CN106421901A - Bone repairing bioactive material capable of being injected and with slow release property and preparation method thereof - Google Patents
Bone repairing bioactive material capable of being injected and with slow release property and preparation method thereof Download PDFInfo
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- CN106421901A CN106421901A CN201610892818.8A CN201610892818A CN106421901A CN 106421901 A CN106421901 A CN 106421901A CN 201610892818 A CN201610892818 A CN 201610892818A CN 106421901 A CN106421901 A CN 106421901A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/12—Phosphorus-containing materials, e.g. apatite
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- A—HUMAN NECESSITIES
- 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
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
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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
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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Abstract
The invention discloses a bone repairing bioactive material capable of being injected and with a slow release property and a preparation method of the bone repairing bioactive material capable of being injected and with the slow release property. The material comprises a curing matrix and curing liquid, while used clinically, the curing matrix and the curing liquid are compounded in a mixed state according to a ratio of slurry to water of (1: 1)-(1: 0.4), and injected to bone defect focuses through a syringe; each gram of the curing matrix contains 0.5 to 5mg of rhBMP-2, 0.1 to 2.0mg of rhFN, 50 to 200mg of HAP, 750 to 900mg of CSH and 10 to 50mg of CSD which are mixed uniformly, packaged, freeze-dried and then subjected to irradiation sterilization by Co<60>; the curing liquid comprises PBS, HA and SA which are mixed uniformly, filled and freeze-dried and then subjected to irradiation sterilization by Co<60>, and redissolved by using water for injection before use. The bone repairing material disclosed by the invention is directly acted on the focuses and avoids an open surgery to reduce the pain of a user, and further carries out bone conduction and bone induction simultaneously to shorten the healing time.
Description
Technical field
The invention belongs to regenerating tissues engineering and biomaterial technical field, more particularly to a kind of injectable have concurrently slow
Release bone restoration bioactive material of performance and preparation method thereof.
Background technology
The bone grafting material of at present clinical practice mainly has autologous bone, homogeneous allogenic bone and various tissue engineerings and bionical
Learn material.Autologous bone is considered as the goldstandard of bone collection, but because of its operating difficulty and takes distortion at bone, not yet obtains extensive
Concern；And homogeneous allogenic bone affects clinical effectiveness because of biological immune, while being limited by originating；Thus come from organizational project
Artificial bone is of great interest.Preferably artificial bone should possess excellent bone conduction and self-bone grafting is made
With.Most domestic product (as the bone cement of new generation with TCP, HAP, BG as main component) only possesses bone conduction work at present
With, and there are problems of that degradation speed is slow, fragility is big, thermal discharge.Another part is, with bone formation, protein BMP -2 occurs
New bone renovating material (the INFUSE Bone Graft of Medtronic, Inc company of the U.S., East China medicine for nucleuses
" self-bone grafting " of group), only possess bone inductive effect during Bone Defect Repari, affected area lacks mechanical strength.The said goods, are facing
Needs operation implantation all there is during bed use, degraded is slow, increase patient suffering's defect.For clinical problem, existing
Research mainly include：Injected bone repairing material with α-half-H 2 O calcium sulphate as core, because of its good biocompatibility, life
Thing Degradation, free of toxic effects, good hemostasis and angiogenesis have arrived extensive approval.But in clinical course, performance
Go out continuous stability poor, bone inductive effect is lacked, repair process is longer.And utilize sodium alginate calcium salt system load BMP-2
Gel-type bone renovating material, the Bone Defect Repari time is short, good biocompatibility, but have that hardening time is short, lack mechanical strength, negative
The problems such as loading system degradation time is long, while there is fluid electrolyte imbalances equivalent risk, (alginate easily chelate the bivalence in body fluid
Metal ion).
In sum, to there is continuous stability poor for existing bone renovating material, lacks bone inductive effect, and repair process is longer；
Lack mechanical strength, the problem of load system degradation time length.
Content of the invention
It is an object of the invention to provide a kind of injectable has the bone restoration bioactive material of sustained release performance and its system concurrently
Preparation Method, it is intended to which solving existing bone renovating material, to there is continuous stability poor, lacks bone inductive effect, and repair process is longer；Lack
Weary mechanical strength, the problem of load system degradation time length.
The present invention is achieved in that a kind of injectable has the bone restoration bioactive material of sustained release performance concurrently, described can
Injection has the bone restoration bioactive material of sustained release performance concurrently by solidifying substrate and solidify liquid constitutes；
During the bone renovating material Clinical practice, substrate will be solidified and solidify liquid will be pressed pulp-water and pressed as 1:1-1:0.4 is mixed
Closing compounding and Cranial defect focus is injected through high pressure injector；
There are albumen, fibronectin, hydroxyapatite, half-H 2 O calcium sulphate, two water by two type bone formation in the solidification substrate
Calcium sulfate constitutes；Per gram of solidification substrate contains bis- type bone formation of 0.5-5mg and albumen, 0.1-2.0mg fibronectin, 50- occurs
200mg hydroxyapatite；750-900mg half-H 2 O calcium sulphate, 10-50mg calcium sulphate dihydrate.
The solidify liquid is made up of hyaluronic acid, sodium alginate, phosphate buffer；Solidify liquid contains 20-100mM phosphoric acid
Salt buffer solution, while add 0.01-0.1% sodium alginate, 0.01-0.1% hyaluronic acid.
Further, the two types bone formation occurs albumen be divided into adult form, truncated-type and mutein.
Further, the truncated-type is that two type bone formation of encoding human source occurs 104 aminoacid sequence SEQ of protein C-terminal
ID NO：1；Mutein is the mutant SEQ ID NO of truncated-type：2；SEQ ID NO：1、SEQ ID NO：2 corresponding bases
Because coded sequence is：SEQ ID NO：3、SEQ ID NO：4；
The fibronectin is extracted from animals and plants or prepared by genetic engineering；
Further, the truncated-type fibronectin that prepared by the fibronectin preferred gene engineering, its aminoacid sequence such as SEQ
ID NO：5；Corresponding gene coded sequence is SEQ ID NO：6.
Further, above-mentioned encoding gene all inserts self-built prokaryotic expression carrier, and proceeds to e. coli bl21 and carry out table
Reach；Self-built carrier, is framework based on prokaryotic expression system PET serial carrier, is oriented in Bgl I, Xba I site respectively slotting
Enter recombination sequence SEQ ID NO：7.
Further, the hydroxyapatite is in coral hydroxyapatite, calcined bone powder, porous hydroxyapatite
Any one.
Further, the half-H 2 O calcium sulphate is α type or β type；
Further, the phosphate buffered solution concentration is 6.0-8.0 for 20-100mM, pH；
The hyaluronan molecule amount is 150 ten thousand to 200 ten thousand；
The sodium alginate molecular weight 1,000,000-200 ten thousand.
Another object of the present invention is to providing the bone restoration bioactive material that a kind of injectable has sustained release performance concurrently
The preparation method of material, the injectable has the preparation method of the bone restoration bioactive material of sustained release performance concurrently includes following step
Suddenly：
Step one, builds rhBMP-2* and rhFN genetic engineering bacterium, through recombinant expressed purification, obtains after lyophilization
RhBMP-2* and rhFN lyophilized powder；Redissolving rhBMP-2*, rhFN protein freeze-dried powder with 10-100mM phosphate buffer makes
Final concentration of 0.1-5mg/mL, the rhFN final concentration of protein of rhBMP-2* is 100-1000ug/mL；In this solution of 1L, 5-50g is added
Porous hydroxyapatite fully suspends, and is homogenized using high-pressure cell crusher, 12,000rpm centrifugation simultaneously lyophilizations, chela
In porous hydroxyapatite after hop protein, rhBMP-2* content is 1-10mg/g for 10-50mg/g, rhFN content, whole 4
Complete under the conditions of DEG C；By porous hydroxyapatite, α-half-H 2 O calcium sulphate and calcium sulphate dihydrate, by 1:4:0.1-1:8.5:0.5 ratio
Example is mixed and is disperseed with high-energy ball milling, is sub-packed in 5mL cillin bottle, through Co60Irradiation sterilization, this is bone restoration bioactive
The solidification substrate (component A) of material；
Step 2, prepares 20-100mM phosphate buffered solution, and HA, the SA for adding；Wherein HA content is 0.01-
0.1%, SA concentration is 0.01-0.1%；Fill lyophilizing through Co in the 5mL cillin bottle60Irradiation sterilization, this is Bone Defect Repari biology
Active material solidify liquid (component B), is redissolved with water for injection using front.
Another object of the present invention is to providing the bone restoration bioactive material that a kind of injectable has sustained release performance concurrently
The using method of material, the injectable has the using method of the bone restoration bioactive material of sustained release performance concurrently to be included：Using front,
Using front, the 1.05-1.2 times of volume according to Cranial defect volume prepares this material, and wherein solidification substrate and solidify liquid are pressed by pulp-water
1:1-1:0.4 carries out ratio mixing compounding use.
Another object of the present invention is to providing the bone restoration bioactive material that a kind of injectable has sustained release performance concurrently
Bone grafting material prepared by material.
The injectable that the present invention is provided has bone restoration bioactive material of sustained release performance and preparation method thereof concurrently, possesses and keeps away
Exempting from perform the operation through focus, rapid shaping, necessary mechanical strength is maintained, while bone conduction, bone is had concurrently in Bone Defect Repari overall process luring
Lead effect.The material of the present invention, it is possible to achieve by the through focus of injection, it is to avoid exploitation operation, reduce patient's pain；BMP-2
Release repairs overall process with bone injury, realizes bone conduction and self-bone grafting while carrying out, and shortens the bone injury healing time.
Description of the drawings
Fig. 1 is pOTO carrier core fragment schematic diagram provided in an embodiment of the present invention.
Fig. 2 is Protein expression and purification renaturation schematic diagram provided in an embodiment of the present invention；
In figure：A is the protein expression figure in embodiment two, wherein swimming lane 1, low molecular weight protein (LMWP) marker, from top to bottom
It is followed successively by：80KDa、60KDa、40KDa、30KDa、20KDa、12KDa；Swimming lane 2 is the escherichia coli bulk sample not induced；Swimming lane 3
It is the full bacterium for inducing 12h through 0.6mM IPTG；Swimming lane 4 is bacteria break supernatant liquid；Swimming lane 5 is for breaking bacterium precipitation；
B is rhBMP-2* purification renaturation figure, and wherein swimming lane 1 is rhBMP-2* monomeric protein after purification, and swimming lane 2 is renaturation
RhBMP-2* dimer afterwards.
Fig. 3 is truncated-type FN protein expression provided in an embodiment of the present invention and purification figure；
In figure：Swimming lane 1 is the escherichia coli bulk sample not induced；Swimming lane 2 is the full bacterium for inducing 12h through 0.6mM IPTG；Swimming
Road 3 is bacteria break supernatant liquid；Swimming lane 4 is for breaking bacterium precipitation；Swimming lane 5,6 is rhFN after purification.
Fig. 4 and Fig. 5 are the bone restoration bioactive materials that the injection for providing in the embodiment of the present invention has sustained release performance concurrently
Preparation method detail flowchart.
Specific embodiment
In order that the objects, technical solutions and advantages of the present invention become more apparent, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that specific embodiment described herein is not used to limit only in order to explain the present invention
Determine the present invention.
Below in conjunction with the accompanying drawings the application principle of the present invention is explained in detail.
The injectable for providing in the embodiment of the present invention has the bone restoration bioactive material of sustained release performance concurrently by solidifying substrate
(component A) and solidify liquid (component B) constitute；Component A presses pulp-water ratio for 1 with component B:1-1:0.4 carries out mixing compounding use, its
In preferably 1:0.9-1:0.6.SEQ ID NO：
The injectable has the bone restoration bioactive material of sustained release performance concurrently, and wherein component A contains two type bone formation and sends out
Raw albumen (rhBMP-2), fibronectin (rhFN), hydroxyapatite (HAP), half-H 2 O calcium sulphate (CSH), calcium sulphate dihydrate
(CSD)；Component B is made up of hyaluronic acid (HA), sodium alginate (SA), phosphate buffer etc..
Especially, per gram of solidification substrate contains 0.5-5mg rhBMP-2,0.1-2.0mg rhFN, 50-200mg HAP；
750-900mg CSH, 10-50mg CSD.
Especially, solidify liquid contains 20-100mM phosphate buffered solution (pH 6.0-8.0), while adding 0.01-
0.1%SA, 0.01-0.1%HA.
Especially, rhBMP-2 can derive from the modes, wherein preferred gene engineering such as animal bone is extracted, prepared by genetic engineering
Prepare.
Especially, people source BMP-2 can be divided into adult form (rhBMP-2-114aa), truncated-type (104 aminoacid, rhBMP-2-
104aa) and mutein (rhBMP-2*), wherein preferred mutein.Per gram of biological bone renovating material component A consumption
For 0.5-5mg rhBMP-2, wherein preferably 1-3mg.
Wherein truncated-type BMP-2, is 104 aminoacid sequence (SEQ ID NO of encoding human source BMP-2 PROTEIN C end：1).
RhBMP-2* is mutant (the SEQ ID NO of truncated-type BMP-2：2), its 65th isoleucine (I) sports Glycine
(G), the 75th Leucine for being (L) sports alanine (A).By prokaryotic expression system codon preference Sexual behavior mode, above-mentioned egg
Bai Xulie, SEQ ID NO：1、SEQ ID NO：2 corresponding gene coded sequences are：SEQ ID NO：3、SEQ ID NO：4.
Especially, fibronectin (FN), can derive from animals and plants and extract or genetic engineering preparation, wherein preferred gene engineering
Truncated-type fibronectin is prepared, per gram of biological bone renovating material component A content is 0.1-2.0mg, wherein preferably 0.5-1mg.
Wherein truncated-type Fibronectin sequence such as SEQ ID NO：5.By prokaryotic expression system codon preference Sexual behavior mode,
Above-mentioned protein sequence, corresponding gene coded sequence is：SEQ ID NO：6.
Especially, above-mentioned encoding gene all inserts self-built prokaryotic expression carrier, and proceeding to e. coli bl21 (DE3) is carried out
Expression.Wherein self-built carrier, is framework based on prokaryotic expression system PET serial carrier, in Bgl I, Xba I site respectively
Orientation insertion recombination sequence (SEQ ID NO：7), structure such as Fig. 2, forms new expression vector pOTO.
Especially, hydroxyapatite can derive from coral hydroxyapatite (CHAP), calcined bone powder, porous hydroxyapatite
Deng wherein preferred porous hydroxyapatite；Per gram of biological bone renovating material component A content is 50-200mg, wherein preferably 100-
150mg.
Especially, half-H 2 O calcium sulphate can be α type or β type, wherein preferably using calcium sulphate dihydrate in high temperature, condition of high voltage
Under be dehydrated at a slow speed, hydrone overflows the α type half-H 2 O calcium sulphate for generating after calcium sulphate dihydrate lattice in liquid form.Per gram of biological bone
Repair materials component A content is 750-900mg, wherein preferably 800-900mg.
Especially, calcium sulphate dihydrate is medical grade, and per gram of biological bone renovating material component A content is 10-50mg, wherein excellent
Elect 15-20mg as.
Especially, biological bone renovating material component B is made up of PBS, HA, SA, wherein phosphate buffered solution preferably phosphoric acid
Sodium salt buffer system, it is 6.0-8.0, wherein preferably 6.8-7.2 that concentration is 20-100mM wherein preferably 50mM, pH, while adding
0.01-0.1%SA, 0.01-0.1%HA.HA preferred molecular weight is 150 ten thousand to 200 ten thousand.SA preferred molecular weight 1,000,000-200 ten thousand.
The injectable for providing in the embodiment of the present invention has the preparation method of the bone restoration bioactive material of sustained release performance concurrently
Comprise the following steps：
Build rhBMP-2* and rhFN genetic engineering bacterium, through recombinant expressed purification, after lyophilization obtain rhBMP-2* and
RhFN lyophilized powder；Redissolving rhBMP-2*, rhFN protein freeze-dried powder with 10-100mM phosphate buffer makes rhBMP-2* final concentration
It is 100-1000ug/mL for 0.1-5mg/mL, rhFN final concentration of protein.In this solution of 1L, 5-50g porous hydroxyapatite is added
(porosity 50%-70%, aperture be 200 μm) fully suspends, and is homogenized using high-pressure cell crusher, 12,000rpm from
The heart lyophilization, it is 1- for 10-50mg/g, rhFN content to chelate rhBMP-2* content in the porous hydroxyapatite after albumen
10mg/g, whole process is completed under the conditions of 4 DEG C.By above-mentioned porous hydroxyapatite, α-half-H 2 O calcium sulphate and calcium sulphate dihydrate, by 1:
4:0.1-1:8.5:0.5 ratio is mixed and is disperseed with high-energy ball milling, is sub-packed in 5mL cillin bottle, through Co60Irradiation sterilization,
This is the solidification substrate of bone restoration bioactive material；Detailed step is as shown in Figure 4.
20-100mM phosphate buffered solution is prepared, and adds appropriate HA, SA.Wherein HA content is 0.01-0.1%,
SA concentration is 0.01-0.1%, fill lyophilizing through Co in the 5mL cillin bottle60Irradiation sterilization, this is bone restoration bioactive material
Material component B, is redissolved with water for injection using front.Detailed step is as shown in Figure 5.
The Bone Defect Repari biology of sustained release performance is had concurrently to a kind of injectable of the announcement of the present invention with reference to specific embodiment
The preparation technology of active material is further described.
Embodiment one：Expression plasmid pOTO builds
Carry out double digestion, and carry out purification returning with restricted enzyme Bgl I and Xba I to prokaryotic expression carrier pET28a
Receive, form a pOTO plasmid backbone.Synthetic fragment (SEQ ID NO：7) structure such as Fig. 2, while to this fragment with identical
Restricted enzyme carries out enzyme action and purification is reclaimed.This two fragments T4 ligase, 16 DEG C of connections overnight, are formed new expression
Carrier pOTO.
Embodiment two：The preparation of rhBMP-2*
According to people BMP-2 maturation peptide sequence (KC294426.1) in GenBank, from 104 aminoacid sequence of C-terminal truncate, shape
Become truncated-type rHBMP-2 (No 1).The isoleucine (I) of the 65th of this aminoacid sequence is sported Glycine (G), the 75th
For Leucine (L) sport alanine (A), form mutant rhBMP-2* (SEQ ID NO：2).By prokaryotic expression system
Codon preference Sexual behavior mode, obtains its gene coded sequence SEQ ID NO：4.And add initiation codon at its 5 ' end and 3 ' ends
Son, termination codon and restriction enzyme site, and synthesize this genetic fragment.
By the fragment of synthetic and new expression vector, double digestion, purification is carried out with identical restriction restriction endonuclease and return
Receive, and under T4 connection enzyme effect, connect under the conditions of 16 DEG C overnight, form expression plasmid pOTO-rhBMP-2*.Proceeded to table
Host e. coli BL21 (DE3) is reached, rhBMP-2* genetic engineering bacterium is formed, and is verified with T7-T7t primer.
Genetic engineering bacterium after picking conversion, is seeded to the LB culture medium that 10mL contains 20-100 μ g/mL kanamycin
In, 30-37 DEG C is cultivated to OD600Reach 0.4-0.7.100-300mL is seeded to by 1%-5% inoculum concentration and contains 20-100 μ g/mL
In the LB culture medium of kanamycin, 30-37 DEG C is cultivated to OD600After reaching 0.4-0.7, IPTG is added to make its final concentration of 0.1-
0.8mM, adjustment cultivation temperature is 15-28 DEG C, continues culture 8-12h.
After the completion of culture, at 4 DEG C, under the conditions of 12,000rpm, 30min collects thalline is centrifuged, and is washed with physiological saline solution
Wash 1-2 time.1 is pressed with broken bacterium buffer (50mM Tris-HCl, 1mM EDTA, pH 8.0):5-1:The resuspended thalline of 10 ratio,
And under 800-1,200bar pressure, bacterium 1-3 time is broken by biomixer, and at 4 DEG C, centrifugation receipts respectively under the conditions of 10,000rpm
Collection supernatant and precipitation, supernatant is for being labeled as solution A.Precipitation is urinated with 0.1%-0.3%Triton X-100,0.5M-1.5M respectively
Element washed once, 4 DEG C, and precipitation is collected by centrifugation under the conditions of 12,000rpm respectively.Precipitation and lysate (7M guanidine hydrochloride, 50mM
Tris, 1mM EDTA, 10mM DTT) press 1:1 ratio is mixed and is stirred and is completely dissolved to precipitation, and in 4 DEG C, 12,000rpm bar
Under part, it is centrifuged 20 minutes, collects supernatant and be labeled as solution B.Merge solution A and B.
Hydroxyapatite is soaked overnight with 2-3 times of column volume level pad (5mM phosphate buffer, pH 8.0)；Stir
Mix and mix and fill post, natural subsidence simultaneously avoids the generation of bubble, and column volume controls in 50-100mL.Rinsed to base with balance liquid
Line, sample concentration maintains 20-50mg/mL, and applied sample amount is 1-5mL.Loading step is forward and backward respectively to add 8M carbamide 7.5-15mL mono-
Secondary, and with the unconjugated albumen of balance liquid eluting.With the phosphate buffer of 5-300mM, pH 8.0 carries out gradient elution, merges
Eluting peak.
Above-mentioned eluent gradient dilution to protein content is 0.05-5mg/ml, and diluent is phosphate buffered solution pH value
For 7.8-8.6, and add 0.2mM GSSH and 2mMGSH, maintain 4-9 days under the conditions of 4 DEG C.4 DEG C, 12,000rpm centrifugations are removed
Precipitation, supernatant sephadex G 75 separates and collects first eluting peak, merges lyophilizing preservation, the as rhBMP- of purification
2*.
Embodiment two：The preparation of rhFN
According to successor's fibronectin complete sequence (X02761.1) on GenBank data base, its complete protein sequence is entered
Row structural analyses, select the partial order of the cell-binding domain, heparin binding domain and second fibronectin binding domain of the albumen
The truncated-type fibronectin rhFN as the present invention is arranged, its aminoacid sequence such as NO5, according to prokaryotic expression system codon preference
Sexual behavior mode, obtains its gene coded sequence NO 6.And add start codon, termination codon and limit at its 5 ' end and 3 ' ends
Property restriction enzyme site processed, and synthesize this fragment.
Fibronectin expression plasmid construction, abduction delivering, cell breakage, crude protein liquid are collected process and are walked with rhBMP-2*
Suddenly.Protein purification adopts the affine absorption of Heparin-Agarose post, and carries out gradient with the sodium chloride solution of 100-300mM and wash
De-, eluting peak is collected and combined, lyophilizing is standby.
Embodiment three：The preparation of biological bone renovating material
1st, bone restoration bioactive material solidification substrate (component A)：
By two scheme of embodiment, rhBMP-2* and rhFN lyophilized powder is obtained, dissolved with 10-100mM phosphate buffer
RhBMP-2*, rhFN protein freeze-dried powder makes final concentration of 0.1-5mg/mL, the rhFN final concentration of protein of rhBMP-2* for 100-
1000ug/mL.In this solution of 1L, 5-50g porous hydroxyapatite (porosity 50%-70%, aperture is 200 μm) is added fully
Suspend, be homogenized using high-pressure cell crusher, 12,000rpm centrifugation simultaneously lyophilizations, chelate the porous hydroxyapatite after albumen
In apatite, rhBMP-2* content is 1-10mg/g for 10-50mg/g, rhFN content, and whole process is completed under the conditions of 4 DEG C.Will be above-mentioned
Porous hydroxyapatite, α-half-H 2 O calcium sulphate and calcium sulphate dihydrate, by 1:4:0.1-1:8.5:0.5 ratio is mixed and is used height
Energy ball milling dispersion, is sub-packed in 5mL cillin bottle, and through Co60Irradiation sterilization, this is the solidification substrate of bone restoration bioactive material
(component A).
2nd, bone restoration bioactive material solidify liquid (component B)：
20-100mM phosphate buffered solution is prepared, and adds appropriate HA, SA.Wherein HA content is 0.01-0.1%,
SA concentration is 0.01-0.1%.Fill lyophilizing in the 5mL cillin bottle, and through Co60Irradiation sterilization, this is bone restoration bioactive material
The solidify liquid (component B) of material, is redissolved with water for injection using front.
3rd, the Clinical practice of bone restoration bioactive material
Using front, enough bone restoration bioactive materials are prepared according to 1.05-1.2 times of volume of Cranial defect focus capacity
Material.A, B component are compared 1 by pulp-water:0.5-1:1 ratio is mixed, and is poured in high pressure injector, after draining air, is installed 27# pin additional
Head.Cranial defect focus being injected under X-ray machine auxiliary, also focus note can be determined by contacting for experienced orthopedist
Penetrate, fixing 10-15 minute, external Macromolecular splint or binder are fixed.
With reference to testing, the biocompatibility to the present invention, physical property are characterized and application effect is explained in detail
1st, biocompatibility (cell toxicity test)
By three method of embodiment, bone restoration bioactive material is prepared.Wherein porous hydroxyapatite from porosity is
70%, aperture is 100 μm, according to 1:8.8:0.2 ratio is mixed with α-half-H 2 O calcium sulphate that particle diameter is 100 μm and calcium sulphate dihydrate
Even, and per gram of bone restoration bioactive material solidification substrate contains 2mg rhBMP-2*, 0.5mg rhFN.By pulp-water ratio it is
1:0.8 with solidify liquid (50mM sodium phosphate buffer, pH 7.0；It is 0.1% that HA content is 0.05%, SA content) mix.Solidification
Afterwards this material is pulverized, according to ratio 1640 cell culture fluids or the hyclone cell culture medium of 0.2g/ml, soak
Carry 24 hours, sampling liquid is according to ISO10993-5:Specified in 2009, mtt assay is tested, parallel three groups of experiments.
Experimental result, cell relative growth rate, minima is more than 80%, and meansigma methodss are 85%, to reach ISO ISO10993-
5:2009 required standards, show this kind of biology stock repair materials good biocompatibility, nontoxic, safety.
2nd, physical property is characterized
By the bone restoration bioactive material for preparing in biocompatibility experiment as physical property sign test for examination
Sample.
1) maximum temperature and setting time when solidifying：
Before test, test sample, hybrid instrument, solidification mould and thermocouple resistance are positioned over 23 ± 1 DEG C, 40% relative humidity
Under the conditions of place 2h.Test sample is mixed and is injected in mould, while recording reaction temperature.Press《YY 0459-2003 surgery
Implant bone cement standard》Appendix C method, the solidification maximum temperature of analysis of material and setting time, test parallel 5 samples,
Average, as a result as following table.
2) comprcssive strength
The material injecting height for preparing is that in mould of 12mm, the internal diameter for 6mm, two sides clamps, and is placed in 37 ± 1 DEG C,
100% humidity prepares specimen sample, after solidification 1h, the demoulding, and two ends polishing, and 37 ± 1 DEG C are placed in, in 100% humidity environment,
24 ± 1h of constant temperature.Press《YY 0459-2003 surgical implant bone cement standard》Annex E method determines the comprcssive strength of material, examination
Test parallel 5 samples.
As a result show, the bone renovating material mean clotting time that the present invention is announced is 14.2min, solidifies center final set highest
Temperature is 38.5 DEG C, and after solidification 24h, mean compressive strength is 43.9Mpa.Physical property is it is also shown that when bone renovating material solidifies
Between can by adjust bone renovating material in each several part ratio adjustment, meet clinical requirement；While the Bone Defect Repari material that the present invention is announced
During the solidification of material, thermal discharge is less, focus of will not burning surrounding tissue, and mechanical strength meets clinical demand.
3rd, Bioactivity
By the bone restoration bioactive material for preparing in biocompatibility test as material to be tested, and with pH's 7.0
The phosphate buffered solution dialysed overnight of 50mM, through 0.22 μm of micro-filtrate membrane filtration, and is mixed with calf serum, while adopting MEM
Basal medium mixes as test liquid.With C2C12 cell line as alkali phosphatase for examination cell strain.Cell culture is to 40%
After converging (about 24 hours), test sample is replaced, continue culture 72 hours, abandon cell culture fluid, and with brine two
Secondary.Detected with alkaline phosphatase detecting reagent box.As a result show alkaline phosphatase activitieses with rhBMP-2* changes of contents in positive
Close, and the alkaline phosphatase activitieses of per gram of biological bone renovating material are more than 1 × 106, illustrate that the present invention has clearly biological living
Property.
4th, ectopic osteogenesis test
Control sample 1：Adopt Japanese Olympus Terumo biomaterial Co., Ltd. production with bata-tricalcium phosphate be
The bone renovating material of main component；
Control sample 2：Certain bone material manufacturer production domestic with hydroxyapatite and tricalcium phosphate constituted many
The bone renovating material of hole bioceramic；
Control sample 3：Certain bone material manufacturer production domestic based on I-type collagen and hydroxyapatite
Bone renovating material
Control sample 4：The bone renovating material based on bovine cancellous bone of certain bone material manufacturer production domestic.
Control sample 5：Composite bone repairing material (BMP content and test group based on soybean lecithin, gelatin load BMP
Identical)；
Test specimen：Bone restoration bioactive material is prepared using biocompatibility test.
Matched group tests white mouse with 1.5% anaesthetized with pentobarbital, after its left hind unhairing, medical ethanol sterilization, at which
At muscle nest, ostomy of the length for 0.5cm is cut, with mosquito forcepss separation skin, blunt separation muscle, fall in muscle
100mg reference substance bone renovating material, go forward side by side whereabouts blood and stitching processing are implanted at nest respectively.Per the parallel 5 groups of examinations of group controlled trial
Test.
Test group tests white mouse with 1.5% anaesthetized with pentobarbital, after its left hind unhairing, medical ethanol sterilization, by 1:
0.8 pulp-water ratio mixes the bone renovating material of test group in the syringe for loading 27# syringe needle, is injected to left hind muscle nest
Place, injects 100mg, parallel 5 test group per only white mouse.
In order to test with comparability, test group occurs protein content to be consistent with the bone formation of matched group 5.
All experimental animal Post operation, all normally revive and recover feed drinking-water, observe postoperative white mouse wound situation, sign
(heating, diarrhoea, appetite and activity)；And 4 weeks after surgery, method of craning one put to death white mouse, and solution takes bone, weighs, averages.
Experimental result such as following table：
| Group | Wound situation | Sign | Ectopic osteogenesis effect |
| Control 1 | Heal, no infect | Normally | Nothing or micro skeletonization |
| Control 2 | Heal, no infect | Normally | Nothing or micro skeletonization |
| Control 3 | Heal, no infect | Normally | Nothing or micro skeletonization |
| Control 4 | Heal, no infect | Normally | Nothing or micro skeletonization |
| Control 5 | Heal, no infect | Normally | Ectopic osteogenesis, average weight gain 270mg |
| Test group | Heal, no infect | Normally | Ectopic osteogenesis, average weight gain 380mg |
Test result indicate that the injectable bone restoration bioactive material that the present invention is announced, has obvious ectopic osteogenesis to imitate
Really, and biocompatibility is preferable.
Presently preferred embodiments of the present invention is the foregoing is only, not in order to limit the present invention, all essences in the present invention
Any modification, equivalent and improvement that is made within god and principle etc., should be included within the scope of the present invention.
Claims (10)
1. a kind of injectable has the bone restoration bioactive material of sustained release performance concurrently, it is characterised in that the injectable has concurrently slow
The bone restoration bioactive material of performance is released by solidifying substrate and solidify liquid constitutes；
During the bone renovating material Clinical practice, solidification substrate and solidify liquid press pulp-water ratio for 1:1-1:0.4 carries out mixing compounds；
The solidification substrate contains two type bone formation and albumen, fibronectin, hydroxyapatite, half-H 2 O calcium sulphate, calcium sulphate dihydrate occurs；
Per gram of solidification substrate contains bis- type bone formation of 0.5-5mg and albumen, 0.1-2.0mg fibronectin, 50-200mg hydroxy-apatite occurs
Stone；750-900mg half-H 2 O calcium sulphate, 10-50mg calcium sulphate dihydrate；
The solidify liquid is made up of hyaluronic acid, sodium alginate, phosphate buffer；Solidify liquid contains 20-100mM phosphate and delays
Solution is rushed, while adding 0.01-0.1% sodium alginate, 0.01-0.1% hyaluronic acid.
2. injectable as claimed in claim 1 has the bone restoration bioactive material of sustained release performance concurrently, it is characterised in that described
Two type bone formation occur albumen be divided into adult form, truncated-type and mutein, and wherein preferably saltant type bone formation occurs
Albumen rhBMP-2*.
3. injectable as claimed in claim 2 has the bone restoration bioactive material of sustained release performance concurrently, it is characterised in that described
Truncated-type is that two type bone formation of encoding human source occurs 104 aminoacid sequence SEQ ID NO of PROTEIN C end：1；Mutein is for cutting
The mutant of short, its aminoacid sequence is SEQ ID NO：2；SEQ ID NO：1、SEQ ID NO：2 corresponding gene codes
Sequence is：SEQ ID NO：3、SEQ ID NO：4；
The fibronectin is extracted from animals and plants or prepared by genetic engineering；Truncated-type fibronectin, its aminoacid sequence is such as
SEQ ID NO：5；Gene coded sequence is accordingly：SEQ ID NO：6.
4. injectable as claimed in claim 3 has the bone restoration bioactive material of sustained release performance concurrently, it is characterised in that
RhBMP-2* and rhFN encoding gene all inserts self-built prokaryotic expression carrier, proceeds to e. coli bl21 and is expressed；Self-built
Carrier, is framework based on prokaryotic expression system PET serial carrier, orients insertion restructuring respectively in Bgl I, Xba I site
Sequence SEQ ID NO：7.
5. injectable as claimed in claim 1 has the bone restoration bioactive material of sustained release performance concurrently, it is characterised in that described
Hydroxyapatite is from coral hydroxyapatite, calcined bone powder, any one in porous hydroxyapatite.
6. injectable as claimed in claim 1 has the bone restoration bioactive material of sustained release performance concurrently, it is characterised in that described
Half-H 2 O calcium sulphate is α type or β type.
7. injectable as claimed in claim 1 has the bone restoration bioactive material of sustained release performance concurrently, it is characterised in that described
It is 6.0-8.0 that phosphate buffered solution concentration is 20-100mM, pH；
The hyaluronan molecule amount is 1,500,000-200 ten thousand；The sodium alginate molecular weight 1,000,000-200 ten thousand.
8. a kind of as claim 1, the injectable has the preparation method of the bone restoration bioactive material of sustained release performance concurrently, its
It is characterised by, the injectable has the preparation method of the bone restoration bioactive material of sustained release performance concurrently and comprises the following steps：
Step one, builds rhBMP-2* and rhFN genetic engineering bacterium, through recombinant expressed purification, obtains rhBMP-2* after lyophilization
And rhFN lyophilized powder；Redissolving rhBMP-2*, rhFN protein freeze-dried powder with 10-100mM phosphate buffer makes rhBMP-2* dense eventually
It is 100-1000ug/mL to spend for 0.1-5mg/mL, rhFN final concentration of protein；In this solution of 1L, 5-50g porous hydroxyapatite phosphorus ash is added
Stone is fully suspended, chelates absorption, is homogenized using high-pressure cell crusher, 12,000rpm centrifugation simultaneously lyophilizations, chelates egg
In porous hydroxyapatite after white, rhBMP-2* content is 1-10mg/g, whole process in 4 DEG C of bars for 10-50mg/g, rhFN content
Complete under part；By porous hydroxyapatite and α-half-H 2 O calcium sulphate, by 1:4:0.1-1:8.5:0.5 ratio is mixed and is used height
Energy ball milling dispersion, is sub-packed in 5mL cillin bottle, through Co60Irradiation sterilization, this is the solidification substrate of bone restoration bioactive material；
Step 2, prepares 20-100mM phosphate buffered solution, and HA, the SA for adding；Wherein HA content is 0.01-0.1%, SA
Concentration is 0.01-0.1%；It is sub-packed in 5mL cillin bottle lyophilizing and through Co60Irradiation sterilization, this is bone restoration bioactive material
Solidify liquid, redissolved with water for injection using front.
9. a kind of injectable as claimed in claim 1 has the using method of the bone restoration bioactive material of sustained release performance concurrently, its
It is characterised by, the injectable has the using method of the bone restoration bioactive material of sustained release performance concurrently to be included：Using front, according to
1.05-1.2 times of volume of Cranial defect volume prepares this material, and wherein solidification substrate and solidify liquid press pulp-water ratio for 1:1-1:0.4.
10. a kind of injectable as described in claim 1～7 has bone shifting prepared by the bone restoration bioactive material of sustained release performance concurrently
Plant material material.
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