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Bone repair material containing rH-BMP2 and phosphate, and preparation method and applications thereof

A bone repair and phosphate technology, applied in the field of regenerative medicine, can solve problems such as difficulty in achieving balance, poor mechanical properties, and rapid degradation, and achieve the effect of reducing allergies

Active Publication Date: 2019-01-29
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 containing rH-BMP2 and phosphate, and preparation method and applications thereof
  • Bone repair material containing rH-BMP2 and phosphate, and preparation method and applications thereof
  • Bone repair material containing rH-BMP2 and phosphate, and preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0054] Example 1 Preparation of BMP-2

[0055] In order to facilitate the experiment process and quality control, the BMP-2 used in the experiment was prepared by the applicant:

[0056] According to the rH-BMP2 amino acid and gene sequence in Genbank, construct the rH-BMP2 gene sequence suitable for expression in E. coli. Its nucleotide sequence (including partial restriction sites) is SEQ ID NO. 1 in the sequence table; Gene fragments were digested and inserted into pET plasmid vector, ligated and transformed into E. coli host; positive clones were screened, and rH-BMP2 expression was induced; fermentation, broken 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). The amino acid sequence of the product is SEQ ID NO. 2 in the sequence table.

[0057] The detection ...

Embodiment 2

[0058] Example 2 Preparation of modified chitosan

[0059] Modified chitosan with allyltrimethylammonium chloride:

[0060] Dissolve 3g of chitosan in 250ml of 5% (volume) acetic acid solution; under the protection of argon, heat to 90℃, add 6ml of 0.08mol / L cerium nitrate, and react for 30min; add 9ml of 50% (mass) allyltrimethyl Ammonium chloride aqueous solution, reaction for 2h; cooling, ethanol precipitation, washing, suction filtration, vacuum drying to obtain allyltrimethylammonium chloride modified chitosan.

[0061] Product infrared spectrum detection shows 1415cm -1 (-NH), 1555cm -1 The characteristic absorption peak of (C=O) indicates that allyltrimethylammonium chloride has been grafted onto chitosan.

[0062] Modified chitosan with acrylamide:

[0063] Dissolve 3g of chitosan solution in 250ml of 5% (volume) acetic acid solution; under the protection of argon, heat to 50℃, add 5ml of 0.06mol / L cerium nitrate, and react for 30min; add 8g of acrylamide and react for 2h; cool...

Embodiment 3

[0065] Example 3 Preparation and basic properties of bone repair materials

[0066] Bone repair materials, including tetracalcium phosphate, tetracalcium phosphate, allyltrimethylammonium chloride or acrylamide-modified chitosan, and rH-BMP2 polypeptide ingredients should be used as ingredients. When not in use, they should be refrigerated at 4°C. save.

[0067]

[0068] The aforementioned bone repair material method includes the following steps:

[0069] 1) Grind tetracalcium phosphate and calcium hydrogen phosphate uniformly, mix, fill, and sterilize by irradiation to make A bottle;

[0070] 2) Mix chitosan and rH-BMP2, mix, fill, and irradiate to sterilize bottle B;

[0071] 3) Prepare disodium hydrogen phosphate and sodium dihydrogen phosphate into a 5%-15% aqueous solution, fill, and sterilize with high temperature steam to make C bottle;

[0072] 4) Mix bottles A, B, and C in a sterile container before use, and knead them into the required shape to obtain the bone repair material....

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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 present invention belongs to the field of biomedical materials containing active proteins and the field of regenerative medicine. Specifically, the present application provides a bone repair material containing rH-BMP2 and phosphate, including modified chitosan, phosphate and rH- BMP2. The material can be used to repair bone injuries. Background technique [0002] Bone morphogenetic protein-2 (BMP-2) is a key factor that promotes bone formation discovered in 1965 by Marshall R. Urist in an osteogenic experiment. BMP-2 belongs to the TGF-α family, which is secreted by osteoblasts and acts on osteoblasts. BMP-2 is the main signal molecule for cell differentiation into mineral deposit osteoblasts, which can induce osteoblast differentiation. . It is expressed during limb growth, endochondral ossification and fracture, and plays an important role in bone growth, development and regeneration. Because BMP-2 has a significant effect of stimulating new bone...

Claims

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Application Information

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Patent Type & Authority Applications(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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