CN105536067B - The method for building high osteogenic activity bone - Google Patents
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Abstract
The present invention relates to a kind of methods of the high osteogenic activity bone of structure, there is following steps：1) homogeneous allogenic bone decalcification bone supporting stand is taken；Fusogenic peptide is dissolved in sucrose solution, until final concentration of the 1% of solution, it is ultrasonically treated, obtains peptide solution；2) it takes allogeneic decalcification bone supporting stand to be fully immersed in peptide solution 48 minutes, takes out, rinsed with phosphate buffered saline solution, obtain the allogeneic de-calcificated bone carriage of self-assembling peptides modification；3) marrow injection is filled in the bone uptake enricher of homogeneous allogenic bone de-calcificated bone carriage of self-assembling peptides modification, marrow is enriched with several times, obtains high osteogenic activity bone.This method can make the enrichment of allogeneic bone holder material high efficiency that can promote osteanagenesis cell, meet the needs of skeletonization, what is obtained is high osteogenic activity bone grafting material.
Description
Technical field
The present invention relates to a kind of extracellular matrix material of biomedicine, more particularly to a kind of side of the high osteogenic activity bone of structure
Method.
Background technology
Bone defect caused by a variety of causes such as wound, infection, tumour is very common, Bone Defect Repari process, is egg more than one
In vain, the complicated regulation process of multiple-factor coordinative role.
Homogeneous allogenic bone (DBM) is current most common bone tissue engineering scaffold, by Cryopreserved, degreasing,
After a series of processing such as de- functional protein, decalcification, gamma-ray irradiation, there is minimum immunogenicity, because of its natural three dimensions
Structure, certain bone-inducting active etc. are a kind of ideal bone tissue engineering scaffolds.
In tissue engineered bone at the concentration of the cell and cell factor of osteanagenesis in bone holder in Bone Defect Repari, can be promoted,
There is very important effect for skeletonization.
It can promote the cell and cell factor of osteanagenesis in the tissue fluid such as marrow, blood containing some.These cells and because
Son is other than with certain volume also with certain surface charge.In general, in order to improve above-mentioned composition in timbering material
Concentration, bioaccumulation efficiency is improved using the method for reducing timbering material aperture.But though this method bioaccumulation efficiency is improved,
But the multiple of enrichment is confined to 1.5-2 times or so, it is difficult to meet the needs of skeletonization, and to the cell factor of volume very little richness
Collect ineffective.
Invention content
The object of the present invention is to provide a kind of method of the high osteogenic activity bone of structure, this method can make homogeneous allogenic bone holder
The enrichment of material high efficiency can promote osteanagenesis cell, meet the needs of skeletonization, what is obtained is high osteogenic activity bone grafting material.
The technical scheme is that：
The method for building high osteogenic activity bone, there is following steps：
1) preparation of enrichment material
Take homogeneous allogenic bone decalcification bone supporting stand；
The fusogenic peptide of purity >=95% is dissolved in 20% sucrose solution, until final concentration of the 1% of solution, it is ultrasonically treated
30 minutes, obtain peptide solution；
2) it takes de- allogeneic calcium bone holder to be fully immersed in peptide solution 4-8 minutes, takes out, with the phosphoric acid of pH value 7.4
Buffer salt solution rinses, and obtains the allogeneic de-calcificated bone carriage of self-assembling peptides modification；
3) it is enriched with
Marrow injection is filled with to the bone uptake enricher of the homogeneous allogenic bone de-calcificated bone carriage of self-assembling peptides modification
In, marrow is enriched with several times, obtains building high osteogenic activity bone material.
The fusogenic peptide includes RADA16-I peptides and is coupled to the osteostatin peptides of the C-terminal of RADA16-I peptide sequences
(TRSAW), amino acid sequence such as SEQ ID NO:Shown in 2；Or the fusogenic peptide includes the C-terminal by RADA16-I peptide sequences
It is formed via amino acid residue GG and osteostatin peptides (TRSAW) coupling, amino acid sequence such as SEQ ID NO:Shown in 3.
The amino acid sequence of the RADA16-I peptides is SEQ ID NO:1.
The sequence of the osteostatin peptides such as SEQ ID NO:Shown in 4.
Marrow enrichment described in step 3) is 4 times.
The present invention using nanoscale self-assembling peptides RADA16-I, is waited with certain charge using " the double modifications of physical chemistry "
Material modify the larger timbering material of homogeneous allogenic bone equal aperture, the material solidifications such as nanoscale self-assembling peptides are in timbering material
Surface can reduce the aperture of material, improve bioaccumulation efficiency；Meanwhile being set in the nanoscale self-assembling peptides on timbering material surface
The group of certain charge is also carried, can be improved by the interaction between charge to cell again with different charges and thin
The suction-operated of intracellular cytokine further increases concentration effect, improves the concentration of the cell and the factor in enrichment material, further carries
High osteogenic ability.
Description of the drawings
Fig. 1 is that blank DBM materials are in tridimensional network figure；
Fig. 2 is that fusogenic peptide/DBM composite materials form laminated structure figure inside the surfaces DBM and mesh；
Fig. 3 is that fusogenic peptide/DBM composite materials form filament figure inside the surfaces DBM and mesh；
Fig. 4 is the scanning electron microscopic picture of unmodified DBM holders；
Fig. 5 is that a large amount of enrichment of structure and its inside of fusogenic peptide/visible mesh of DBM composite inners of modification is thin
Born of the same parents；
Fig. 6 is the enlarged drawing of fusogenic peptide/DBM composite material enrichment of cell of modification.
Specific implementation mode
One, reagents and instrument
1. reagent：
Allogeneic decalcification bone supporting stand sample (Beijing great Qing Technology Co., Ltd., China)
RADA16-I peptides and fusogenic peptide (the strong biological Co., Ltd of credit, Shanghai, China)
FACSCal ibur stream type cell analyzers (BD companies, the U.S.)
Hemolysin Nanchang Baxter Bio-tech Co., Ltd
Mixed reagent C D34-PE, CD45-Per CP, nucleic acid dye, (karyocyte)
Nucleic acid dye (BD companies, the U.S.)
2. instrument
Ultrasonic cleaner K5100 (Kunshan Co., Ltd, Shanghai, China)
Bone uptake enricher (Fu Wosi Medical Devices Co., Ltd.s of Chongqing City, China)
Biochip scanner (Axon instrument companies of the U.S.)
Embodiment 1The design of polypeptide
With RADA16-I peptides, sequence is SEQ ID NO:1(AcN-RADARADARADARADA-CONH2) based on,
Its C-terminal is directly coupled one section of osteostatin peptide (TRSAW) by means of peptide bond, which is SEQ
ID NO:2 (RADARADARADARADAGGTRSAW), molecular weight 2387.54；Or RADA16-I peptides C-terminal through two
Amino acid residue GG is coupled with osteostatin peptides (TRSAW), which is SEQ ID NO:3
(RADARADARADARADAGGWASRT), aforementioned polypeptides are synthesized by Suzhou Chinapeptides Co., Ltd..
The preparation of 2 enrichment material of embodiment
By homogeneous allogenic bone decalcification bone supporting stand (DBM) sample be cut into cube (10 × 10 × 5mm, it is spare.Example 1
The polypeptide (polypeptide can reach technique effect of the present invention), the present embodiment use SEQ ID NO:2, purity
>=95%, it is dissolved in 20% sucrose solution, ultimate density is 1% (w/v), cleans ultrasonically device and carries out being ultrasonically treated 30 points
Clock is to reduce the viscosity of peptide.DBM is fully immersed in peptide solution about 5 minutes.It is molten with enough phosphate-buffered salts after taking-up DBM
Liquid (PBS, pH value 7.4) rinses, that is, completes the self assembly of peptide, forms the DBM materials (SAP/DBM) of self-assembling peptides modification.
Embodiment 3 is enriched with
In the vertebra cup that marrow injection has been loaded to bone renovating material, outer cover is covered tightly, uniformly firmly presses bone uptake enricher
It is unclamped after handle to concordant top, this product is automatically performed 1 marrow enrichment behaviour under the suction function that constant force spring provides
Make, and marrow resorption is entered in the cylinder that flows back.After marrow total reflux, handle are restored to initial position, handle is pressed again and is opened
Begin to recycle for second.Add up 4 above-mentioned enrichment procedures, that is, completes the structure of high osteogenic activity bone grafting material, total process about 5-
10 minutes.
The aperture index observing of enrichment material：Enrichment material is surveyed after preparing by 3 D video microscopic image analysis system
Fixed unmodified DBM pore sizes are (414.32 ± 175.00) μm (sample quantity is n=60), and the timbering material after modification melts
It is (220.55 ± 158.54) μm (n=60) to close peptide/DBM material pore sizes, has significant difference therebetween.Specific figure
As seeing Fig. 1 (unmodified DBM) and Fig. 2 (DBM of fusogenic peptide modification).After the aperture of timbering material is reduced, filter can be formed
The same effect of sieve can significantly improve the delay that timbering material promotes cell in marrow etc. the ingredient of skeletonization.
4 concentration effect of embodiment is evaluated
1. the counting (FACSCal ibur stream type cell analyzers) of enrichment of cell：
Fluidic cell operating process：
Sample to be tested (marrow before enrichment and after enrichment) is diluted 10 times by 1.1；
1.2 take BD absolute number counting tubes, are loaded 50ul；
1.3 mix reagent is added into absolute counting pipe, and mixing is protected from light and is incubated 15min；
1.4 are added 10ul hemolysins, then add 440ul transparent liquids (BD FACS), and mixing is protected from light and is incubated 30min
Machine testing on 2..
As a result：As a contrast with DBM, the enrichment times of karyocyte are 2.2 times in the marrow of SAP/DBM, cytode
It is 1.62 times.The enrichment times of monocyte are 3.63 times wherein in karyocyte；The enrichment times of red blood cell are 4.2 times；Between
The enrichment times of mesenchymal stem cells are 2.6 times.
The counting of the 5 enrichment of cell factor of embodiment
Using protein biochip technology (RayBio companies of the U.S.)
A is closed and is incubated
(1) kit is opened, glass-chip is taken out, room temperature air-dries 2h.The 4 DEG C of preservations of remaining reagent.
(2) glass-chip is assembled to and is incubated cell and is incubated in frame.
(3) 2X Block buffers are diluted with distilled water.The Block buffer of 100ul 1X is added in per hole, is incubated at room temperature
30min closes slide.Pay attention to：Ensure bubble-free in every hole, only the reagent adding into the hole for be coated with antibody.
(4) the closing buffer in being sucked out per hole, every group of detection sample take 100ul to be added to 6,7,8,9,10 chip battle arrays
The 1-7 sample apertures of row.It is added in the Block buffer to internal reference pipe of 100ul, 1ul mixed liquors is pipetted after mixing, each array is added
No. 8 internal reference holes in.2h is incubated after film seal at room temperature.Pay attention to：Adherent unhurried current when sample-adding generates to avoid bubble.
(5) 20 times of cleaning buffer solution 1 is diluted with distilled water obtains 1 times of buffer solution.The sample in every hole is fallen off,
It is cleaned 3 times with 1 times of cleaning buffer solution 1 at room temperature, each 2min.Pay attention to：Sample is avoided to flow into neighbouring aperture.
(6) glass-chip is put into togerther in the box for filling 1X cleaning buffer solutions 1 (buffer solution will cover completely together with holder
Build entire slide and holder), slight concussion cleaning 2 times at room temperature, each 10min.
(7) 20 times of cleaning buffer solution 2 is diluted with distilled water obtains 1 times of buffer solution.Cleaning buffering in absorbing per hole
Glass-chip is put into the box for the cleaning buffer solution 2 for filling 1 times by liquid together with holder, at room temperature slight concussion cleaning 2 times,
Each 5min.Thoroughly absorb remaining buffer solution 2 after washing.
(8) Antibody preparation antibody working solution is diluted with 1 times of Block buffer.It is diluted it to be separately added into 70ul in per hole
The antibody (kit included) that biotin combines is incubated 2h after film seal at room temperature.
(9) cleaning 3 times, each 2min are shaken at room temperature with 1 times of cleaning buffer solution 2 after being washed according to step 5.Thoroughly
Absorb remaining buffer solution 2 after washing.
(10) it is added in the Streptavidin (kit is included) to each array that 70ul fluorescent dyes combine, film is complete
All standing is incubated cell, is incubated 2h in darkroom at room temperature.
(11) it according to step 5, is cleaned 2 times with 1X cleaning buffer solutions 1.
B fluoroscopic examinations
(1) cleaning buffer solution in aperture is absorbed.
(2) slide is removed from incubation cell and holder.
(3) entire slide is placed in the centrifuge tube of 30ml, enough 1X cleaning buffer solutions 1 is added and cover entire slide,
10min is slightly shaken at room temperature, shakes cleaning 10min with 1X cleaning buffer solutions 2 are slight at room temperature after being repeated twice.
(4) 1000rpm centrifuges 3min, and slide is placed in air thoroughly dry at least 20min (being protected from light).Ensure that fluorescence is swept
Slide is completely dried before retouching.
(5) the green light aperture acquisition signal pattern (excitation spectrum 532nm) of biochip scanner is used.
As a result：Compared with the concentration in marrow, in the marrow being enriched in SAP/DBM materials with closely related thin of skeletonization
Intracellular cytokine：The multiple of BMP-6, PDGF-BB, VEGF, and EGF point is than being 1.38,1.30,1.65, and 3.95.
Shown in Fig. 1-6：The colloid substance of fusogenic peptide/visible water white transparency of DBM materials attaches to inside mesh stent and DBM tables
Face.
3 D video microscopic image analysis system measurement DBM pore sizes are (414.32 ± 175.00) μm (n=60),
Fusogenic peptide/DBM material pore sizes are (220.55 ± 158.54) μm (n=60), and fusogenic peptide/DBM is overall equal with the apertures DBM
Number significant difference (p ＜ 0.05).Blank DBM materials are in tridimensional network (Fig. 1), and pore interior is interconnected, and aperture is opposite
It is larger；Fusogenic peptide/DBM composite materials not only maintain the three-dimensional mesh structure of natural bone material, and in the surfaces DBM and net
Sheet, filament (Fig. 2, Fig. 3) are formed inside hole, are crossed-over into mesh-like structure, aperture is relatively small, Kong Yukong
Between have small hole be interconnected.It can be seen that material (Fig. 5,6, wherein the composite material after modifying as seen from Figure 5 after modification
Inside is mesh and has a large amount of enrichment of cell figure) than the material before modification, (Fig. 4, cell is to be dispersed in material as seen from Figure 4
Surface), there is higher bioaccumulation efficiency.
Claims (5)
1. a kind of method of the high osteogenic activity bone of structure, which is characterized in that there is following steps：
1）The preparation of enrichment material
Take homogeneous allogenic bone decalcification bone supporting stand；
The fusogenic peptide of purity >=95% is dissolved in 20% sucrose solution, until final concentration of the 1% of solution, it is ultrasonically treated 30 minutes,
Obtain peptide solution；The fusogenic peptide includes RADA16-I peptides and is coupled to the osteostatin of the C-terminal of RADA16-I peptide sequences
Peptide, amino acid sequence such as SEQ ID NO:Shown in 2；
2）It takes allogeneic decalcification bone supporting stand to be fully immersed in peptide solution 4-8 minutes, takes out, with the phosphoric acid buffer of pH value 7.4
Flushed obtains the allogeneic de-calcificated bone carriage of self-assembling peptides modification；
3）Enrichment
Marrow injection is filled in the bone uptake enricher of the homogeneous allogenic bone de-calcificated bone carriage of self-assembling peptides modification, bone
Marrow is enriched with several times, obtains high osteogenic activity bone.
2. a kind of method of the high osteogenic activity bone of structure, which is characterized in that there is following steps：
1）The preparation of enrichment material
Take homogeneous allogenic bone decalcification bone supporting stand；
The fusogenic peptide of purity >=95% is dissolved in 20% sucrose solution, until final concentration of the 1% of solution, it is ultrasonically treated 30 minutes,
Obtain peptide solution；The fusogenic peptide includes the C-terminal by RADA16-I peptide sequences via amino acid residue GG and osteostatin peptides
Coupling is formed, amino acid sequence such as SEQ ID NO:Shown in 3；
2）It takes allogeneic decalcification bone supporting stand to be fully immersed in peptide solution 4-8 minutes, takes out, with the phosphoric acid buffer of pH value 7.4
Flushed obtains the allogeneic de-calcificated bone carriage of self-assembling peptides modification；
3）Enrichment
Marrow injection is filled in the bone uptake enricher of the homogeneous allogenic bone de-calcificated bone carriage of self-assembling peptides modification, bone
Marrow is enriched with several times, obtains high osteogenic activity bone.
3. method according to claim 1 or 2, it is characterised in that：The sequence of the osteostatin peptides such as SEQ ID
NO:Shown in 4.
4. method according to claim 1 or 2, it is characterised in that：The amino acid sequence of the RADA16-I peptides is SEQ
ID NO:1。
5. method according to claim 1 or 2, it is characterised in that：Step 3）The marrow enrichment is 4 times.
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| CN103877616A (en) * | 2014-03-18 | 2014-06-25 | 北京大学第三医院 | Cartilage tissue engineering repair bracket and preparation method thereof |
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