CN114246984A

CN114246984A - Bone repair material with rhBMP-2 carried by gelatin sponge and preparation method thereof

Info

Publication number: CN114246984A
Application number: CN202111409403.8A
Authority: CN
Inventors: 曹建新; 邓平
Original assignee: Zhejiang Ruigu Biotechnology Co ltd
Current assignee: Zhejiang Ruigu Biotechnology Co ltd
Priority date: 2021-11-19
Filing date: 2021-11-19
Publication date: 2022-03-29
Anticipated expiration: 2041-11-19
Also published as: CN114246984B

Abstract

The invention provides a bone repair material using gelatin sponge to carry rhBMP-2 and a preparation method thereof, which comprises bone morphogenetic protein rhBMP-2, a drug carrier gelatin sponge and other auxiliary materials, wherein the gelatin sponge is prepared from gelatin and a cross-linking agent. According to the bone repair material with the rhBMP-2 carried by the gelatin sponge, the bone morphogenetic protein BMP-2 is loaded into the gelatin sponge, the gelatin sponge is used as a bracket material, and the gelatin sponge is modified by adopting carbodiimide and 1, 6-hexamethylene diisocyanate cross-linking agent, so that the mechanical stress strain and the enzymolysis stability of the gelatin sponge are improved, and the sufficient degradation time of the gelatin sponge in a human body is ensured.

Description

Bone repair material with rhBMP-2 carried by gelatin sponge and preparation method 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 with rhBMP-2 carried by gelatin sponge and a preparation method thereof.

Background

The treatment of orthopedic disorders generally requires bone grafting as a bridge for new bone growth, such as bone tumors, nonunions, herniated cervical discs, osteoporotic fractures of the spine, and numerous spinal disorders requiring fusion surgery. In China, tens of thousands of patients suffer from bone defects caused by lesions such as trauma, infection, tumor and the like every year, and hundreds of thousands of patients undergo spinal fusion surgery, and both of the patients need bone transplantation treatment. Bone transplantation has many problems of newly added operation trauma, limited source, immunological rejection, disease transmission, high cost and the like. With the development of material science, the replacement of bone graft with biomaterial has become a new trend. The ideal bone repair material not only needs to be capable of actively inducing new bone formation and rapidly repairing bone defects, but also needs to have good biocompatibility and degradability, and needs to be safe and nontoxic.

Bone morphogenetic protein-2 (BMP-2) is a key factor for promoting osteogenesis, belongs to a TGF-alpha family member, and BMP-2 is a main signal molecule for differentiating cells into mineral deposition osteoblasts and plays a role in inducing osteoblast differentiation. The expression is performed during limb growth, endochondral ossification and fracture, plays an important role in bone growth and development and regeneration repair, and has good application prospect in clinical treatment of bone injury. In addition, the common bone repair material carrying the rhBMP often intensively releases the rhBMP-2 in the early stage of the use of the product, and the rhBMP-2 can not be controlled to be continuously released in a trace manner, so that the content of the rhBMP-2 in local tissues is enriched in a period of time, and unnecessary ectopic bone formation can be caused when the rhBMP-2 flows to other tissues except the tissue area to be repaired along with blood flow or tissue fluid flow. In addition, rhBMP-2 has a short half-life in vivo, is metabolized and absorbed quickly upon topical application, is difficult to work with, often does not provide a sustained effective concentration at the target site even when administered in large doses continuously, does not provide a good therapeutic effect for bones that require long-term growth and recovery, and is prone to low bone quality, inflammatory reactions and other potential risks in patients. The above reasons suggest that how to control the sustained, stable and slow release of the rhBMP-2, which not only can achieve the effect of effectively promoting the bone growth, but also can avoid and prevent the generation of adverse reactions is a key technology of using the rhBMP-2.

Gelatin sponge is a good medical drug carrier, is obtained by gelatin crosslinking, has higher porosity inside, and has good drug loading and drug release capacity, formaldehyde, glutaraldehyde or genipin and the like are adopted as the crosslinking agent of the currently used gelatin sponge, but the obtained gelatin sponge has poor water absorption or is easy to collapse when meeting water, and has insufficient mechanical strength, in practical application, after BMP-2 is loaded, the gelatin sponge is implanted into a human body, and the gelatin sponge collapses easily due to the influence of blood water, so that BMP-2 is released from the gelatin sponge, and the problem of ectopic osteogenesis is easily caused along with the blood flow.

Disclosure of Invention

The invention aims to provide a special cross-linking agent combination and a cross-linking mode, so that gelatin is compounded with rhBMP-2 when being cross-linked into gelatin sponge, and the bone repair material which is loaded with the rhBMP-2 by the gelatin sponge has strong support property, good drug loading performance and good rhBMP-2 distribution uniformity.

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

a bone repair material using gelatin sponge to carry rhBMP-2 comprises bone morphogenetic protein rhBMP-2, a drug carrier gelatin sponge and other auxiliary materials, wherein the gelatin sponge is prepared from gelatin and a cross-linking agent.

Furthermore, the other auxiliary materials are tris buffer solution, trehalose, bovine serum albumin and PEG 6000.

Further, the cross-linking agent is one or more of carbodiimide, hydroxysuccinimide and 1, 6-hexamethylene diisocyanate.

Further, the bone morphogenetic protein rhBMP-2 freeze-dried powder comprises, by weight, 0.1% -1% of bone morphogenetic protein rhBMP-2 freeze-dried powder, 0.5% -1.5% of tris buffer solution, 1% -2% of trehalose, 0.5% -2% of bovine serum albumin, 0.5% -3% of cross-linking agent, 1% -5% of gelatin, 0.5% -2% of PEG6000 and the balance of water.

Further, the cross-linking agent is carbodiimide and 1, 6-hexamethylene diisocyanate in a weight ratio of 1-2: 1, mixing; firstly adding carbodiimide into gelatin-PEG solution, adding bone morphogenetic protein rhBMP-2 and other auxiliary materials, and then adding 1, 6-hexamethylene diisocyanate.

The invention also discloses a preparation method of the bone repair material with the rhBMP-2 carried by the gelatin sponge, which comprises the following steps:

(1) preparing gelatin-PEG 6000 solution;

(2) firstly adding carbodiimide into the gelatin-PEG solution, controlling the temperature until the crosslinking is not solidified, and stirring the mixture to be semi-colloidal;

(3) adding rhBMP-2 freeze-dried powder, tris buffer solution, trehalose and bovine serum albumin, stirring, adding 1, 6-hexamethylene diisocyanate, and crosslinking after stirring;

(4) and (3) sequentially washing the colloid obtained by crosslinking with 0.1mol/L Na2HPO4, 4mol/L NaCl and deionized water, and freeze-drying in a freeze dryer to obtain the rhBMP-2-loaded gelatin sponge.

Furthermore, in the gelatin-PEG 6000 solution, the concentration of gelatin is 3.0%, and the concentration of PEG6000 is 1.0%.

Further, in the step (2), a crosslinking agent carbodiimide is added to a concentration of 0.75%, and 1, 6-hexamethylene diisocyanate is added to a concentration of 0.5%.

Further, the parameters of the freeze-drying process are as follows:

the bone repair material with the rhBMP-2 carried by the gelatin sponge, disclosed by the invention, has the advantages that the bone morphogenetic protein BMP-2 is loaded into the gelatin sponge, the gelatin sponge is used as a bracket material, the rhBMP-2 is subjected to sustained and controlled release, the requirement of the bone at the initial induction growth stage is ensured, the gelatin sponge is modified, carbodiimide and a 1, 6-hexamethylene diisocyanate cross-linking agent are adopted, the mechanical stress strain and the enzymolysis stability of the gelatin sponge are improved, and the sufficient degradation time of the gelatin sponge in a human body is ensured. Meanwhile, in the preparation process, carbodiimide is added for crosslinking reaction to form a semi-colloid, then rhBMP-2 and other auxiliary materials are added, and then the crosslinking agent 1, 6-hexamethylene diisocyanate is added to complete crosslinking, so that the rhBMP-2 and the gelatin sponge are integrated, and the problems that the rhBMP-2 is not uniformly distributed in the sponge and is easily attached to the surface of the sponge due to the fact that the rhBMP-2 is loaded in a sponge soaking rhBMP-2 solution at present are solved.

Drawings

FIG. 1: gelatin sponge carrying rhBMP-2 and obtained by different cross-linking agents; wherein A is the rhBMP-2 loaded gelatin sponge obtained in example 1; b is sample 1; c is sample 2; d is sample 3; e is sample 4; f is sample 5.

Detailed Description

Example 1: preparation of bone repair material of gelatin sponge carrying rhBMP-2

The rhBMP-2 is produced by enterprises by self, the specific production process refers to the production method (expression in escherichia coli, purification by ion exchange chromatography and detection of escherichia coli host residual protein < 0.005%) in the Chinese patent application No. CN201910030649.0 of the applicant, and the antibiotic residue <0.1ppm is detected by a bacteriostatic circle method;

gelatin, carbodiimide, 1, 6-hexamethylene diisocyanate, tris buffer, trehalose, bovine serum albumin are all commercially available.

The preparation method comprises the following steps:

(1) preparing 200ml of gelatin-PEG 6000 solution, wherein the concentration of gelatin is 3.0%, and the concentration of PEG6000 is 1.0%;

(2) firstly adding carbodiimide into gelatin-PEG solution to the concentration of 0.75%, controlling the temperature to 37 ℃ for crosslinking without solidification, and stirring to be semi-colloidal;

(3) adding rhBMP-2 lyophilized powder 1mg, tris buffer solution 20ml, trehalose 4mg and bovine serum albumin 2mg, stirring, adding 1, 6-hexamethylene diisocyanate until the concentration is 0.5%, stirring, and crosslinking at 25 deg.C for 24 h;

(4) the colloid obtained by crosslinking is sequentially added with 0.1mol/L Na²HPO4, 4mol/L NaCl and deionized water, and then placing the mixture into a freeze dryer for freeze drying to obtain rhBMP-2-loaded gelatin sponge; the resulting sample is shown as a in fig. 1.

The freeze-drying process parameters are as follows:

serial number	Control process	Slab control temperature (. degree. C.)	Controlled vacuum (mbar)	Process time (min)
					1	Loading	8±1	---	---
2	Prefreezing	-4	---	20
					3	Prefreezing	-10	---	35
4	Prefreezing	-15	---	60
					5	Prefreezing	-35	---	120
7	Evacuation	---	0.1	---
					8	Drying	-10～-5	0.15±0.01	60
9	Drying	-10～-5	0.15±0.01	1200
					10	Drying	20	0.15±0.01	60
11	Drying	20	＜0.01	300
					12	Pressure rise	---	≤0.011	---
13	Pre-deflation	---	0.4-0.5bar	---
					14	Press plug	---	---	120 seconds
15	Air release	---	---	---
					16	Storing	20	---	---

Example 2: preparation of gelatin sponge rhBMP-2-loaded bone repair samples with different crosslinking agents

Sample 1: the cross-linking agent adopts carbodiimide and 1, 6-hexamethylene diisocyanate, and is added and stirred simultaneously in the preparation process step (2); the rest preparation method is the same as the embodiment;

sample 2: the cross-linking agent adopts carbodiimide and 1, 6-hexamethylene diisocyanate, the amount of the cross-linking agent is the same as that in the example 1, 6-hexamethylene diisocyanate is firstly added in the step (2) to the concentration of 0.5 percent; adding carbodiimide to the concentration of 0.75% in the step (3); the rest of the procedure was the same as in example 1.

Sample 3: the carbodiimide in example 1 was crosslinked with hydroxysuccinimide by the same procedure as in example 1.

Sample 4: the same procedure as in example 1 was repeated except that 1, 6-hexamethylene diisocyanate in example 1 was replaced with hydroxysuccinimide.

Sample 5: the carbodiimide concentration in example 1 was 0.5%, and the 1, 6-hexamethylene diisocyanate concentration was 0.75%, and the other preparation methods were the same as in example 1.

The resulting samples are individually shown in figure 1 as B, C, D, E, F.

In the research process, the applicant tries other various cross-linking agents and combinations of the cross-linking agents, but the actual effect is not ideal, the drug loading is low, or the gelatin sponge is easy to collapse when meeting water and cannot be molded. Only the rhBMP-2-loaded gelatin sponge obtained in example 1 was the best in each index.

Example 3: appearance and physical Properties of rhBMP-2-Supported gelatin sponge

The samples obtained in example 1 and example 2 are shown in FIG. 1.

Example 4: mechanical stress test of rhBMP-2-loaded gelatin sponges prepared with different crosslinking agents

And testing the stress-strain performance of the gelatin sponge by a mechanical drawing machine. The sponges were cut into sponge pieces (5 mm. times.5 mm) of equal size, measured in both the dry and wet states, respectively. The two ends of the sample are wrapped and fixed by medical adhesive tape so as to prevent the sample from being damaged when the sample is fixed by the clamp. The sample was fixed and straightened out with a force of 0.05g, a sensor force of 100N was applied, and the dry state was tested with a 5mm/min wet state at a tensile rate of 1mm/min, wherein the wet state was equilibrated for 30min in a PBS (pH7.2) solution (20 ℃) before the measurement.

Stress-strain performance table of gelatin sponge with different cross-linking agents

The tensile stress-strain test compares the change in load, breaking strength, breaking strain and elastic modulus for different sets. The average values of the samples of each group are calculated and shown in the table, and through SPSS11.5 statistical analysis, the states of the sponge (dry and wet) and the influence of different cross-linking agents on the mechanical strength of the sponge have significant differences (Two-factor ANOVA; p is less than 0.0001).

Maximum load: the difference in maximum load was significant in both dry and wet states (p < 0.0001). The example 1 sample gave a significant increase in the maximum load the sponge was subjected to (p < 0.0001), while in the wet state its corresponding maximum load value was greatly reduced, but the maximum load was optimal for the gelatin sponge of example 1 relative to the other comparative samples.

Breaking stress: the breaking stress is significantly reduced in the wet state compared to the dry state (p < 0.0001). The breaking strength of the example 1 group in the dry state was significantly enhanced (p < 0.0001) compared to the control group; the stress at break was reduced for each group in the wet state and the group of example 1 was slightly higher than the other sample groups, but the difference was not statistically significant.

Breaking strain: the strain at break is significantly increased in the wet state compared to the dry state. This indicates that gelatin sponges can withstand greater deformation without breaking in the wet state (p < 0.0001).

Modulus of elasticity: the elastic modulus difference between the matrix state and different groups is obvious, and the elastic modulus of the group of example 1 is obviously improved.

The maximum load, the breaking stress and the elastic modulus of the gelatin sponge obtained in the example 1 in a dry state are obviously enhanced; whereas the maximum load and breaking stress in the wet state are higher for the group of example 1 than for the other groups of samples, which may be related to the choice of cross-linking agent and the weight ratio between the two cross-linking agents during the cross-linking treatment.

Example 5: enzymatic stability test

Test samples: example 1 and samples 1, 2, 3, 4, 5;

the main reagents are as follows: gelatinase.

Preparing a 5CU/ml gelatinase solution by using fresh DMEM culture solution, placing about 5mg of sterile samples into 1ml of gelatinase solution, incubating at the constant temperature of 37 ℃, replacing fresh gelatinase solution every day, and sampling at time points of digestion for 4h, 1d, 2d, 3d, 6d and 9 d. Adding 0.25mol/L EDTA200 μ L into the enzyme solution to stop digestion, carefully taking out with ophthalmologic operation forceps, washing with a large amount of PBS and deionized water, placing in a clean tubule, freezing and storing at-20 ℃, vacuum freeze-drying for 36h, weighing the mass of the residual sample, and expressing the enzymolysis stability of the collagen sponge matrix by the mass of the residual sponge matrix.

The difference of the enzymolysis rates of the gelatin sponge scaffolds crosslinked by different crosslinking agents is determined through a gelatinase degradation experiment. After 9 days of enzymatic hydrolysis, the mass of the remaining sponge pieces of the group of example 1 was 83.5% of the original mass, respectively. The mass of the remaining sponge pieces of the groups of sample 1, sample 2, sample 3, sample 4, sample 5 was below 60.0%, 55.8%, 57.1%, 48.9%, 59.4%, 60.1%, respectively. In contrast, the gelatin sponges of the example 1 group and the enzyme action time had a significant effect on the degradation of the sponge mass, greatly enhancing the enzymatic stability of the gelatin sponges.

Example 6: in vitro release assay for rhBMP-2-loaded gelatin sponges

Test groups: example 1, sample 2, sample 3, sample 4, sample 5;

control group: the rhBMP-2 bone repair material produced by Hangzhou Jiuyuan genetic engineering company consists of rhBMP-2 and carrier material, wherein the carrier material comprises hydroxyapatite, soybean phospholipid and medicinal gelatin.

The method comprises the following steps: the test group and the control group were placed in dialysis bags, which were placed in 20ml of PBS buffer 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 substantial release of rH-BMP2 in the control material was completed within 168h, and the drug release of the rhBMP-2 loaded gelatin sponge of example 1 could be extended to around 5 weeks, corresponding substantially to the initial phase of production in bone repair; samples 1, 2, 3, 4, and 5 were also able to release for 4-5 weeks, but the drug release of example 1 was more stable.

Claims

1. A bone repair material using gelatin sponge to carry rhBMP-2 comprises bone morphogenetic protein rhBMP-2, a drug carrier gelatin sponge and other auxiliary materials, and is characterized in that the gelatin sponge is prepared from gelatin and a cross-linking agent.

2. The bone repair material of claim 1, wherein the other excipients are tris buffer, trehalose, bovine serum albumin, and PEG 6000.

3. The bone repair material according to claim 1, wherein the cross-linking agent is one or more selected from the group consisting of carbodiimide, hydroxysuccinimide, and hexamethylene-1, 6-diisocyanate.

4. The bone repair material of claim 2, which comprises, by weight, 0.1-1% of rhBMP-2 lyophilized powder, 0.5-1.5% of tris buffer, 1-2% of trehalose, 0.5-2% of bovine serum albumin, 0.5-3% of cross-linking agent, 1-5% of gelatin, 0.5-2% of PEG6000, and the balance water.

5. The bone repair material carrying rhBMP-2 with gelatin sponge according to claim 4, wherein the cross-linking agent is carbodiimide and 1, 6-hexamethylene diisocyanate in a weight ratio of 1-2: 1, mixing; firstly adding carbodiimide into gelatin-PEG solution, adding bone morphogenetic protein rhBMP-2 and other auxiliary materials, and then adding 1, 6-hexamethylene diisocyanate.

6. The method for preparing a bone repair material carrying rhBMP-2 with gelatin sponge according to any one of claims 1 to 5, comprising the steps of:

(1) preparing gelatin-PEG 6000 solution;

(4) the colloid obtained by crosslinking is sequentially added with 0.1mol/L Na₂HPO₄And 4mol/L NaCl and deionized water, and then placing the mixture into a freeze dryer for freeze drying to obtain the rhBMP-2-loaded gelatin sponge.

7. The method for preparing a bone repair material carrying rhBMP-2 with gelatin sponge according to claim 6, wherein the gelatin concentration in the gelatin-PEG 6000 solution is 3.0%, and the PEG6000 concentration is 1.0%.

8. The method for preparing a bone repair material carrying rhBMP-2 with gelatin sponge as claimed in claim 6, wherein in the step (2), carbodiimide is added to the gelatin-PEG solution to a concentration of 0.75%, and the temperature is controlled to 37 ℃ to crosslink without solidification; adding 1, 6-hexamethylene diisocyanate in the step (3) until the concentration is 0.5%, stirring, and crosslinking for 24 hours at 25 ℃.

9. The method for preparing a bone repair material carrying rhBMP-2 by using gelatin sponge according to claim 6, wherein the parameters of the freeze-drying process are as follows: