CN109316629A - A kind of injected bone repairing material and its preparation method and application - Google Patents

A kind of injected bone repairing material and its preparation method and application Download PDF

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CN109316629A
CN109316629A CN201811443147.2A CN201811443147A CN109316629A CN 109316629 A CN109316629 A CN 109316629A CN 201811443147 A CN201811443147 A CN 201811443147A CN 109316629 A CN109316629 A CN 109316629A
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chitosan
bone
phosphate
bmp2
injected
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CN109316629B (en
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曹建新
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Zhejiang Ruigu Biotechnology Co Ltd
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Zhejiang Ruigu Biotechnology Co Ltd
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/222—Gelatin
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
    • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
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    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
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    • A61L2430/24—Materials or treatment for tissue regeneration for joint reconstruction
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Abstract

The invention belongs to field of biomedical materials and regenerative medicine field containing activated protein, and specifically, this application provides a kind of injected bone repairing materials, include modified chitosan, phosphate and rH-BMP2.The material can be used for the reparation of bone injury.A kind of injected bone repairing material, by mass percentage, the bone renovating material include ingredient below: rH-BMP2:1%-10%；Chitosan: 10%-40%；Tetracalcium phosphate: 10%-50%；Calcium monohydrogen phosphate: 5%-15%；Gelatin: 5%-10%；Disodium hydrogen phosphate: 0.2%-5；Sodium dihydrogen phosphate 0.2%-5%.The present invention realizes huge progress using allyl trimethyl ammonium chloride or acrylic amide modified chitosan and gelatin as slow-released carrier on degradation/drug release time, and significantly reduces anaphylaxis.

Description

A kind of injected bone repairing material and its preparation method and application
Technical field
The invention belongs to field of biomedical materials and regenerative medicine field containing activated protein, and specifically, the application mentions A kind of injected bone repairing material has been supplied, modified chitosan, phosphate and rH-BMP2 are included.The material can be used for bone damage The reparation of wound.
Background technique
Bone morphogenesis protein-2 (bone morphogenetic protein-2, BMP-2) be nineteen sixty-five by Marshall R.Urist carries out the key factor of the promotion ostosis of discovery when heterotopic Osteogenesis.BMP-2 belongs to TGF- α family, It is secreted by osteoblast, and acts on osteoblast, and BMP-2 is the main letter that cell differentiation metallogenic material deposits osteoblast Number molecule, plays the role of induced osteogenesis cell differentiation.It is expressed in limbs growth, endochondral ossification, fracture, in bone growth Development and Regeneration and Repair play an important role.It is considered as treatment bone since BMP-2 plays the role of significantly stimulating new bone formation The promising active constituent of the diseases such as folding, bone defect is also used as bone shifting when spinal fusion and joint fusion The substitution medical scheme of plant (such application has already passed through FDA approval).
The major defect of BMP-2 is its half-life short, after intravenous injectiont1/2Only 6-7min, and be easy because new old Metabolic process, physiological environment variation, or with the effect of enzyme and be denaturalized.Even if therefore the administration routes large dosage such as injection, oral is held Continuous administration also tends to the concentration that cannot be supplied to target area continuous and effective, for the bone for needing to grow and restore for a long time Good therapeutic effect can not be provided, and easily cause low patient's skeletonization quality, inflammatory reaction and other potential risks. Therefore, suitable delivering mode is selected, such as it is to realize that BMP-2 is widely applied, and improve that BMP-2, which is placed in bone renovating material, The key of its curative effect.
The natural macromolecular materials such as chitosan, hyaluronic acid, alginic acid, cellulose due to its good biocompatibility and Degradation characteristic becomes the good carrier of BMP-2, but there are poor mechanical property and the fast problem of degradation in when it is as bone renovating material, It is difficult to obtain good balance cooperating other materials.
Summary of the invention
Inventor attempts to be modified to solve the above technical problems natural polymer-chitosan.Inventor discovery with Allyl trimethyl ammonium chloride or acrylic amide modified chitosan and gelatin are real on degradation/drug release time as slow-released carrier Show huge progress, and significantly reduces anaphylaxis.It is achieved in release experiment and zoopery in vitro significant excellent In the effect of chitosan and existing similar material.Cytotoxicity experiment also its safety of preliminary proof.
The first purpose of the invention is to provide a kind of injected bone repairing materials, by mass percentage, the Bone Defect Repari Material includes ingredient below:
RH-BMP2:1%-10%；
Chitosan: 10%-40%；
Tetracalcium phosphate: 10%-50%；
Calcium monohydrogen phosphate: 5%-15%；
Gelatin: 5%-10%；
Disodium hydrogen phosphate: 0.2%-5；
Sodium dihydrogen phosphate 0.2%-5%.
Further, by mass percentage, which includes ingredient below:
RH-BMP2:2%-8%；
Chitosan: 20%-35%；
Tetracalcium phosphate: 20%-40%；
Calcium monohydrogen phosphate: 8%-12%；
Gelatin: 5%-10%；
Disodium hydrogen phosphate 0.5%-2.5%；
Sodium dihydrogen phosphate 0.5%-2.5%.
Further, the chitosan use modification of chitosan, modification of chitosan be allyl trimethyl ammonium chloride or Acrylic amide modified chitosan.
Further, wherein modification of chitosan is prepared as follows:
3g chitosan is dissolved in 250ml 5% (volume) acetum；Under argon gas protection, 90 DEG C are heated to, 6ml is added 0.08mol/L cerous nitrate reacts 30min；9ml 50% (quality) allyl trimethyl ammonia chloride aqueous ammonium is added, reacts 2h； Cooling, ethanol precipitation is washed, and is filtered, is dried in vacuo to obtain allyl trimethyl ammonium chloride modification of chitosan.
Further, wherein modification of chitosan is prepared as follows:
3g chitosan solution is dissolved in 250ml 5% (volume) acetum；Under argon gas protection, 50 DEG C are heated to, is added 5ml0.06mol/L cerous nitrate reacts 30min；8g acrylamide is added, reacts 2h；Cooling, adding sodium hydroxide solution adjusts pH To 10, must precipitate, acetone washing precipitating is dried in vacuo to obtain acrylic amide modified chitosan.
Further, rH-BMP2 of the invention, which is used, has the active BMP-2 albumen of bon e formation, protein mutant or egg White active fragment can be extracted from source or be obtained with gene engineering method from the expression of various host's strains.
Further, the amino acid sequence of rH-BMP2 of the invention is SEQ ID NO.2, edits the amino acid sequence Gene order is SEQ ID NO.1.
Further, the amino acid sequence of rH-BMP2 of the invention is SEQ ID NO.4, edits the amino acid sequence Gene order is SEQ ID NO.3.
Chitosan of the invention can come from various sources, and molecular weight can be by those skilled in the art according to reparation portion The needs of position are adjusted in the range of 1 ten thousand to 80 ten thousand, and modifying and decorating voluntarily can advance or buy modified finished product by implementer.
A second object of the present invention is to provide the preparation methods of above-mentioned bone renovating material, method includes the following steps:
1) tetracalcium phosphate and calcium monohydrogen phosphate are ground uniformly, mixing, filling, irradiation sterilization is made A bottles；
2) it by chitosan and rH-BMP2, mixes, filling, irradiation sterilization is made B bottles；
3) disodium hydrogen phosphate and sodium dihydrogen phosphate are configured to 5%-15% aqueous solution, filling, C is made in high-temp steam sterilizing Bottle；
4) A, B, C bottles are mixed before use in sterile chamber, is filled in Bone cement gun, being injected into operative site makes With.
Third object of the present invention is to provide above-mentioned bone renovating materials to repair the application in bone injury.
Further, wherein bone injury is bone defect, bone nonunion, knitting delay.
In addition to bone injury, the composition of the application can be also used for the orthopedic reparation of therapeutic bone, esthetics orthopedic reparation, ridge The filling of the needs such as column fusion, joint fusion and the situation for promoting bone uptake.
The present invention due to the adoption of the above technical solution, with allyl trimethyl ammonium chloride or acrylic amide modified shell Glycan realizes huge progress as slow-released carrier with gelatin on degradation/drug release time, and significantly reduces anaphylaxis. The effect for being significantly better than chitosan and existing similar material is achieved in release experiment and zoopery in vitro.Cytotoxicity Experiment also its safety of preliminary proof.The present invention is also added into gelatin, can promote Bone Defect Repari effect.
Detailed description of the invention
Fig. 1 is that the mouse of experimental group 1 carries out radiological examination figure.
Fig. 2 is that the mouse of experimental group 2 carries out radiological examination figure.
Fig. 3 is that the mouse of experimental group 3 carries out radiological examination figure.
Fig. 4 is that the mouse of control group carries out radiological examination figure.
Specific embodiment
Reagent and instrument:
Tetracalcium phosphate, calcium monohydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate are mentioned by Shanghai Ruibang Biological Material Co., Ltd. For；
Gelatin is provided by Shandong Province Yangxin east Biotechnology Co., Ltd；
(intermediate molecular weight, ten thousand) about 40 are produced chitosan by Shanghai Ai Yan Biotechnology Co., Ltd；
Allyl trimethyl ammonium chloride, acrylamide (analysis is pure) are produced by Shanghai nation at Chemical Co., Ltd.；
Escherichia coli host residual protein detection kit is produced by Shanghai Cheng Shao Biotechnology Co., Ltd；
RH-BMP2ELISA detection kit is produced by Tianjin An Nuoruikang Bioisystech Co., Ltd；
CCK-8 cell Proliferation toxicity detection kit is produced by Dalian U.S. logical sequence Science and Technology Ltd.；
5700 infrared spectrometer of Nicolet is produced by Nicolet；
Moieties biologic operation trust money Si Rui Biotechnology Co., Ltd carries out；
Partial chemical modification operation commission Shanghai KaiTuoZhe Chemical Research Management Co., Ltd carries out；
Some Animals experiment commission Beijing University of Chinese Medicine's scientific experiment center/Experimental Animal Center carries out；
Remaining reagent and instrument are domestic conventional brand and model.
The preparation of 1 BMP-2 of embodiment
To control convenient for experiment process and quality, BMP-2 used in experiment is voluntarily prepared by applicant:
According to the rH-BMP2 amino acid and gene order in Genbank, building is suitble to the rH- in expression in escherichia coli BMP2 gene order, nucleotide sequence (containing partially digested site) is SEQ ID NO.1 in sequence table；By genetic fragment digestion It is inserted into pET plasmid vector afterwards, is ligated and transformed into escherichia coli host；Screening positive clone, induction rH-BMP2 expression；Fermentation is broken Broken thallus collects inclusion body, inclusion body cracking and protein renaturation；Anion and cation-exchange chromatography purifying obtain rH-BMP2, The single 12kDa of electrophoresis showed or so band (consistent with the rH-BMP2 of report), product amino acid sequence are SEQ in sequence table ID NO.2。
(it is lower than with escherichia coli host residual protein detection kit detection escherichia coli host residual protein 0.005%), meet the demand of further experiment with inhibition zone method detection antibiotic residue (being lower than 0.1ppm).
The preparation of 2 modification of chitosan of embodiment
With allyl trimethyl ammonium chloride modification of chitosan:
3g chitosan is dissolved in 250ml 5% (volume) acetum；Under argon gas protection, 90 DEG C are heated to, 6ml is added 0.08mol/L cerous nitrate reacts 30min；9ml 50% (quality) allyl trimethyl ammonia chloride aqueous ammonium is added, reacts 2h； Cooling, ethanol precipitation is washed, and is filtered, is dried in vacuo to obtain allyl trimethyl ammonium chloride modification of chitosan.
Product infrared spectroscopy detection display 1415cm-1(-NH)、1555cm-1(C=O) characteristic absorption peak, shows allyl The scion grafting of base trimethyl ammonium chloride is on chitosan.
With acrylic amide modified chitosan:
3g chitosan solution is dissolved in 250ml 5% (volume) acetum；Under argon gas protection, 50 DEG C are heated to, is added 5ml0.06mol/L cerous nitrate reacts 30min；8g acrylamide is added, reacts 2h；Cooling, adding sodium hydroxide solution adjusts pH To 10, must precipitate, acetone washing precipitating is dried in vacuo to obtain acrylic amide modified chitosan.
Product infrared spectroscopy detection display 1670cm-1(-CONH2)、1415cm-1(-NH)、1574cm-1(-NH2) feature Absorption peak shows acrylamide scion grafting on chitosan.
The preparation and its basic performance of 3 bone renovating material of embodiment
Tetracalcium phosphate, tetracalcium phosphate, allyl trimethyl ammonium chloride or the acrylic amide modified shell of bone renovating material are poly- Sugar and rH-BMP2 polypeptide moiety formula below carry out ingredient, and 4 DEG C of difference is stored refrigerated when being not used.
The method of above-mentioned bone renovating material the following steps are included:
1) tetracalcium phosphate and calcium monohydrogen phosphate are ground uniformly, mixing, filling, irradiation sterilization is made A bottles；
2) it by chitosan and rH-BMP2, mixes, filling, irradiation sterilization is made B bottles；
3) disodium hydrogen phosphate and sodium dihydrogen phosphate are configured to 5%-15% aqueous solution, filling, C is made in high-temp steam sterilizing Bottle；
4) A, B, C bottles are mixed before use in sterile chamber, pinches into required shape to get bone renovating material.
The CCK-8 cell Proliferation toxicity test being carried out according to kit specification shows that the cytotoxicity of the material is CTS 0-I grades, its safety of tentative confirmation.
Another preparation control bone renovating material, according to the formula of example 1, wherein modification of chitosan is substituted using chitosan, other Process and components unchanged.
The rH-BMP2 extracorporeal releasing experiment of 4 material of embodiment
Bone renovating material and control each 3g of bone renovating material (142mg containing rH-BMP2) are prepared according to the method for embodiment 3, It places it in bag filter, bag filter is placed in 20ml containing 0.2% sodium azide, in the PBS buffer solution of pH7.0.It is statically placed in 37 At DEG C, in 12h, for 24 hours, the separately sampled 1ml of 120h, 168h, 336h, 504h, 672h, 840h (then supplement contain 0.2% Azide Sodium, the PBS buffer solution 1ml of pH7.0), rH-BMP2 concentration is measured with ELISA method according to the explanation of kit, Conversion Calculation is tired Meter release percentage.As a result as shown in the table.
Table 1, the accumulative release percentage of rH-BMP2
RH-BMP2 in the control material release substantially in 168h completes that (while chitosan in material and collagen are basic Disappear), and the pharmaceutical release time of modification of chitosan material extends to 672 (4 weeks) Zuo You, substantially with the growth in Bone Defect Repari at the beginning of Phase is corresponding (while chitosan in material and gelatin disappear substantially), micro- no less than many polymer provided in existing literature Ball material, and can to avoid polymer microballoon degradation when acidic environment and allergic problem.
5 chitosan allergic experiment of embodiment
The Hartley hero cavy 30 for choosing weight about 400g, is equally divided into 3 groups.In three groups of difference on the the 1st, 4 of experiment The trimethyl ammonium chloride of allyl containing 2.0mg modification of chitosan, acrylic amide modified chitosan and unmodified chitosan is injected intraperitoneally Aqueous suspensions, experiment the 7th day three groups respectively inject the trimethyl ammonium chloride of allyl containing 3.0mg modification of chitosan, acryloyl The aqueous suspensions of amine modification of chitosan and unmodified chitosan.Testing weighing cavy weight on the 1st, 4,10.In experiment 7-10 days Observe allergic reaction situation, evaluation index includes that allergic reaction is negative (normal), allergic reaction weakly positive (it is restless, tremble, scratch Nose), the allergic reaction positive (sneeze is short of breath, excrement of urinating, sheds tears), allergic reaction strong positive (expiratory dyspnea, wheezing sound, purple Purplish or white patches on the skin, instability of gait, spasm, rotation), allergic reaction is extremely strong positive (death).
2 chitosan allergic experiment of table
The results show that three groups of cavy average weight situations of change are almost the same (to the 10th Average weight increasing a day about 30-35g)；Three Be in group cavy there is strong allergic reaction, but the cavy of unmodified chitosan group higher proportion occur weak allergic symptom (with Prior document result of study is similar), two kinds of modification of chitosan allergic conditions are substantially better than unmodified chitosan, wherein allyl three Ammonio methacrylate modification of chitosan does not observe that allergic conditions shows that two kinds of modification of chitosan also achieve in sensitization completely It is apparent to improve.
The experiment of the practical Bone Defect Repari of embodiment 6
One, it designs:
Grouping comparison, multi-angle assess observation experiment.
Two, material:
Mouse strain is ICR or ICR hero mouse, age of mouse 30 days or so；The bone renovating material of rH-BMP2 and carrier material preparation It is provided by Zhejiang Rui Gu Biotechnology Co., Ltd.
Three, method:
35 normal ICR or KM male mices are randomly divided into 7 groups, every group 3.
After mouse is anaesthetized with 6% yellow Jackets, skin is cut in unhairing disinfection in hind leg thigh, separates spatium intermusculare, implantation is not Same carrier material, and certain antibiotic is added to prevent from infecting, then layer-by-layer suture muscle and skin, sterilize wound, normal to raise It supports.
Four, it observes:
After the completion of heeling-in experiment after 4 weeks, mouse is subjected to radiological examination (X-ray), checks whether mouse hind leg bone occurs Tissue.After 4 weeks progress radiological examinations, mouse is put to death, takes out the bone tissue in heeling-in area, weighs the wet of bone contained by the inside Weight, and carry out the assessment of HE stained tissue.
Five, the standards of grading of X-ray observation knitting
1. 1962 Chai Benfu union measurement standards:
2. Lane-Sandhu X-ray and histological score standard:
Six, result
The mouse of experimental group and control group progress radiological examination is as shown in Figure 1 to 4, scoring such as table 1, shown in table 2.
Table is measured in 1 1962 Chai Benfu union of table
Number Broken ends of fractured bone edge Periosteal reaction Yield of callus Poroma density Poroma edge
Example 1 ++++ ++++ ++++ ++++ +++
Example 2 +++ ++++ ++++ ++++ +++
Example 3 ++++ ++++ ++++ ++++ ++++
Example 4 ++ ++ ++ ++ ++
2. Lane-Sandhu X-ray of table and histological score table
Number Bon e formation Bone connection Bone moulding
Example 1 4 3 4
Example 2 4 3 4
Example 3 4 4 4
Example 4 2 2 2
The above are the descriptions to the embodiment of the present invention to keep this field special by the foregoing description of the disclosed embodiments Industry technical staff can be realized or using the present invention.Various modifications to these embodiments carry out those skilled in the art Saying will be apparent.The general principles defined herein can be the case where not departing from the spirit or scope of the present invention Under, it realizes in other embodiments.Therefore, the present invention is not intended to be limited to these implementation columns shown in this article, but to accord with Close the widest scope consistent with principles disclosed herein and novel point.
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<110>Zhejiang Rui Gu Biotechnology Co., Ltd
<120>a kind of injected bone repairing material and its preparation method and application
<160> 4
<170> SIPOSequenceListing 1.0
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<211> 348
<212> DNA
<213>bone morphogenesis protein-2 (bone morphogenetic protein-2)
<400> 1
atgcaggcga aacacaaaca gcgtaaacgt ctgaaaagca gctgcaaacg tcacccgctg 60
tacgttgatt tcagcgatgt tggctggaac gattggatcg ttgcgccgcc gggctaccac 120
gcgttctact gccacggtga atgcccgttc ccgctggcgg atcacctgaa cagcaccaac 180
cacgcgatcg ttcagaccct ggttaacagc gttaacagca aaatcccgaa agcgtgctgc 240
gttccgaccg aactgtctgc gatcagcatg ctgtaccttg atgaaaacga gaaagtagtg 300
ctgaaaaact atcaagacat ggtggtggaa ggctgtggct gccgttaa 348
<210> 2
<211> 115
<212> PRT
<213>bone morphogenesis protein-2 (bone morphogenetic protein-2)
<400> 2
Met Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys
1 5 10 15
Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp
20 25 30
Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys
35 40 45
Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val
50 55 60
Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys Cys
65 70 75 80
Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn
85 90 95
Glu Lys Val Val Leu Lys Asn Tyr Gln Asp Met Val Val Glu Gly Cys
100 105 110
Gly Cys Arg
115
<210> 3
<211> 348
<212> DNA
<213>bone morphogenesis protein-2 (bone morphogenetic protein-2)
<400> 3
atgggtgcaa aacagaaaca acgtaaacgc ctgaagtcct cttgcaagcg tcacccgctg 60
tacgtggatt tttctgacgt tggttggaac gactggattg tggctcctcc gggctatcac 120
gcattctact gtcacggcga gtgcccgttc ccgctggccg atcatctgaa cagcaccaac 180
cacgcgatcg tccagactct ggttaactct gttaactcca aaatcccgaa agcttgttgt 240
gtgccaaccg aactgtccgc gatcagcatg ctgtacctgg acgaaaatga aaaagtagta 300
ctgaaaaact atcaggatat ggttgttgaa ggctgcggtt gccgttaa 348
<210> 4
<211> 115
<212> PRT
<213>bone morphogenesis protein-2 (bone morphogenetic protein-2)
<400> 4
Met Gly Ala Lys Gln Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys
1 5 10 15
Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp
20 25 30
Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys
35 40 45
Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val
50 55 60
Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys Cys
65 70 75 80
Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn
85 90 95
Glu Lys Val Val Leu Lys Asn Tyr Gln Asp Met Val Val Glu Gly Cys
100 105 110
Gly Cys Arg
115

Claims (10)

1. a kind of injected bone repairing material, which is characterized in that by mass percentage, the bone renovating material is by ingredient below It constitutes:
RH-BMP2:1 %-10 %；
Chitosan: 10 %-40 %；
Tetracalcium phosphate: 10%-50%；
Calcium monohydrogen phosphate: 5%-15%；
Gelatin: 5%-10%；
Disodium hydrogen phosphate: 0.2%-5；
Sodium dihydrogen phosphate 0.2%-5%.
2. a kind of injected bone repairing material according to claim 1, which is characterized in that by mass percentage, the bone Repair materials are made of ingredient below:
RH-BMP2:2%-8 %；
Chitosan: 20 %-35 %；
Tetracalcium phosphate: 20%-40%；
Calcium monohydrogen phosphate: 8%-12%；
Gelatin: 5%-10%；
Disodium hydrogen phosphate 0.5%-2.5%；
Sodium dihydrogen phosphate 0.5%-2.5%.
3. a kind of injected bone repairing material according to claim 1 or 2, which is characterized in that chitosan is using modified shell Glycan, modification of chitosan are allyl trimethyl ammonium chloride or acrylic amide modified chitosan.
4. a kind of injected bone repairing material according to claim 3, which is characterized in that modification of chitosan is by the following method Preparation:
3g chitosan is dissolved in 5% volume acetum of 250ml；Under argon gas protection, 90 DEG C are heated to, 6ml 0.08mol/ is added L cerous nitrate reacts 30min；50% mass allyl trimethyl ammonia chloride aqueous ammonium of 9ml is added, reacts 2h；Cooling, ethyl alcohol is heavy It forms sediment, washs, filter, be dried in vacuo to obtain allyl trimethyl ammonium chloride modification of chitosan；
Alternatively, modification of chitosan is prepared as follows:
3g chitosan solution is dissolved in 5% volume acetum of 250ml；Under argon gas protection, 50 DEG C are heated to, 5ml is added 0.06mol/L cerous nitrate reacts 30min；8g acrylamide is added, reacts 2h；It is cooling, adding sodium hydroxide solution adjust pH to 10, it must precipitate, acetone washing precipitating is dried in vacuo to obtain acrylic amide modified chitosan.
5. a kind of injected bone repairing material according to claim 1 or 2, which is characterized in that rH-BMP2, which is used, has bone Active BMP-2 albumen, protein mutant or protein active fragment are formed, can be extracted from source or with genetic engineering Method is obtained from the expression of various host's strains.
6. a kind of injected bone repairing material according to claim 5, which is characterized in that the amino acid sequence of rH-BMP2 For SEQ ID NO.2, the gene order for editing the amino acid sequence is SEQ ID NO.1.
7. a kind of injected bone repairing material according to claim 5, which is characterized in that the amino of the rH-BMP2 Acid sequence is SEQ ID NO.4, and the gene order for editing the amino acid sequence is SEQ ID NO.3.
8. a kind of method for preparing bone renovating material described in claim 1 ~ 7 any one claim, which is characterized in that should Method the following steps are included:
1) tetracalcium phosphate and calcium monohydrogen phosphate are ground uniformly, mixing, filling, irradiation sterilization is made A bottles；
2) it by chitosan and rH-BMP2, mixes, filling, irradiation sterilization is made B bottles；
Disodium hydrogen phosphate and sodium dihydrogen phosphate are configured to 5%-15% aqueous solution, filling, high-temp steam sterilizing is made C bottles；
4) A, B, C bottles are mixed in sterile chamber before use, it is filled in Bone cement gun, is injected into operative site use.
9. bone renovating material described in claim 1 ~ 7 any one claim repairs answering in bone injury drug in preparation With.
10. application according to claim 9, which is characterized in that bone injury is bone defect, bone nonunion or knitting delay.
CN201811443147.2A 2018-11-29 2018-11-29 Injection type bone repair material and preparation method and application thereof Active CN109316629B (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109692344A (en) * 2019-02-18 2019-04-30 浙江瑞谷生物科技有限公司 One kind bone renovating material containing rH-BMP2 and its preparation method and application
CN110665060A (en) * 2019-10-18 2020-01-10 浙江瑞谷生物科技有限公司 Bone repair material and preparation method and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105749356A (en) * 2016-03-02 2016-07-13 浙江瑞谷生物科技有限公司 Active polysaccharide composite bone repair material
CN108273131A (en) * 2018-03-19 2018-07-13 深圳市中科海世御生物科技有限公司 A kind of composite bone cement, preparation method and applications and a kind of bone renovating material

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105749356A (en) * 2016-03-02 2016-07-13 浙江瑞谷生物科技有限公司 Active polysaccharide composite bone repair material
CN108273131A (en) * 2018-03-19 2018-07-13 深圳市中科海世御生物科技有限公司 A kind of composite bone cement, preparation method and applications and a kind of bone renovating material

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109692344A (en) * 2019-02-18 2019-04-30 浙江瑞谷生物科技有限公司 One kind bone renovating material containing rH-BMP2 and its preparation method and application
CN109692344B (en) * 2019-02-18 2021-04-20 浙江瑞谷生物科技有限公司 rH-BMP 2-containing bone repair material and preparation method and application thereof
CN110665060A (en) * 2019-10-18 2020-01-10 浙江瑞谷生物科技有限公司 Bone repair material and preparation method and application thereof
CN110665060B (en) * 2019-10-18 2021-09-21 浙江瑞谷生物科技有限公司 Bone repair material and preparation method and application thereof

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