CN109692344B

CN109692344B - rH-BMP 2-containing bone repair material and preparation method and application thereof

Info

Publication number: CN109692344B
Application number: CN201910122921.8A
Authority: CN
Inventors: 曹建新
Original assignee: Zhejiang Ruigu Biotechnology Co ltd
Current assignee: Zhejiang Ruigu Biotechnology Co ltd
Priority date: 2019-02-18
Filing date: 2019-02-18
Publication date: 2021-04-20
Anticipated expiration: 2039-02-18
Also published as: CN109692344A

Abstract

The invention belongs to the field of biomedical materials containing active protein and the field of regenerative medicine, and particularly provides 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.

Description

rH-BMP 2-containing bone repair material and preparation method and application thereof

Technical Field

The invention belongs to the field of biomedical materials containing active protein and the field of regenerative medicine, and particularly provides a bone repair material containing rH-BMP2, which comprises modified gelatin and rH-BMP 2. The material can be used for repairing bone injury.

Background

Bone morphogenetic protein-2 (BMP-2) is a key factor found to promote osteogenesis in Marshall R.Urist in 1965. BMP-2 belongs to TGF-alpha family, is secreted by osteoblasts and acts on the osteoblasts, and BMP-2 is a main signal molecule for differentiation of cells into mineral-deposited osteoblasts and plays a role in inducing differentiation of the osteoblasts. It is expressed in limb growth, endochondral ossification and fracture, and plays an important role in bone growth and development and regeneration repair. Because of its remarkable effect of stimulating the formation of new bone, BMP-2 is considered as a promising active ingredient for the treatment of diseases such as bone fractures, bone defects, etc., and can be used as an alternative medical treatment for bone grafting in the case of spinal fusion, joint fusion, etc. (such application has been approved by the FDA).

The main disadvantage of BMP-2 is its short half-life, after intravenous injection_t1/2It is only 6-7min, and is easily denatured by metabolic process, physiological environment change, or action with enzyme. Therefore, even if a large dose of the drug is continuously administered, the drug administration routes such as injection and oral administration often cannot provide a continuously effective concentration for a target site, cannot provide a good treatment effect for bones which need to grow and recover for a long time, and is easy to cause low bone formation quality, inflammatory reaction and other potential risks for patients. Therefore, the selection of a suitable delivery means, such as placement of BMP-2 in a bone repair material containing rH-BMP2, is critical to achieving widespread use of BMP-2 and improving its therapeutic efficacy.

Natural polymer materials such as gelatin, chitosan, hyaluronic acid, alginic acid, cellulose and the like become good carriers of BMP-2 due to good biocompatibility and degradation characteristics, but when used as bone repair materials containing rH-BMP2, the natural polymer materials have the problems of poor mechanical properties and rapid degradation, and even when used with other materials, good balance is difficult to achieve.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a bone repair material containing rH-BMP2, which improves the mechanical property, realizes great progress in degradation/drug release time as a slow release carrier and obviously reduces the anaphylaxis. 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.

The invention provides a bone repair material containing rH-BMP2, which comprises the following components in percentage by mass:

as a further improvement, the bone repair material comprises the following components in percentage by weight:

as a further improvement, the modified gelatin adopts linoleic acid modified gelatin and chitosan modified gelatin or linoleic acid chitosan modified gelatin.

As a further improvement, the modified gelatin is linoleic acid chitosan modified gelatin, and the weight ratio of the gelatin: and (3) chitosan: linoleic acid is 5-20: 2-4: 0.5-1.5.

As a further improvement, the linoleic acid chitosan modified gelatin comprises the following components in percentage by mass: and (3) chitosan: linoleic acid was 10:2: 0.5.

As a further improvement, the preparation method of the linoleic acid chitosan modified gelatin comprises the following steps: heating and dissolving gelatin in a water bath at 60-80 ℃, and preparing a 5% (w/v) solution of the gelatin by using distilled water; preparing chitosan into a 2% (w/v) solution using 2% acetic acid (pH about 5); adding the chitosan solution into the gelatin solution, stirring uniformly, then adding 0.5% (w/v) linoleic acid into the gelatin solution, and stirring for 1-2 hours at 40-50 ℃ for full crosslinking.

As a further improvement, the nucleotide sequence table of the rH-BMP2 is shown in SEQ NO.1, and the amino acid sequence is shown in SEQ NO. 2; or the nucleotide sequence table of the rH-BMP2 is shown in SEQ NO.3, and the amino acid sequence is shown in SEQ NO. 4.

The second purpose of the invention is to provide a preparation method of the bone repair material containing rH-BMP2, which comprises the following steps: mixing the calcium chloride with a half of the modified gelatin according to the prescription amount, adding water to dissolve, adding rH-BMP2 to freeze-dry, mixing the freeze-dried powder with the hydroxyapatite and the rest of the modified gelatin, adding a small amount of water to dissolve, filling and freeze-drying.

Alternatively, the method of the present invention comprises the steps of: mixing the calcium chloride and the gelatin according to the prescription amount, adding water to dissolve, adding the hydroxyapatite and the rH-BMP2, uniformly mixing, filling and freeze-drying.

The third purpose of the invention is to provide the application of the bone repair material containing rH-BMP2 in repairing bone injury; further, the bone injury is bone defect, bone nonunion, and delayed bone healing.

In addition to bone injury, the compositions of the present application may also be used in therapeutic orthopedic bone repair, cosmetic orthopedic repair, spinal fusion, joint fusion, and the like, where filling and promotion of bone growth is desired.

Due to the adoption of the technical scheme, the linoleic acid modified gelatin and the chitosan modified gelatin or the linoleic acid chitosan modified gelatin are used as slow release carriers to realize great progress in degradation/drug release time, and the anaphylaxis is obviously reduced. 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.

Drawings

FIG. 1 is a photograph of a radiology examination of the mice of example 1.

FIG. 2 is a radiology examination of the mice of example 2.

FIG. 3 is a radiology view of the mouse of example 3.

FIG. 4 is a radiology examination of the mice of example 4.

FIG. 5 is a photograph showing a radiological examination of a mouse in a control example.

Detailed Description

Reagents and instrumentation:

gelatin is supplied by Yangxin east Biotechnology Ltd of Shandong province;

chitosan (medium molecular weight, about 40 million) is produced by shanghai ai gay biotechnology limited;

linoleic acid is produced by Shanghai gold company pharmaceutical Co., Ltd;

the detection kit for the residual protein of the escherichia coli host is produced by Shanghai Chengxao biological science and technology limited;

the rH-BMP2ELISA detection kit is produced by Tianjin Anorikang biotechnology, Inc.;

the CCK-8 cell proliferation toxicity detection kit is produced by Dalian Meilun biological technology limited company;

nicolet 5700 an infrared spectrometer is produced by Nicolet;

partial molecular biology operations are entrusted to Kingsry Biotechnology, Inc;

partial chemical modification operation is entrusted to Shanghai pioneer chemical research management company;

part of animal experiments are carried out by the scientific research experiment center/experimental animal center of Beijing university of traditional Chinese medicine;

the rest reagents and instruments are all made by conventional brands and models.

EXAMPLE 1 preparation of BMP-2

For the convenience of experimental progress and quality control, BMP-2 used in the experiment was prepared by the applicant:

constructing an rH-BMP2 gene sequence suitable for being expressed in escherichia coli according to an rH-BMP2 amino acid and gene sequence in Genbank, wherein the nucleotide sequence (containing partial enzyme cutting sites) is SEQ ID NO.1 in a sequence table; the gene fragment is inserted into a pET plasmid vector after enzyme digestion, and is transformed into an escherichia coli host after connection; screening positive clones, and inducing rH-BMP2 to express; fermenting, breaking thallus, collecting inclusion body, cracking inclusion body and renaturing protein; purifying by anion and cation exchange chromatography to obtain rH-BMP2, electrophoretically displaying a single band (identical with reported rH-BMP 2) about 12kDa, and the amino acid sequence of the product is SEQ ID NO.2 in the sequence table.

The detection of residual protein (less than 0.005%) in colibacillus host by using colibacillus host residual protein detection kit and the detection of antibiotic residue (less than 0.1ppm) by using bacteriostatic loop method all meet the requirements of further experiments.

Example 2 preparation of modified gelatin

The preparation method of the chitosan modified gelatin comprises the following steps: according to the mass ratio, gelatin: the ratio of chitosan is 10: 2; heating and dissolving gelatin in a water bath at 70 ℃ to prepare a 5% (w/v) solution by using distilled water; preparing chitosan into a 2% (w/v) solution using 2% acetic acid (pH about 5); adding the chitosan solution into the gelatin solution, stirring uniformly, and stirring at 30 ℃ for 1 hour for full crosslinking.

The preparation method of the linoleic acid modified gelatin comprises the following steps: according to the mass ratio, gelatin: linoleic acid is 10: 1; heating and dissolving gelatin in a water bath at 70 ℃ to prepare a 5% (w/v) solution by using distilled water; linoleic acid was formulated as a 0.5% (w/v) solution using 2% acetic acid (pH about 5); the two solutions were mixed as follows 1: 1, adding 0.5% (w/v) linoleic acid, and stirring at 30 ℃ for 1 hour to fully crosslink.

The preparation method of the linoleic acid chitosan modified gelatin comprises the following steps: according to the mass ratio, gelatin: and (3) chitosan: linoleic acid is 10:2: 0.5; heating and dissolving gelatin in a water bath at 70 ℃ to prepare a 5% (w/v) solution by using distilled water; preparing chitosan into a 2% (w/v) solution using 2% acetic acid (pH about 5); adding the chitosan solution into the gelatin solution, stirring uniformly, then adding 0.5% (w/v) linoleic acid into the gelatin solution, and stirring for 1 hour at 30 ℃ to fully crosslink.

Example 3

The bone repair material containing the rH-BMP2 comprises the following components in percentage by weight:

mixing the calcium chloride with a half of the modified gelatin according to the prescription amount, adding water to dissolve, adding rH-BMP2 to freeze-dry, mixing the freeze-dried powder with the hydroxyapatite and the rest of the modified gelatin, adding a small amount of water to dissolve, filling and freeze-drying. And (5) drying, and then irradiating for sterilization to obtain the bone repair material containing the rH-BMP 2. When not in use, the product is stored at 4 ℃ in a refrigeration way.

CCK-8 cell proliferation toxicity experiments carried out according to the kit instructions show that the cytotoxicity of the material is CTS 0-I grade, and the safety of the material is preliminarily verified.

A control rH-BMP 2-containing bone repair material was also prepared in which chitosan was used instead of modified gelatin, with the other processes and ingredients unchanged.

EXAMPLE 4 in vitro rH-BMP2 Release assay of Material

3g each of the rH-BMP 2-containing bone repair material and the control rH-BMP 2-containing bone repair material (containing rH-BMP 260 mg) were prepared according to the procedure of example 3, and placed in a dialysis bag, which was placed in 20ml of PBS buffer solution containing 0.2% sodium azide and having a pH of 7.0. After standing at 37 ℃, 1ml of each of 12h, 24h, 120h, 168h, 336h, 504h, 672h and 840h was sampled (followed by 1ml of PBS buffer containing 0.2% sodium azide and having pH 7.0) and the rH-BMP2 concentration was measured by ELISA method according to the kit instructions and converted to the cumulative release percentage. The results are shown in the following table.

TABLE 1 cumulative percent release of rH-BMP2

The rH-BMP2 in the control material is basically released within 168h (meanwhile, gelatin in the material is basically disappeared), the drug release time of the modified gelatin material is prolonged to about 4-5 weeks, the modified gelatin material basically corresponds to the initial growth stage in bone repair (meanwhile, the gelatin in the material is basically disappeared), the modified gelatin material is not inferior to many polymer microsphere materials provided in the prior art, and the problems of acidic environment and allergy during the degradation of the polymer microspheres can be avoided.

Example 5 gelatin allergy test

30 Hartley male guinea pigs weighing about 400g were selected and divided into 3 groups on average. Three groups on days 1 and 4 of the experiment are respectively injected with aqueous suspensions containing 2.0mg of linoleic acid chitosan modified gelatin, linoleic acid modified gelatin, chitosan modified gelatin and unmodified gelatin in the abdominal cavity, and three groups on day 7 of the experiment are respectively injected with aqueous suspensions containing 3.0mg of linoleic acid chitosan modified gelatin, linoleic acid modified gelatin, chitosan modified gelatin and unmodified gelatin. Weight of guinea pigs was weighed on days 1, 4, and 10 of the experiment. The allergic reaction condition is observed on the 7 th to 10 th days of the experiment, and the evaluation indexes comprise negative (normal) allergic reaction, weak positive (restlessness, shivering and nasal pruritus), positive (sneezing, tachypnea, urination and lacrimation), strong positive (dyspnea, wheezing, purpura, unstable gait, spasm and rotation) allergic reaction and strong positive (death) allergic reaction.

TABLE 2 gelatin allergy test

The results show that the average weight change of three groups of guinea pigs is basically consistent (the average weight gain is about 30-35g by day 10); strong allergic reactions occurred in all three groups of guinea pigs, but a higher proportion of guinea pigs in the unmodified gelatin group exhibited symptoms of weak allergy (similar to previous literature studies), with two modified gelatins being significantly more allergic than the unmodified gelatin, with no allergic observed at all.

Example 6 actual bone repair experiment

Firstly, designing:

grouping comparison and multi-angle evaluation observation experiment.

II, materials:

the mouse breed is ICR or ICR male mouse, and the mouse age is about 30 days; the bone repair material prepared from the rH-BMP2 and the carrier material is provided by Zhejiang Ruigao Biotechnology GmbH.

Thirdly, the method comprises the following steps:

35 normal ICR or KM male mice were randomly divided into 7 groups of 3 mice each.

After the mice are anesthetized by 6% sodium pentobarbital, the hind limb and the thigh are unhaired and disinfected, the skin is cut open, the muscle gap is separated, different carrier materials are implanted, a certain amount of antibiotic is added to prevent infection, then the muscle and the skin are sutured layer by layer, the wound is disinfected, and the mice are normally raised.

Fourthly, observation:

4 weeks after completion of the implantation experiment, the mice were subjected to a radiological examination (X-ray) to see whether or not bone tissue was present in the hind limbs of the mice. After 4 weeks of radiologic examination, the mice were sacrificed, bone tissues in the implanted region were taken out, wet weights of bones contained therein were measured, and HE staining histological evaluation was performed.

Five, scoring standard for bone healing observed by X-ray

1.1962 Chaibenfu fracture healing criteria:

Lane-Sandhu X-ray and histological scoring criteria:

sixthly, the results

The mouse scores of the experimental group and the control group are shown in table 1 and table 2.

TABLE 11962 measurement Table for fracture healing

Numbering	Edge of broken end	Periosteal reaction	Amount of callus	Density of callus	Callus margin
						Example 1	++++	++++	++++	++++	++++
Example 2	+++	+++	++++	++++	+++
						Example 3	+++	+++	++++	++++	+++
Example 4	++++	+++	++++	++++	+++
						Comparative example	++	++	++	++	++

TABLE 2 Lane-Sandhu X-ray and histological grading Table

Numbering	Bone formation	Bone connection	Bone shaping
				Example 1	4	4	4
Example 2	4	3	4
				Example 3	4	3	4
Example 4	4	4	3
				Comparative example	2	2	2

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention, including any reference to the above-mentioned embodiments. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

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Claims

1. The bone repair material containing rH-BMP2 is characterized by comprising the following components in percentage by weight:

rH-BMP2 1 %-5 %

60 to 86 percent of modified gelatin

5 to 20 percent of hydroxyapatite

8 to 15 percent of calcium chloride;

the modified gelatin is linoleic acid chitosan modified gelatin.

2. The rH-BMP 2-containing bone repair material according to claim 1, which is characterized by comprising the following components in percentage by weight:

rH-BMP2 2%-4 %

70 to 80 percent of modified gelatin

8 to 15 percent of hydroxyapatite

10 to 12 percent of calcium chloride.

3. The rH-BMP 2-containing bone repair material according to claim 1, wherein the modified gelatin is linoleic acid chitosan modified gelatin, and the mass ratio of gelatin: and (3) chitosan: linoleic acid is 5-20: 2-4: 0.5-1.5.

4. The rH-BMP 2-containing bone repair material according to claim 3, wherein the linoleic acid chitosan modified gelatin comprises, by mass: and (3) chitosan: linoleic acid was 10:2: 0.5.

5. The rH-BMP 2-containing bone repair material according to claim 3 or 4, wherein the linoleic acid chitosan modified gelatin is prepared by the following method: heating and dissolving the gelatin in a water bath at the temperature of 60-80 ℃, and preparing the gelatin into a solution with the weight volume ratio of 5% by using distilled water; preparing chitosan into a solution with the weight volume ratio of 2% by using 2% acetic acid; adding the chitosan solution into the gelatin solution, uniformly stirring, adding linoleic acid with the weight volume ratio of 0.5%, and stirring at 40-50 ℃ for 1-2 hours for full crosslinking.

6. The rH-BMP 2-containing bone repair material of claim 1, wherein the nucleotide sequence table of the rH-BMP2 is shown in SEQ NO.1, and the amino acid sequence is shown in SEQ NO. 2; or the nucleotide sequence table of the rH-BMP2 is shown in SEQ NO.3, and the amino acid sequence is shown in SEQ NO. 4.

7. The method for preparing a rH-BMP 2-containing bone repair material according to any one of claims 1 to 6, wherein the method comprises the following steps: mixing the calcium chloride with a half of the modified gelatin according to the prescription amount, adding water to dissolve, adding rH-BMP2 to freeze-dry, mixing the freeze-dried powder with the hydroxyapatite and the rest of the modified gelatin, adding a small amount of water to dissolve, filling and freeze-drying.

8. The method for preparing a rH-BMP 2-containing bone repair material according to any one of claims 1 to 6, wherein the method comprises the following steps: mixing the calcium chloride and the modified gelatin according to the prescription amount, adding water to dissolve, adding the hydroxyapatite and the rH-BMP2, uniformly mixing, filling and freeze-drying.

9. The use of the rH-BMP 2-containing bone repair material according to any one of claims 1 to 6 in the preparation of a medicament for repairing bone damage, wherein the bone damage is bone defect, nonunion, and delayed bone healing.