Bone repair material as well as preparation method and application thereof

A bone repair and reaction technology, applied in the field of regenerative medicine, can solve problems such as difficulty in achieving balance, poor mechanical properties, and fast degradation, and achieve the effect of reducing allergies and improving degradation/release time

Active Publication Date: 2019-03-15
ZHEJIANG RISING BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Chitosan, hyaluronic acid, alginic acid, cellulose and other natural polymer materials have become good carriers of BMP-2 due to their good biocompatibility and degradation characteristics, but they have poor mechanical properties and poor mechanical properties when used as bone repair materials. The problem of fast degradation, it is difficult to achieve a good balance even with other materials

Method used

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  • Bone repair material as well as preparation method and application thereof
  • Bone repair material as well as preparation method and application thereof
  • Bone repair material as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0043] The preparation of embodiment 1BMP-2

[0044] In order to facilitate the experiment process and quality control, the BMP-2 used in the experiment was prepared by the applicant:

[0045] According to the rH-BMP2 amino acid and gene sequence in Genbank, the rH-BMP2 gene sequence suitable for expression in Escherichia coli was constructed, and its nucleotide sequence (including partial restriction site) was SEQ ID NO.1 in the sequence table; Insert the gene fragment into the pET plasmid vector after digestion, and transform the E. coli host after ligation; screen the positive clones and induce the expression of rH-BMP2; ferment, break up the bacteria, collect the inclusion bodies, lyse the inclusion bodies and refold the protein; anion and cation exchange layer The rH-BMP2 was obtained by analysis and purification, and electrophoresis showed a single band of about 12kDa (consistent with the reported rH-BMP2), and the amino acid sequence of the product was SEQID NO.2 in the...

Embodiment 2

[0047] The preparation of embodiment 2 modified chitosan

[0048] Chitosan modified with allyltrimethylammonium chloride:

[0049] Dissolve 3g of chitosan in 250ml of 5% (volume) acetic acid solution; under the protection of argon, heat to 90°C, add 6ml of 0.08mol / L cerium nitrate, and react for 30min; add 9ml of 50% (mass) allyl trimethyl Ammonium chloride aqueous solution, reacted for 2h; cooling, ethanol precipitation, washing, suction filtration, and vacuum drying to obtain allyltrimethylammonium chloride modified chitosan.

[0050] Product infrared spectrum detection shows 1415cm -1 (-NH), 1555cm -1 The characteristic absorption peak of (C=O) shows that allyltrimethylammonium chloride has been grafted on chitosan.

[0051] Chitosan modified with acrylamide:

[0052] Dissolve 3g chitosan solution in 250ml 5% (volume) acetic acid solution; under the protection of argon, heat to 50°C, add 5ml0.06mol / L cerium nitrate, react for 30min; add 8g acrylamide, react for 2h; cool...

Embodiment 3

[0054] Preparation of embodiment 3 bone repair material and its basic properties

[0055] Gelatin, allyltrimethylammonium chloride or acrylamide-modified chitosan, and rH-BMP2 polypeptide components (mass ratio 1000:1000:100) of the bone repair material were stored in cold storage at 4°C when not in use.

[0056] The gelatin is dissolved in ultrapure water, the modified chitosan is added into the gelatin solution, and ground evenly; rH-BMP2 is added, mixed evenly; filled in a mold, sterilized by irradiation after vacuum freeze-drying, and the bone repair material is obtained.

[0057] The CCK-8 cell proliferation toxicity test conducted according to the kit instructions shows that the cytotoxicity of the material is CTSO-I grade, which preliminarily confirms its safety.

[0058] A control bone repair material was also prepared, in which chitosan was used instead of modified chitosan, and other processes and components remained unchanged.

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Abstract

The invention belongs to the field of active protein-containing biomedical materials and the field of regenerative medicine and particularly provides a bone repair material. The bone repair material contains modified chitosan, gelatin and rH-BMP2 (bone morphogenetic protein-2). The material can be used for repairing bone injury. The bone repair material is prepared from the following components inpercentage by mass: 1%-10% of rH-BMP2, 20%-70% of modified chitosan and 20%-70% of gelatin. A tremendous advance is made in degradation / drug release time by using allyltrimethylammonium chloride or acrylamide modified chitosan and gelatin as sustained release carriers, and allergy is reduced substantially. The material has the effects obviously better than those of chitosan and the existing similar materials in in-vitro release tests and animal tests, and safety of the material is proved preliminarily by cytotoxicity tests.

Description

technical field [0001] The invention belongs to the field of biomedical materials containing active protein and the field of regenerative medicine. Specifically, the application provides a bone repair material, which includes modified chitosan, gelatin and rH-BMP2. The material can be used for repairing bone damage. Background technique [0002] Bone morphogenetic protein-2 (bone morphogenetic protein-2, BMP-2) is a key factor for promoting bone formation discovered by Marshall R. Urist in 1965 when conducting osteogenesis experiments. BMP-2 belongs to the TGF-α family, which is secreted by osteoblasts and acts on osteoblasts. BMP-2 is the main signaling molecule for cell differentiation into mineral deposition osteoblasts and plays a role in inducing osteoblast differentiation. . It is expressed during limb growth, endochondral ossification, and fracture, and plays an important role in bone growth and development, regeneration and repair. Since BMP-2 has a significant ef...

Claims

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Application Information

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IPC IPC(8): A61L27/26A61L27/54A61L27/58C08B37/08
CPCA61L27/26A61L27/54A61L27/58A61L2300/252A61L2300/412A61L2300/604C08B37/003C08L89/00C08L5/08
Inventor 曹建新
Owner ZHEJIANG RISING BIOTECH CO LTD
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