CN102475916A - Preparation of bone morphogenetic protein and hydroxyapatite composite dosage form - Google Patents
Preparation of bone morphogenetic protein and hydroxyapatite composite dosage form Download PDFInfo
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- CN102475916A CN102475916A CN2010105534435A CN201010553443A CN102475916A CN 102475916 A CN102475916 A CN 102475916A CN 2010105534435 A CN2010105534435 A CN 2010105534435A CN 201010553443 A CN201010553443 A CN 201010553443A CN 102475916 A CN102475916 A CN 102475916A
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- CN
- China
- Prior art keywords
- bone morphogenetic
- morphogenetic protein
- hydroxyapatite
- carbamide
- dosage form
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 102000007350 Bone Morphogenetic Proteins Human genes 0.000 title claims abstract description 36
- 108010007726 Bone Morphogenetic Proteins Proteins 0.000 title claims abstract description 36
- 229940112869 bone morphogenetic protein Drugs 0.000 title claims abstract description 36
- 229910052588 hydroxylapatite Inorganic materials 0.000 title claims abstract description 31
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 title claims abstract description 31
- 239000002552 dosage form Substances 0.000 title claims description 13
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 239000002131 composite material Substances 0.000 title abstract 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims abstract description 53
- 239000004202 carbamide Substances 0.000 claims abstract description 28
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 8
- 239000000203 mixture Substances 0.000 claims abstract description 6
- 235000013877 carbamide Nutrition 0.000 claims description 25
- 150000001875 compounds Chemical class 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 15
- 239000002244 precipitate Substances 0.000 claims description 8
- 239000008363 phosphate buffer Substances 0.000 claims description 7
- 238000011010 flushing procedure Methods 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 4
- 230000001476 alcoholic effect Effects 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 abstract description 5
- 239000002245 particle Substances 0.000 abstract description 4
- 238000004140 cleaning Methods 0.000 abstract description 2
- 238000001035 drying Methods 0.000 abstract 1
- 238000009472 formulation Methods 0.000 abstract 1
- 239000008055 phosphate buffer solution Substances 0.000 abstract 1
- 238000000926 separation method Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 abstract 1
- 210000000988 bone and bone Anatomy 0.000 description 14
- 230000000694 effects Effects 0.000 description 5
- 238000001179 sorption measurement Methods 0.000 description 5
- 229960000789 guanidine hydrochloride Drugs 0.000 description 4
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 4
- 239000007943 implant Substances 0.000 description 4
- 230000011164 ossification Effects 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 230000008439 repair process Effects 0.000 description 4
- 239000003462 bioceramic Substances 0.000 description 2
- 239000012876 carrier material Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000002980 postoperative effect Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- 229910000391 tricalcium phosphate Inorganic materials 0.000 description 2
- 208000017234 Bone cyst Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 208000028528 solitary bone cyst Diseases 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Materials For Medical Uses (AREA)
Abstract
The invention belongs to the field of biomedical materials. Mainly prepares a bone morphogenetic protein and hydroxyapatite composite preparation. The method of the invention firstly dissolves the bone morphogenetic protein in urea containing phosphate buffer solution, then adds hydroxyapatite in the urea, then adds ethanol to separate the bone morphogenetic protein from the urea solution and attach the bone morphogenetic protein on the surface of hydroxyapatite particles, and obtains the required bone morphogenetic protein and hydroxyapatite composite formulation after centrifugal separation, cleaning and drying.
Description
Technical field
The invention belongs to field of biomedical materials.Relate generally to and produce bone morphogenetic protein and the compound dosage form of hydroxyapatite.
Background technology
Bone morphogenetic protein (bone morphogenefic protein is called for short BMP) is one
Plant the protein in body factor, in body osteanagenesis and repair process, bring into play pivotal role, knochenbruch is connect fast again, lack the quick Regeneration and Repair of bone, in the orthopaedics and the department of stomatology, have extensively and application prospect with unique induced osteogenesis function.
At present, preparation BMP has two kinds of methods: a kind of is to utilize biochemical extraction method directly from beast bone such as cattle, horse, pig, sheep, rabbit, Mus or osteosarcoma, to extract mixed type BMP; Another kind is the people BMP that utilizes the synthetic unitary type of method of gene recombination.Gene recombinaton BMP lures bone or bone active strong, and is higher 10 3-106 times than the BMP of biochemical extraction method preparation.Though it is active that BMP has very strong induced osteogenesis, during implant into body, it is fast to absorb metabolism, and action time is short, is difficult to give full play to its induced osteogenesis effect; , in addition, BMP does not have the support effect when reparation large scale bone is damaged.Therefore, BMP also need be compound with a certain carrier material when clinical practice, just so that himself obtain slow release and support.
Hydroxyapatite (Hydoxyapatite; Be called for short HA) be a kind of of calcium orthophosphate base bioceramic, its chemical formula is Ca10 (P04) 6 (0H) 2, it is the main inanimate matter that constitutes bone and tooth; Behind the implant into body; Can form chemical bonding with bone and soft tissue on its surface, have good biological activity and biocompatibility, be one of ideal carrier material of BMP.BMP and HA is compound, both helped giving full play to of BMP induced osteogenesis effect, the bone repair of hydroxyapatite is strengthened.
In the prior art, " material science and technology encyclopaedia gold book " the 742-74th 3 page reported calcium orthophosphate base bioceramic and bone morphogenetic protein compound material and preparation method thereof.Its preparation method is that BMP is dissolved in the guanidine hydrochloride of 4M, again the porous hydroxyapatite ceramic particle is placed the BMP guanidine hydrochloride solution, then mixture is dialysed with deionized water, eliminates guanidine hydrochloride; Mixture after the dialysis is drained through negative pressure in container, after lyophilizing, uses oxirane disinfection again, and is subsequent use.
This manufacturing approach is not introduced its technological process and corresponding technological parameter in detail, and on the other hand, also there is following weak point in this method: promptly the ionic strength of guanidine hydrochloride is high, is difficult to make.BMP adsorbs in hydroxyapatite surface, causes the BMP adsorption rate low, and during this compound material implant into body, the BMP amount of bringing into is also few, and the good effect of BMP is not in full use.
Summary of the invention
The objective of the invention is to overcome above-mentioned technical problem, a kind of bone morphogenetic protein and compound dosage form of hydroxyapatite produced is provided.
According to above-mentioned purpose, the present invention is that medium are realized the well compound of bone morphogenetic protein and hydroxyapatite with carbamide, and concrete skill step is following:
(1) bone morphogenetic protein is dissolved in the carbamide that contains phosphate buffer, forms the urea liquid that contains bone morphogenetic protein;
The concentration of phosphate buffer is 5-50mM, and PH is PH6-8, and the concentration of carbamide is 5-8M.
The addition of phosphate buffer (volume ratio) is the 1-1 0% of urea amount,
The addition that bone morphogenetic protein adds in the carbamide is: 0.01-O.05mg/ml;
(2) granulous hydroxyapatite is added in the formed urea liquid that contains bone morphogenetic protein of above-mentioned steps, and after stirring under one 30 ℃ of temperature of 2 o, left standstill 0.5-2 hours;
Addition in the adding bone morphogenetic protein urea liquid of hydroxyapatite is: 0.1-1.Og/ml;
Hydroxyapatite is a graininess, and its granularity is 50-500um.
Stirring and leave standstill certain hour after the adding, is to make bone morphogenetic protein as much as possible all be adsorbed on the surface of hydroapatite particles, improves adsorption rate.
(3) ethanol is added in the formed urea liquid that contains hydroxyapatite and bone morphogenetic protein of above-mentioned technology, behind the mix homogeneously, at-60--80 ℃; StorE O.5-2 hour; Adopt centrifugal method subsequently, remove the liquid part in the solution, obtain precipitate.It is the hydroxyapatite of surface adsorption bone morphogenetic protein.Amount of alcohol added be urea liquid 1.5-3 times, adding alcoholic acid purpose is for accelerated bone morphogenetic proteins separating out from urea liquid, adheres to then or is deposited on hydroxyapatite surface, further improves adsorption efficiency,
(4) with above-mentioned through centrifugalize gained precipitate use temperature as 1-0 ℃, concentration is 85% alcoholic solution flushing 2-3 times; The reuse temperature is 1-10 ℃, the alcohol flushing of concentration 100% one time afterwards; And airing; Aseptic preservation promptly becomes the compound dosage form of bone morphogenetic protein of the present invention and hydroxyapatite.
The ethanol of twice usefulness different concentration washes precipitate, is to fall for the carbamide complete flushing that will mix in the precipitate.Remaining then is the compound dosage form of pure bone morphogenetic protein and hydroxyapatite.
Compared with prior art, the present invention has following advantage:
1, behind the compound dosage form implant into body of prepared bone morphogenetic protein and hydroxyapatite, can make bone morphogenetic protein obtain effective slow release, the repair process of bone is accelerated.Experiment shows that postoperative just had a large amount of new bones and cartilage to form in 7-21 days, and visible ripe bone in postoperative 21 backs and bone marrow form.
2, carbamide is low from falling intensity, makes bone morphogenetic protein be easy to be adsorbed on the surface of hydroapatite particles, and promptly the BMP adsorption efficiency is high, and adsorbance is big, makes the effect of BMP be able to give full play to.
The specific embodiment
Adopt manufacturing approach of the present invention, prepared three batches of bone morphogenetic proteins and the compound dosage form of hydroxyapatite.
At first a certain amount of bone morphogenetic protein is dissolved in the carbamide that contains phosphate buffer; Then in above-mentioned urea liquid, add granulous hydroxyapatite; After stirring, leave standstill certain hour at-60--80 ℃; Centrifugalize is partly separated the liquid in the solution, obtains precipitate.Precipitate with gained washes twice with Different concentrations of alcohol at last, and airing, promptly obtains the compound dosage form of bone morphogenetic protein and hydroxyapatite.Adopt prepared BMP of the present invention and the compound dosage form of HA to be used for, after cleaning,, sew up, promptly basic healing after month in this compound dosage form of bone defect filling because the bone that bone cyst causes is damaged.
Claims (1)
1. produce the compound dosage form of bone morphogenetic protein and hydroxyapatite, it is characterized in that this method comprises following processing step:
(1) bone morphogenetic protein is dissolved in the carbamide that contains phosphate buffer, forms and contain the bone morphogenetic protein urea liquid;
The concentration of phosphate buffer is 5-50mM, and PH is PH6-8,
The concentration of carbamide is 4-9M,
The addition of phosphate buffer (volume ratio) is 1-10% of a urea amount,
The addition that bone morphogenetic protein adds in the carbamide is 0.O1-0.05mg/ml;
(2) granulous hydroxyapatite is added in the formed urea liquid that contains bone morphogenetic protein of above-mentioned steps, and under 20-30 ℃ of temperature, stirs, and left standstill 0.5-2 hours:
The addition of hydroxyapatite is carbamide: 0.1-1.Og/ml;
(3) ethanol is added in the formed urea liquid that contains hydroxyapatite and bone morphogenetic protein of above-mentioned technology, behind the mix homogeneously, storE 0.5-2 hours, adopt centrifugal method subsequently, remove the liquid part in the solution, obtain precipitate at-60--80 ℃.The addition that its ethanol adds above-mentioned gained urea liquid is 1.5-3 times (volume ratios) of urea liquid;
(4) with above-mentioned through centrifugalize gained precipitate use temperature for foretell 10 ℃, concentration is 85% alcoholic solution flushing 2-3 times; The reuse temperature is the alcohol flushing one time of 0 ℃ of 1-1, concentration 1 00% afterwards; And airing; Aseptic preservation promptly becomes the compound dosage form of bone morphogenetic protein of the present invention and hydroxyapatite;
(5), the granularity of hydroxyapatite is 50-500 u m.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105534435A CN102475916A (en) | 2010-11-22 | 2010-11-22 | Preparation of bone morphogenetic protein and hydroxyapatite composite dosage form |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105534435A CN102475916A (en) | 2010-11-22 | 2010-11-22 | Preparation of bone morphogenetic protein and hydroxyapatite composite dosage form |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102475916A true CN102475916A (en) | 2012-05-30 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010105534435A Pending CN102475916A (en) | 2010-11-22 | 2010-11-22 | Preparation of bone morphogenetic protein and hydroxyapatite composite dosage form |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102475916A (en) |
-
2010
- 2010-11-22 CN CN2010105534435A patent/CN102475916A/en active Pending
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| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20120530 |