solubility and stability of the BMP-2 protein are enhanced, and on the other hand, the recombinant protein MBMP2 can be covalently connected to a biological material through the T4 lysozyme at the nitrogen tail end to achieve the effect of slowly releasing growth factors for a long time;

(3) the preparation process steps are simplified by adopting TG enzyme to replace a chemical cross-linking agent, and the MBMP2 protein composite hydrogel has good biological safety; meanwhile, the hydrogel prepared under specific conditions can be cooperated with agarose microspheres to doubly release a recombinant protein BMP2C carrier, so that the purpose of long-term sustained release of BMP2C can be realized while the protein activity of the carrier is maintained;

(4) the special agarose microspheres have good biocompatibility and uniform particle size distribution, and can be efficiently combined with the recombinant protein BMP2C to realize slow release.

Drawings

FIG. 1 is an SEM scanning electron micrograph of the agarose-MBMP 2 composite microspheres provided in preparation example 1;

FIG. 2 is an EDS energy spectrum of agarose-MBMP 2 composite microspheres provided in preparation example 1;

FIG. 3 is an SEM scanning electron micrograph of the internal micro-pore structure of the MBMP2 protein composite hydrogel provided in example 1;

FIG. 4 is a graph showing the results of the slow release test of the MBMP2 protein composite hydrogel provided in example 1;

FIG. 5 is a graph showing the results of the sustained-release effect test of the BMP-2 protein composite hydrogel provided in comparative example 1.

Detailed Description

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

In the following preparation examples, comparative examples and effect tests, a DNA fragment of T4L-BMP2 was synthesized by Kinseri;

BMP-2, T4 lysozyme protein, agarose microspheres, type A pig skin gelatin, ELISA kit purchased from sigma.

The pET28a expression vector was purchased from GE Healthcare;

BL21(DE3) is available from GE Healthcare.

Preparation example 1

The preparation example provides an agarose-MBMP 2 composite microsphere, and the preparation method comprises the following steps:

(1) plasmid construction: subcloning the DNA fragment of T4L-BMP2 into a pET28a expression vector through NdeI and BamHI sites to obtain an MBMP2 recombinant plasmid;

(2) and (3) transformation: using competent cells of BL21(DE3), introducing the MBMP2 recombinant plasmid into the competent cells, culturing the bacterial liquid on an LB solid plate (containing 100 ug/mL ampicillin) at 37 ℃ overnight, and preparing a successfully transformed monoclonal plate for later use;

(3) and (3) amplification culture: picking out small protuberant bacteria with neat edges, translucency and smooth surfaces on an LB flat plate, transferring the small protuberant bacteria into 5mL of LB liquid culture medium, carrying out shake culture for 4 hours at 37 ℃, then transferring the small protuberant bacteria into 800mL of LB liquid culture medium, continuing to culture for about 5 hours, and reducing the culture temperature to 16 ℃ and adding 0.4mM IPTG inducer to induce and express target protein when OD600 of the bacteria liquid reaches 0.4-0.6;

(4) collecting bacteria: transferring 800mL of culture solution into a bacteria collection bottle the next day, centrifuging at 5500rpm for 15 minutes, removing supernatant liquid, and collecting bacteria;

(5) breaking the bacteria: suspending the bacterial liquid with protein buffer solution 20mM HEPES, pH 7.5, 500mM NaCl and 0.6mM protease inhibitor PMSF (phenylmethylsulfonyl fluoride), ultrasonically crushing the bacterial liquid by an ultrasonic cell crusher for 3 cycles, and placing the bacterial liquid on ice in the whole process;

(6) high-speed centrifugation: centrifuging the ultrasonically crushed bacteria liquid in a centrifugal tube of a high-speed centrifugal machine at 20000rpm for 50 minutes at 4 ℃, and collecting insoluble precipitates;

(7) resuspending: the insoluble pellet fraction was resuspended in buffer (20mM HEPES, pH 7.5, 500mM NaCl, 30mM imidazole, 2M urea, 0.6mM protease inhibitor PMSF) and incubated overnight;

(8) hanging columns: after centrifugation at 20000rpm for 50 min again, MBMP2 was purified from the supernatant using a Ni-NTA column. The supernatant was loaded onto a Ni-NTA column and washed with 40mL of buffer (20mM HEPES, pH 7.5, 500mM NaCl, 30mM imidazole, 2M urea);

(9) and (3) purification: the protein supernatant and the Ni purification medium were mixed at 4 ℃ and the suspension was hung on a Ni column 3 times more. A6-0M urea gradient was then linearly folded, washing with the above wash buffer was started and completed at buffer B (20mM HEPES, pH 7.5, 500mM NaCl, 30mM imidazole). Elution of the protein using another linear gradient, starting with buffer B and ending with the same buffer containing 500mM imidazole;

(10) further purification by molecular sieve: fractions containing the protein of interest were combined, concentrated and further purified using a Superdex 200 size exclusion chromatography column. Purified MBMP2 was lyophilized and stored at-80 ℃ for later use;

(11) and mixing the agarose microspheres and MBMP2, wherein the concentration of the agarose microspheres is 10mg/mL, the concentration of the MBMP2 is 2mg/mL, and the agarose-MBMP 2 composite microspheres are obtained, wherein the mixing temperature is 4 ℃, and the mixing time is 12 h. The SEM scanning electron micrograph is shown in figure 1, and the EDS energy spectrum is shown in figure 2.

Preparation example 2

The preparation example provides an agarose-MBMP 2 composite microsphere, and the preparation method comprises the following steps:

steps (1) to (10) correspond to preparation example 1;

(11) and mixing the agarose microspheres and MBMP2, wherein the concentration of the agarose microspheres is 50mg/mL, the concentration of the MBMP2 is 2mg/mL, and the agarose-MBMP 2 composite microspheres are obtained, wherein the mixing temperature is 4 ℃, and the mixing time is 4 hours.

Preparation example 3

The preparation example provides an agarose-MBMP 2 composite microsphere, and the preparation method comprises the following steps:

steps (1) to (10) correspond to preparation example 1;

(11) and mixing the agarose microspheres and MBMP2, wherein the concentration of the agarose microspheres is 50mg/mL, the concentration of the MBMP2 is 15mg/mL, and the agarose-MBMP 2 composite microspheres are obtained, wherein the mixing temperature is 4 ℃, and the mixing time is 8 h.

Preparation example 4

The preparation example provides an agarose-MBMP 2 composite microsphere, and the preparation method comprises the following steps:

steps (1) to (10) correspond to preparation example 1;

(11) and mixing the agarose microspheres and MBMP2, wherein the concentration of the agarose microspheres is 10mg/mL, the concentration of the MBMP2 is 15mg/mL, and the agarose-MBMP 2 composite microspheres are obtained, wherein the mixing temperature is 8 ℃, and the mixing time is 10 hours.

Comparative preparation example 1

This preparation provides an agarose-BMP-2 mixture, which is prepared by the following steps:

and mixing the agarose microspheres and the BMP-2, wherein the concentration of the agarose microspheres is 10mg/mL, the concentration of the BMP-2 is 2mg/mL, and the agarose-BMP-2 mixture is obtained, wherein the mixing temperature is 4 ℃, and the mixing time is 12 h.

Example 1

The embodiment provides an MBMP2 protein composite hydrogel, which is prepared by the following steps:

(1) dissolving the type A pigskin gelatin in deionized water to prepare a gelatin solution with the mass fraction of 20 wt%, and adjusting the pH to 8.0 by using Tri buffer solution.

(2) And (2) adding tannic acid into the gelatin solution obtained in the step (1), wherein the mass fraction of tannic acid in the gelatin solution is 2 wt%, and stirring in a water bath at 55 ℃ for 4h to prepare the tannin-doped gelatin solution.

(3) A TG enzyme solution was prepared by diluting purified transglutaminase (TG enzyme) in a buffer, and the concentration of TG enzyme was 0.5 mg/mL.

(4) And (3) mixing the tannin-doped gelatin solution obtained in the step (2) with the agarose-MBMP 2 composite microspheres obtained in the preparation example 1, wherein the concentration of the agarose-MBMP 2 composite microspheres is 2mg/mL, then adding the TG enzyme solution obtained in the step (3), mixing, and incubating to obtain the MBMP2 protein composite hydrogel. The volume ratio of the tannin-doped gelatin solution to the TG enzyme solution is 5:1, the incubation time is 1min, and the incubation temperature is 35 ℃. The SEM scanning electron micrograph of the internal micro-pore structure is shown in FIG. 3.

Example 2

The embodiment provides an MBMP2 protein composite hydrogel, which is prepared by the following steps:

(1) dissolving the type A pigskin gelatin in deionized water to prepare a gelatin solution with the mass fraction of 20 wt%, and adjusting the pH to 8.0 by using Tri buffer solution.

(2) And (2) adding tannic acid into the gelatin solution obtained in the step (1), wherein the mass fraction of tannic acid in the gelatin solution is 4 wt%, and stirring in a water bath at 55 ℃ for 4h to prepare the tannin-doped gelatin solution.

(3) A TG enzyme solution was prepared by diluting purified transglutaminase (TG enzyme) in a buffer, and the concentration of TG enzyme was 0.5 mg/mL.

(4) And (3) mixing the tannin-doped gelatin solution obtained in the step (2) with the agarose-MBMP 2 composite microspheres obtained in the preparation example 2, wherein the concentration of the agarose-MBMP 2 composite microspheres is 0.5mg/mL, then adding the TG enzyme solution obtained in the step (3), mixing, and incubating to obtain the MBMP2 protein composite hydrogel. The volume ratio of the tannin-doped gelatin solution to the TG enzyme solution is 5:1, the incubation time is 0.5min, and the incubation temperature is 37 ℃.

Example 3

The embodiment provides an MBMP2 protein composite hydrogel, which is prepared by the following steps:

(1) dissolving the type A pigskin gelatin in deionized water to prepare a gelatin solution with the mass fraction of 10 wt%, and adjusting the pH to 8.0 by using Tri buffer solution.

(2) And (2) adding tannic acid into the gelatin solution obtained in the step (1), wherein the mass fraction of tannic acid in the gelatin solution is 1 wt%, and stirring in a water bath at 55 ℃ for 4h to prepare the tannin-doped gelatin solution.

(3) A TG enzyme solution was prepared by diluting purified transglutaminase (TG enzyme) in a buffer, and the concentration of TG enzyme was 1 mg/mL.

(4) And (3) mixing the tannin-doped gelatin solution obtained in the step (2) with the agarose-MBMP 2 composite microspheres obtained in the preparation example 3, wherein the concentration of the agarose-MBMP 2 composite microspheres is 2mg/mL, then adding the TG enzyme solution obtained in the step (3), mixing, and incubating to obtain the MBMP2 protein composite hydrogel. The volume ratio of the tannin-doped gelatin solution to the TG enzyme solution is 8:1, the incubation time is 5min, and the incubation temperature is 40 ℃.

Example 4

The embodiment provides an MBMP2 protein composite hydrogel, which is prepared by the following steps:

(1) dissolving the type A pigskin gelatin in deionized water to prepare a gelatin solution with the mass fraction of 20 wt%, and adjusting the pH to 8.0 by using Tri buffer solution.

(2) A TG enzyme solution was prepared by diluting purified transglutaminase (TG enzyme) in a buffer, and the concentration of TG enzyme was 1 mg/mL.

(3) And (3) mixing the gelatin solution obtained in the step (1) with the agarose-MBMP 2 composite microspheres obtained in the preparation example 1, wherein the concentration of the agarose-MBMP 2 composite microspheres is 1mg/mL, then adding the TG enzyme solution obtained in the step (2), mixing, and incubating to obtain the MBMP2 protein composite hydrogel. The volume ratio of the gelatin solution to the TG enzyme solution is 5:1, the incubation time is 5min, and the incubation temperature is 45 ℃.

Example 5

The embodiment provides an MBMP2 protein composite hydrogel, which is prepared by the following steps:

(1) dissolving the type A pigskin gelatin in deionized water to prepare a gelatin solution with the mass fraction of 20 wt%, and adjusting the pH to 8.0 by using Tri buffer solution.

(2) And (2) adding tannic acid into the gelatin solution obtained in the step (1), wherein the mass fraction of tannic acid in the gelatin solution is 4 wt%, and stirring in a water bath at 55 ℃ for 10h to prepare the tannin-doped gelatin solution.

(3) A TG enzyme solution was prepared by diluting purified transglutaminase (TG enzyme) in a buffer, and the concentration of TG enzyme was 1 mg/mL.

(4) And (3) mixing the tannin-doped gelatin solution obtained in the step (2) with the agarose-MBMP 2 composite microspheres obtained in the preparation example 4, wherein the concentration of the agarose-MBMP 2 composite microspheres is 2mg/mL, then adding the TG enzyme solution obtained in the step (3), mixing, and incubating to obtain the MBMP2 protein composite hydrogel. The volume ratio of the tannin-doped gelatin solution to the TG enzyme solution is 5:1, the incubation time is 2min, and the incubation temperature is 37 ℃.

Comparative example 1

This comparative example provides a BMP-2 protein composite hydrogel, which was prepared in the same manner as in example 1, except that the agarose-MBMP 2 composite microspheres obtained in preparation example 1 were replaced with the same amount of the agarose-BMP-2 mixture obtained in comparative preparation example 1.

And (3) testing the slow release effect:

the MBMP2 protein complex hydrogel provided in example 1 and the BMP-2 protein complex hydrogel provided in comparative example 1 were each soaked in 2mL of phosphate buffer (pH 7.4) at 1g, the leachate was taken out at a fixed time point and stored at-80 ℃, and soaked in the same amount of phosphate buffer at 1, 3, 6, 11, 15, 18, 22, 27, and 37d, respectively, and the release of MBMP2 and BMP-2 was measured with an ELISA kit as a function of time, and the results are shown in fig. 4 to 5.

As can be seen from the results in the figure, the MBMP2 protein composite hydrogel provided by the invention has an excellent slow-release effect of the MBMP2 protein, which indicates that the MBMP2 protein composite hydrogel has excellent effects on bone defect repair and bone regeneration.

The applicant states that the invention is illustrated by the above examples to the MBMP2 protein composite hydrogel and the preparation method and application thereof, but the invention is not limited by the above examples, i.e. the invention is not limited by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.