A kind of bone repair material containing rh-bmp2 and phosphate and its preparation method and application

A bone repair and phosphate technology, applied in the field of regenerative medicine, can solve the problems of difficult to achieve balance, fast degradation, poor mechanical properties, etc., and achieve the effect of reducing allergies

Active Publication Date: 2020-07-14
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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  • A kind of bone repair material containing rh-bmp2 and phosphate and its preparation method and application
  • A kind of bone repair material containing rh-bmp2 and phosphate and its preparation method and application
  • A kind of bone repair material containing rh-bmp2 and phosphate and its preparation method and application

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preparation example Construction

[0054] The preparation of embodiment 1 BMP-2 is for the convenience of experiment process and quality control, and the BMP-2 used in the experiment is obtained by applicant's self-preparation:

[0055] 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; The gene fragment was digested and inserted into pET plasmid vector, and then transformed into E. coli host after ligation; positive clones were screened, and the expression of rH-BMP2 was induced; fermentation, breaking up the bacteria, collection of inclusion bodies, inclusion body lysis and protein renaturation; 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 th...

Embodiment 2

[0057] Preparation of embodiment 2 modified chitosan Modified chitosan with allyltrimethylammonium chloride:

[0058] Dissolve 3g chitosan in 250ml 5% (volume) acetic acid solution; under argon protection, heat to 90°C, add 6ml 0.08mol / L cerium nitrate, react for 30min; add 9ml 50% (mass) allyltrimethyl Ammonium chloride aqueous solution, reacted for 2h; cooling, ethanol precipitation, washing, suction filtration, and vacuum drying to obtain allyltrimethylammonium chloride modified chitosan.

[0059] 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.

[0060] Chitosan modified with acrylamide:

[0061] Dissolve 3g chitosan solution in 250ml 5% (volume) acetic acid solution; under argon protection, heat to 50°C, add 5ml0.06mol / L cerium nitrate, react for 30min; add 8g acrylamide, react for 2h; cool, add Adjust the pH to 10 with sodium hydroxid...

Embodiment 3

[0063] Preparation of embodiment 3 bone repair material and its basic properties

[0064] Tetracalcium phosphate, tetracalcium phosphate, allyltrimethylammonium chloride or acrylamide-modified chitosan, and rH-BMP2 polypeptide ingredients of bone repair materials are used for batching, and refrigerated at 4°C when not in use save.

[0065]

[0066] The method for above-mentioned bone repair material comprises the following steps:

[0067] 1) Grinding tetracalcium phosphate and calcium hydrogen phosphate evenly, mixing, filling, and irradiating to make bottle A;

[0068] 2) mixing chitosan and rH-BMP2, filling, irradiating and sterilizing to make bottle B;

[0069] 3) Disodium hydrogen phosphate and sodium dihydrogen phosphate are configured into a 5%-15% aqueous solution, filled, and sterilized by high-temperature steam to make a C bottle;

[0070] 4) Before use, mix bottles A, B, and C in a sterile container and knead them into desired shapes to obtain bone repair mater...

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Abstract

The invention belongs to the field of biomedical materials containing active proteins and the field of regenerative medicine, and specifically provides a bone repair material containing rH-BMP2 and phosphate, wherein the bone repair material contains modified chitosan, phosphate and rH-BMP2, can be used for the repair of bone injury, and specifically comprises, by mass, 1-10% of rH-BMP2, 10-50% ofchitosan, 10-50% of tetracalcium phosphate, 5-20% of calcium hydrogen phosphate, 0.2-5% of disodium hydrogen phosphate, and 0.2-5% of sodium dihydrogen phosphate. According to the present invention,the allyltrimethylammonium chloride or acrylamide modified chitosan and the gelatin are used as the sustained-release carrier so as to substantially improve the degradation / drug release time and significantly reduce the allergicity; the results of in vitro release experiments and animal experiments show that the bone repair material of the present invention has good effects significantly superiorto chitosan and existing similar materials; and the results of cytotoxicity test initially prove the safety of the bone repair material of the present invention.

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 containing rH-BMP2 and phosphate, including modified chitosan, phosphate 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. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61L27/46A61L27/54A61K38/18A61K47/36A61P19/08
CPCA61K38/1875A61K47/36A61L27/46A61L27/54A61L2300/252A61L2300/412A61L2300/604A61L2430/02A61P19/08C08L89/00C08L5/08
Inventor 曹建新
Owner ZHEJIANG RISING BIOTECH CO LTD
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