CN110522946B

CN110522946B - rhBMP-2-loaded bone repair material microsphere and preparation method thereof

Info

Publication number: CN110522946B
Application number: CN201910896495.3A
Authority: CN
Inventors: 陈春; 曹建新
Original assignee: Zhejiang Ruigu Biotechnology Co ltd
Current assignee: Zhejiang Ruigu Biotechnology Co ltd
Priority date: 2019-09-20
Filing date: 2019-09-20
Publication date: 2021-11-30
Anticipated expiration: 2039-09-20
Also published as: CN110522946A

Abstract

The invention belongs to the field of biomedical materials containing active protein and the field of regenerative medicine. A bone repair material comprises rhBMP-2 (recombinant bone morphogenetic protein-2) and poly (lactide-co-glycolide) (PLGA) microspheres, wherein the rhBMP-2 is wrapped in the poly (lactide-co-glycolide) microspheres, and the surfaces of the microspheres are modified by mussel mucin.

Description

rhBMP-2-loaded bone repair material microsphere 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 rhBMP-2-loaded bone repair material microsphere 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.

The poly (glycolide-co-lactide) (hereinafter PLGA) microspheres are one of the most widely used and studied drug carrier materials due to their good biocompatibility and biodegradability, and have been approved by the FDA in the united states as a raw material for implantable medical devices.

Mussel Adhesive Protein (MAP) is a protein complex secreted by the foot secretory gland of mussels, and the super-strong viscosity of the mussels is the key to firmly attach the mussels to reefs and to resist the impact of sea waves. Mussel mucin has the advantages of wide adhesion range, high strength, good water resistance, good biocompatibility and degradability, etc., and is considered to be a good biological adhesive. The BMP-2 is loaded on the mussel mucin adhesive, and the strong adhesive property and the BMP-2 bone growth promoting function of the mussel mucin adhesive are utilized to treat diseases such as fracture, especially the condition of difficult fixation such as comminuted fracture, and the like, and the mussel mucin adhesive is a promising medicament.

Disclosure of Invention

The invention aims to provide a bone repair material with good biocompatibility and biodegradability, sustained release and targeted controlled release. The invention also aims to provide a preparation method of the bone repair material.

A bone repair material comprises rhBMP-2 (recombinant bone morphogenetic protein-2) and poly (lactide-co-glycolide) (PLGA) microspheres, wherein the rhBMP-2 is wrapped in the poly (lactide-co-glycolide) microspheres, and the surfaces of the microspheres are modified by mussel mucin.

A preparation method of a bone repair material comprises the following steps:

(1) preparing PLGA microspheres loaded with rhBMP-2; (2) polymerizing mussel mucin onto the PLGA microspheres loaded with rhBMP-2.

Further, step (1): dissolving rhBMP-2 freeze-dried powder in an acetic acid buffer solution, adding acetonitrile containing PLGA, emulsifying at the rotating speed of 3000rpm for 30 seconds, adding the mixture into a mixed emulsion of liquid paraffin and Tween-80, stirring at the rotating speed of about 300rpm for 2-4 hours, standing for 30 minutes after completion, alternately washing with petroleum ether and isopropanol, and freeze-drying to obtain the dried microspheres.

Further, suspending the PLGA microspheres loaded with the rhBMP-2 in an acetic acid aqueous solution, taking the mussel mucin solution, adjusting the pH value to 6.0, slowly stirring at 2-8 ℃, adding the PLGA microsphere suspension into the mussel mucin solution, uniformly dispersing the PLGA microsphere suspension into the mussel mucin solution, slowly stirring for 3-4 hours, standing for more than 12 hours, alternately washing the obtained rhBMP-2-loaded mussel mucin-PLGA microspheres with petroleum ether and isopropanol, and freeze-drying to obtain the dry PLGA microspheres with surfaces modified by the mussel mucin.

Further, the mass ratio of the mussel mucin to the PLGA microspheres is 1: 80-120.

The PLGA microspheres which are modified by the adhesion of the surfaces of mussel mucins and loaded with rhBMP-2 are taken as main raw materials, have the functions of slow release, targeted controlled release of the rhBMP-2 and directional adsorption (nerve, blood vessel, cell and the like) of tissue repair factors for promoting bone growth, and also have the function of self-adhesion of broken bone pieces, thereby being an ideal bone tissue repair product.

The invention wraps rhBMP-2 with PLGA microspheres with good biocompatibility and biodegradation, and the surfaces of the PLGA microspheres are modified by mussel adhesive, so that the PLGA microspheres are used for surface modification of the PLGA microspheres by utilizing the surface cross-linking and adsorption characteristics of mussel mucin. The PLGA microspheres adsorbed with the mussel mucin molecules form microprotrusions on the surfaces, and due to the force effect between protein molecules and the interaction between the microspheres, the microspheres can be gathered and form a loose and porous structure, and the adsorption and the penetration growth of growth factors of nerves and blood vessels and other cells can be promoted; and because the surface adsorbs mucin, the chance of degradation reaction is reduced, and the problems of PLGA burst release and sustained and controlled release of the drug are well solved.

The rhBMP-2PLGA microspheres retain the dual functions of adhesion and adhesion of the mussel mucin due to the directional adsorption of the mussel mucin on the surfaces of the microspheres, so that the bone tissue repair material can be directionally adhered to the port needing repair by the directional adsorption, and the materials and effective components are prevented from permeating into other places along with blood flow and tissues to cause ectopic osteogenesis; meanwhile, nerves, blood vessels, mesenchymal cells and the like around the fractured bone port can be adsorbed to the surface of the fractured bone, so that the repair of the fractured bone is better promoted.

Drawings

FIG. 1: a cell electron micrograph; wherein, A is a microsphere group carrying rhBMP-2 PLGA; b is mussel mucin modified rhBMP-2-loaded PLGA microsphere group;

FIG. 2: an ectopic osteogenesis experiment X-ray film in a mouse body; a, B, C is a test group; d is a control group; e is blank control.

Detailed Description

Example 1: preparation of bone repair material microspheres

Natural mussel mucin extract product: BD Bioscience, designated Mytilus edulis products, containing Mefp-2, Mefp-3;

BMP-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 applicant's CN201910030649.0 Chinese patent application, and the antibiotic residue <0.1ppm is detected by a bacteriostatic loop method;

the polyglycolide is produced by DURECT corporation, usa.

The preparation method comprises the following steps:

(1) preparation of rhBMP-2 loaded PLGA microspheres

The preparation method comprises the steps of preparing the rhBMP-2-loaded PLGA microspheres by adopting a W/O/W method, dissolving rhBMP-2 freeze-dried powder in an acetic acid buffer solution, adding acetonitrile containing PLGA, emulsifying at the rotating speed of 3000rpm for 30 seconds, adding the mixture into mixed emulsion of liquid paraffin and Tween-80, stirring at the rotating speed of about 300rpm for 2-4 hours, standing for 30 minutes after completion, washing with petroleum ether and isopropanol alternately, and freeze-drying to obtain the dried rhBMP-2-loaded PLGA microspheres.

(2) Carrying out microsphere surface modification by using mussel mucin;

suspending the microspheres prepared in the above step in an acetic acid aqueous solution, adjusting the pH of a mussel mucin solution to 6.0, slowly stirring at 2-8 ℃, adding a PLGA microsphere suspension into the mussel mucin solution, uniformly dispersing the PLGA microsphere suspension in the mussel mucin solution (the mussel mucin: PLGA microspheres are 1:100), slowly stirring for 3-4 hours, standing for more than 12 hours, alternately washing the obtained rhBMP-2 mussel mucin-PLGA microsphere microspheres with petroleum ether and isopropanol, and freeze-drying to obtain a new dried mussel mucin-containing microsphere.

The PLGA microspheres loaded with rhBMP-2 obtained in this example utilize the property of mussel mucin surface cross-linking adsorption to modify the surface of the PLGA microspheres, and the PLGA microspheres loaded with mussel mucin molecules form microprotrusions on the surface, and due to the force between protein molecules and the interaction between microspheres, the microspheres can be aggregated to form a loose porous structure, which can promote the adsorption and growth of nerve and blood vessel growth factors and other cells; and because the surface adsorbs mucin, the opportunity of degradation reaction is reduced, the biodegradable property is good, the problem of burst release of the existing PLGA drug-loaded microspheres is solved, and the time for gradually releasing the rhBMP is basically consistent with the time for repairing the bone tissue.

The rhBMP-2PLGA microspheres retain the dual functions of adhesion and adhesion of the mussel mucin due to the directional adsorption of the mussel mucin on the surfaces of the microspheres, so that the bone tissue repair material can be directionally adhered to the port needing repair by the directional adsorption, and the materials and effective components are prevented from permeating into other places along with blood flow and tissues to cause ectopic osteogenesis; meanwhile, nerves, blood vessels, mesenchymal cells and the like around the fractured bone port can be adsorbed to the surface of the fractured bone, so that the repair of the fractured bone is better promoted

Test 1: the cell adherence test is used for verifying the adsorption performance of the new microsphere material on bone growth promoting factors such as nerves, blood vessels, mesenchymal cells and the like;

the method comprises the following steps: adding 1.0 ml/hole of suspension containing the rhBMP-2PLGA microspheres obtained in the step (1) of the example 1 and suspension containing the mussel mucin surface modified rhBMP-2PLGA microspheres obtained in the step (2) into 2 culture plates with 6 holes respectively, drying at 55 ℃ to enable the microspheres to be attached to the bottoms of the culture plates, and adding water into the culture plates to wash for 3 times for later use. The cell culture adopts mouse epidermal fibroblasts, removes cells in logarithmic growth phase, respectively adds the cells into prepared 2 6-hole cell culture plates, cultures for 2h at 37 ℃, and observes the growth condition of the cells.

The results are shown in FIG. 1: after 2 hours of culture, the cell anchorage rate of the rhBMP-2PLGA microsphere group carried by the graph 1A is 35%, and the cell anchorage rate of the mussel mucin-modified rhBMP-2PLGA microsphere group carried by the graph 2B is 85%.

Test 2: in vitro release experiment of rhBMP-2-loaded PLGA microsphere bone repair material

Test groups: dividing the PLGA microsphere bone repair material carrying the rhBMP-2 obtained in the embodiment into 3 groups of test groups;

control group: the rhBMP-2 bone repair material produced by Hangzhou Jiuyuan genetic engineering Co., Ltd is named bone induction and consists of rhBMP-2 and carrier materials, wherein the carrier materials comprise hydroxyapatite, soybean phospholipid and medicinal gelatin.

The method comprises the following steps: the 3 test groups and 1 control group were placed in dialysis bags 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 rH-BMP2 in the contrast material is basically released within 168h, and the drug release time of the bone repair material microsphere carrying the rhBMP-2 is prolonged to about 5 weeks, which basically corresponds to the growth initial stage in the bone repair. Test 3: 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;

test groups: the rhBMP-2-loaded PLGA microsphere bone repair material is provided by Zhejiang Ruigao Biotechnology Co., Ltd;

Thirdly, the method comprises the following steps:

10 normal ICR or KM male mice were randomly divided into 5 groups of 2 mice each; of these, test 3, control 1, and blank control 1 groups.

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
						Test group 1	++++	++++	++++	++++	++++
Test group 2	+++	+++	++++	++++	+++
						Test group 3	++++	+++	++++	++++	++++
Control group	++	++	++	++	++
						Blank control	+	+	+	+	+

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

Numbering	Bone formation	Bone connection	Bone shaping
				Test group 1	4	4	4
Test group 2	4	3	4
				Test group 3	4	3	4
Control group	2	3	2
				Blank control	1	2	1

As shown in the figure 2, an in-vivo ectopic osteogenesis experiment X-ray film of a mouse shows that the rhBMP-2-loaded PLGA microsphere bone repair material prepared by the invention is used for a mouse muscle bag osteogenesis experiment, new bone growth can be seen 1 month after injection, and the rhBMP-2-loaded PLGA microsphere bone repair material has good osteogenesis performance.

Claims

1. The rhBMP-2-loaded bone repair material microsphere is characterized by comprising rhBMP-2 (recombinant bone morphogenetic protein-2) and poly (lactide-co-glycolide) (PLGA) microspheres, wherein the rhBMP-2 is wrapped in the poly (lactide-co-glycolide) microspheres, the surfaces of the microspheres are modified by mussel mucin, and the preparation method comprises the following steps: (1) dissolving rhBMP-2 freeze-dried powder in an acetic acid buffer solution, adding acetonitrile containing PLGA, emulsifying at the rotating speed of 3000rpm for 30 seconds, adding the mixture into a mixed emulsion of liquid paraffin and Tween-80, stirring at the rotating speed of about 300rpm for 2-4 hours, standing for 30 minutes after completion, alternately washing with petroleum ether and isopropanol, and freeze-drying to obtain dried microspheres; (2) suspending the PLGA microspheres loaded with the rhBMP-2 in an acetic acid aqueous solution, taking a mussel mucin solution, adjusting the pH value to 6.0, slowly stirring at 2-8 ℃, adding the PLGA microsphere suspension into the solution, uniformly dispersing the PLGA microsphere suspension into the mussel mucin solution, slowly stirring for 3-4 hours, standing for more than 12 hours, alternately washing the obtained rhBMP-2-loaded mussel mucin-PLGA microspheres with petroleum ether and isopropanol, and freeze-drying to obtain the dry PLGA microspheres with surfaces modified by mussel mucin.

2. The microspheres of claim 1, wherein the mass ratio of the mussel mucin to the PLGA microspheres is 1: 80-120.