CN102241738A - Mimic polypeptide of non-collagen protein and application of the mimic peptide in biomimetic mineralization - Google Patents
Mimic polypeptide of non-collagen protein and application of the mimic peptide in biomimetic mineralization Download PDFInfo
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- CN102241738A CN102241738A CN2011101488381A CN201110148838A CN102241738A CN 102241738 A CN102241738 A CN 102241738A CN 2011101488381 A CN2011101488381 A CN 2011101488381A CN 201110148838 A CN201110148838 A CN 201110148838A CN 102241738 A CN102241738 A CN 102241738A
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Abstract
The invention discloses mimic polypeptide of non-collagen protein and application of the mimic polypeptide in biomimetic mineralization. An amino acid sequence of the polypeptide in the invention is represented as SEQ ID NO:1. Function and effect of polypeptide mimic soluble non-collagen protein (BSP and DMP1) in the invention are as follows: controlling deposition and growth of mineral in I type collagen, further simulating formation processes of human body natural hard tissues, promoting mineral nucleation and growth, increasing combination capability of the mineral and collagen fiber, raising mineralization degree of the mineral especially hydroxy apatite in the I type collagen, thereby promoting restoration of bone and dentine.
Description
Technical field
The present invention relates to a kind of mimic peptide of noncollagen protein and the application in biomimetic mineralization thereof.
Background technology
Sclerous tissues such as bone, dentine aligns establishment by mineralized collagen fiber orderly, multistage to constitute.Type i collagen template albumen is bone, Dentinal main matrix albumen, and hydroxyapatite is deposited on the surface and the interstitial area of I type stromatin.These mineral, particularly interstitial area hydroxyapatite mineral play keying action to the mechanical property of bone, Dentinal excellence.
At present to main two kinds of bone, Dentinal repair materials.A kind of mainly is the cement (bone cement etc.) of mainly being had an opportunity to form by inorganic mineral or polymer, fills defective tissue; A kind of is the bionical repair materials of sclerous tissues by hydroxyapatite and collagen blend.The former is different with skeleton after all, after the certain hour, reduces with human body bone, dentin matrix binding ability, causes weighting material loosening.The latter is as a kind of bionical repair materials, though have composition and the structure near with the human body bone photo, hydroxyapatite and collagen substrate binding ability are limited, so compare with human body natural's bone, dentine, its mechanical property is still waiting further raising.Therefore need develop the material that can promote collagen substrate mineralising.
Summary of the invention
An object of the present invention is to provide a peptide species and derivative thereof and application thereof.
Polypeptide provided by the invention, its aminoacid sequence is shown in SEQ ID NO:1.
Polypeptide derivative provided by the invention is the polypeptide derivative that obtains after the serine phosphorylation in the polypeptide shown in the SEQ ID NO:1 is handled, and its structural formula is suc as formula shown in the I.
Formula I
Another object of the present invention provides a kind of mineralizer.
Mineralizer provided by the present invention, its activeconstituents are polypeptide derivative shown in polypeptide shown in the SEQ ID NO:1 or the above-mentioned formula I.
In the above-mentioned mineralizer, described mineralizer is for promoting the mineralizer of collagen protein mineralising.
In above-mentioned arbitrary described mineralizer, described mineralizer carries out the mineralizer of mineralising for promoting hydroxyapatite to collagen protein.
In above-mentioned arbitrary described mineralizer, the mineralizer of described mineralizer in preparation hydroxyapatite and collagen protein matrix material, using.
In above-mentioned arbitrary described mineralizer, the mineralizer of described mineralizer in preparation dentine or ground substance of bone repair materials, using.
In above-mentioned arbitrary described mineralizer, described collagen protein is a type i collagen albumen.
The following application of polypeptide derivative shown in polypeptide or the above-mentioned formula I shown in the SEQ ID NO:1 also belongs to protection scope of the present invention:
Polypeptide derivative shown in polypeptide or the above-mentioned formula I shown in the SEQ ID NO:1 has application in the product that promotes the collagen protein mineralization function in preparation, is specially to have in preparation to promote hydroxyapatite collagen protein to be carried out the application in the product of mineralization function;
The application of polypeptide derivative shown in polypeptide or the above-mentioned formula I shown in the SEQ ID NO:1 in preparation hydroxyapatite and collagen protein matrix material;
The application of polypeptide derivative shown in polypeptide or the above-mentioned formula I shown in the SEQ ID NO:1 in preparation dentine or ground substance of bone repair materials.
In above-mentioned arbitrary described application, described collagen protein is a type i collagen albumen.
Polypeptide of the present invention comprises two portions, a part is continuous 8 L-glutamic acid E, the L-glutamic acid of this repeated arrangement is important aminoacid sequence among the BSP (bone sialoprotein bone sialoprotein), have very strong calcium ion adsorptive power, continuous 8 L-glutamic acid also are the important foundations of its performance regulation and control mineralization function.Another part is DSESSEEDR, and this segment derives from the distortion of middle DSEDDEEDR of DMP1 (dentine matrix protein 1 dentin matrix protein) and sequences such as DSEDDEEDR and SSEDSDSQDSSR.These several fragments also be simultaneously in the DMP1 albumen with type i collagen bonded action site, they can be adsorbed in the interstitial area of collagen.
Sequences polypeptide involved in the present invention has strong electronegativity.When this polypeptide adds in collagen system, its DSESSEEDR partly will be preferential with collagen in the concentrated e1 district of relative positive charge by the electrostatic interaction combination.This process can influence the collagenous fibrosis process, strengthens the assembling of collegen filament, thereby the collegen filament diameter is increased.On the other hand, polypeptide involved in the present invention is rich in a large amount of acidic-groups, and these functional groups provide a large amount of forming core sites, can promote the forming core and the growth of mineral, improves the mineralization degree of collegen filament.
The function and the effect of polypeptide imitation solubility noncollagen protein of the present invention (BSP and DMP1), the regulation and control mineral are in the deposition and the growth of type i collagen, the forming process of the natural sclerous tissues of further bionical human body, promote mineral forming core and growth, increase the binding ability of mineral and collegen filament, mineral, the particularly hydroxyapatite mineralization degree on type i collagen be can better improve, thereby bone and Dentinal reparation promoted.
Description of drawings
Fig. 1 is the stable state gel systems.
Fig. 2 is external biomimetic mineralization collegen filament SEM pattern.
(A) collagen/one day (B) collagen of polypeptide system mineralising system mineralising is one day.
(C) collagen/polypeptide system mineralising one week (D) collagen system one week of mineralising.
Fig. 3 is impaired dentine remineralization (erosional surface) scanning electron microscope result (4 weeks of mineralising) under the polypeptide effect.
(A C) adds polypeptide (B, D) control group.Arrow is depicted as on the type collagen fiber mineral of periodic arrangement and asks among the figure (C).
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The amino acid sequence of polypeptide of using in the following experiment all is that synthetic obtains as EEEEEEEEDSESSEEDR (SEQ ID NO:1), and three Serines have wherein been carried out the phosphorylation processing, and its structural formula is suc as formula shown in the I:
Formula I
The application of embodiment 1, polypeptide
One, polypeptide forming core and mineralization experiments
Adopt stable state gel systems (steady-state agarose gel system) that the mineralization ability of polypeptide is analyzed.Stable state gel reaction system schematic as shown in Figure 1.Take by weighing an amount of agarose (agarose) and in deionized water, be heated to the boiling dissolving, be configured to 3% agarose solution.Be cooled to below 50 ℃, add NaCl, Tris-HCl and polypeptide mother liquor respectively, to ultimate density be: 2% agarose, 150mM NaCl, 10mM Tris-HCl, different experiments peptide concentration that group adopts is respectively 0,0.03,0.3,3,30 and 100 μ g/ml.With the agarose mixing solutions 5ml for preparing pack into rapidly the envelope have in the reactive tank of semi-permeable membranes (molecular weight cut-off is 1000Da).After treating its gelation, begin to feed calcium, phosphorus reaction liquid.Calcium reaction solution composition is: 6.5mM CaCl
2, 150mM NaCl, 20mM HEPES, PH=7.4.Phosphorus reaction liquid composition is: 3.9mM Na
3PO
4150mM NaCl 20mM HEPES, PH=7.4.The inflow of reaction soln is controlled by peristaltic pump, flow velocity 1ml/h.Be reflected at and carried out under 37 ℃ 7 days.After experiment finishes, gel is taken out, inserts in the concentrated nitric acid solution of 10ml, be heated to 80 ℃, treat that it fully dissolves after, volume reduces to till the 1ml.This acid solution is diluted to 10ml, measures the calcium phosphorus content respectively.Wherein, (172-8035 Japan) measures calcium ion content employing atomic absorption spectrum, and phosphate anion content adopts the phospho-molybdic acid colorimetry to analyze.
Adopt the stable state gel systems to analyze the ability of this polypeptide regulation and control calcium phosphate mineralising.Atomic absorption spectrum and phospho-molybdic acid colorimetry are analyzed (table 1) to calcium phosphorus content in its gel, can see: along with the increase of peptide concentration, the calcium microcosmic salt content of its formation obviously increases.This shows that polypeptide has very strong nucleation ability to the calcium microcosmic salt, and the polypeptide solution of high density can be caught more calcium ion and phosphate anion, thereby promotes the deposition of mineral.
The polypeptide mineralization ability is analyzed under the table 1 stable state gel mineralising system
Two, polypeptide is to the mineralising promoter action of collagen protein
The collagen that uses in the experiment is type i collagen albumen, according to the described method of people such as Pins [Pins, G.D., Christiansen, D.L., Patel, R., et al.Self-assembly of collagen fibers.Influence of fibrillar alignment and decorin on mechanical properties.Biophysical Journal.1997,73:2164-2172.], extract sour solvable type i collagen albumen from the mouse tail tendon.
In the small test tube of 2ml, add 1.5ml collagen solution (2mg/ml), drip 30 μ l polypeptide solutions (1mg/ml) again.Thorough mixing is placed on 4 ℃ of refrigerator overnight.Test tube seals with semi-permeable membranes (SpectraPor-3, molecular weight cut-off are 3500Da).It is 2mM CaCl that 200ml concentration is equipped with in the small test tube immersion
2In the beaker of solution, left standstill 1 hour.In beaker, add 200ml damping fluid (1.2mM NaH subsequently
2PO
4-Na
2HPO
4, PH=7.4).Do not add polypeptide in the control group experiment, other conditions are identical.Be reflected under 37 ℃ and carry out.
Polypeptide is added collagen biomimetic mineralization system, and mineralized collagen fiber SEM (scanning electronic microscope) pattern after mineralising 1 day and 1 week as shown in Figure 2.Fig. 2 (B) and 2 (A) are respectively the SEM pattern of collegen filament mineralising after 1 day before and after polypeptide adds.After adding polypeptide, it is collegen filament about 40nm that tropocollagen molecule is assembled into diameter, and these collegen filament are arranged in parallel with each other, further be assembled into collagenous fiber bundle, and do not add in the collagen mineralising system of polypeptide, it is in disorder that collagenous fiber bundle distributes, and arranges unordered.As seen, polypeptide has promoted the molecule assembling of collegen filament, and adjacent collegen filament are more prone to be parallel to each other, and closely arrange, and have formed zonal robust fibre or have been woven into the fibrous bundle net.Fig. 2 (C) and (D) be the pattern that this mineralising system is reacted the collegen filament after 1 week.1 day result is similar with mineralising, and the collegen filament behind the interpolation polypeptide are more prone to form thicker fibrous bundle or mesh grid.Particularly, the collegen filament behind the interpolation polypeptide, the visible significantly mineralized layer in its fibrous bundle surface is shown in asterisk among Fig. 2 (C).This experiment shows that the surperficial mineralising of the many collagens of polypeptide has played promoter action.
Equally, (add polypeptide/do not add polypeptide) collagen mineralization product under two kinds of different systems is carried out Infrared spectroscopy, calculated phosphoric acid salt v1, the integral area ratio of v3 peak and amide I peaks.This area is than the mineral content that has reflected in the collagen system.The result shows that mineralising 1 day and 1 week of mineralising in collagen/polypeptide system, its area ratio is respectively 0.4478 and 0.6998, and pure collagen mineralising system is respectively 0.3509 (1 day) and 0.4601 (1 week).Therefore can judge that behind the interpolation polypeptide, the collagen mineralization degree obviously improves.
Three, polypeptide is to damaged Dentinal remineralization
Testing used human third molar is provided by Cret mouth care research institute of Beijing P﹠G Technology Ltd..The tooth donor is 15～30 years old (all informed consents), and dental health does not have carious tooth or other defect.30 ground one's teeth in sleep be parallel to the face of chewing and be cut into the thin slice that thickness is about 3mm.Adopting EDTA (ethylenediamine tetraacetic acid (EDTA)) to carry out decalcification then handles.The dentine thin slice is immersed in the 0.5M EDTA disodium salt solution (pH=7.0) decalcification 6 hours.Tris-HCl solution with 50mM cleans three times subsequently, thereby obtains the dentine thin slice of decalcification.
Dentine demineralization thin slice is immersed in the polypeptide solution of 0.1mg/ml, place 4 ℃ of refrigerator overnight.Use the deionized water clean surface subsequently 3 times, remove unconjugated polypeptide.A part does not process and gives over to control group.It is 2mM CaCl that 500ml concentration is equipped with in these two groups of samples immersions
2In the solution beaker, static 1 hour.Add 500ml phosphoric acid buffer (1.2mM NaH subsequently
2PO
4-Na
2HPO
4, PH=7.4).Be reflected under 37 ℃ and carried out for 4 weeks, change the calcium phosphorus solution weekly one time.
The result of scanning electronic microscope shows that the dentin surface through polypeptide is handled has deposited thicker layer mineral (Fig. 3 A).After it should be noted that the interpolation polypeptide, formed a large amount of nano-mineral nucleus (Fig. 3 C arrow), and be periodic arrangement along the collegen filament long axis direction, its gap cycle is 64nm.As seen, polypeptide has promoted the interstitial area mineralising of dentin collagen albumen substrate.
Claims (10)
1. a peptide species or derivatives thereof is following 1) or 2) shown in:
1) polypeptide: the polypeptide of aminoacid sequence shown in SEQ ID NO:1.
2) polypeptide derivative: with the polypeptide derivative that obtains after the processing of the serine phosphorylation in the polypeptide shown in the SEQ ID NO:1, its structural formula is suc as formula shown in the I.
Formula I
2. mineralizer, its activeconstituents is described polypeptide of claim 1 or the described polypeptide derivative of claim 1.
3. mineralizer according to claim 2 is characterized in that: described mineralizer is for promoting the mineralizer of collagen protein mineralising.
4. according to claim 2 or 3 described mineralizers, it is characterized in that: described mineralizer carries out the mineralizer of mineralising for promoting hydroxyapatite to collagen protein.
5. according to claim 2 or 3 or 4 described mineralizers, it is characterized in that: the mineralizer of described mineralizer in preparation hydroxyapatite and collagen protein matrix material, using;
Or the mineralizer of described mineralizer in preparation dentine or ground substance of bone repair materials, using.
6. according to arbitrary described mineralizer among the claim 2-5, it is characterized in that: described collagen protein is a type i collagen albumen.
7. described polypeptide of claim 1 or described polypeptide derivative have application in the product that promotes the collagen protein mineralization function in preparation, are specially to have in preparation to promote hydroxyapatite collagen protein to be carried out the application in the product of mineralization function.
8. described polypeptide of claim 1 or the described polypeptide derivative application in preparation hydroxyapatite and collagen protein matrix material.
9. described polypeptide of claim 1 or the described polypeptide derivative application in preparation dentine or ground substance of bone repair materials.
10. according to claim 7 or 8 described application, it is characterized in that: described collagen protein is a type i collagen albumen.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103417386A (en) * | 2012-05-25 | 2013-12-04 | 清华大学 | Calcium phosphate based dental restoration material for treating dentin hypersensitiveness |
| CN104292309A (en) * | 2014-09-29 | 2015-01-21 | 广西中医药大学 | Micro-molecule polypeptide |
| CN106890095A (en) * | 2016-12-07 | 2017-06-27 | 中日友好医院 | A kind of dental prosthetic material and its application for promoting dentine remineralization |
| CN113527425A (en) * | 2021-08-17 | 2021-10-22 | 清华大学 | Polypeptide derivative, medicine and application thereof |
| CN114832155A (en) * | 2022-04-29 | 2022-08-02 | 大连理工大学 | Preparation and application of biomineralization-based hydroxyapatite with controllable hydrophilicity and hydrophobicity |
| US11617711B2 (en) | 2018-08-30 | 2023-04-04 | University Of Washington | Compositions and methods for dental care |
-
2011
- 2011-06-03 CN CN2011101488381A patent/CN102241738A/en active Pending
Non-Patent Citations (4)
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| CHEN,H.F.: "Acellular synthesis of a human enamel-like microstructure", 《ADVANCED MATERIALS》 * |
| PARK,S.: "Romberg,E.,et al.On the brittleness of enamel and selected dental materials", 《DENTAL MATERIALS》 * |
| SAWADA: "Spatial-distribution of enamel proteins and fibronectin at early stages of rat Incisor tooth formation", 《ARCHIVES OF ORAL BIOLOGY》 * |
| 程振江: "蛋白调控骨、牙矿化的研究及模拟", 《中国学术文献网络出版总库》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103417386A (en) * | 2012-05-25 | 2013-12-04 | 清华大学 | Calcium phosphate based dental restoration material for treating dentin hypersensitiveness |
| CN104292309A (en) * | 2014-09-29 | 2015-01-21 | 广西中医药大学 | Micro-molecule polypeptide |
| CN104292309B (en) * | 2014-09-29 | 2017-04-12 | 广西中医药大学 | Micro-molecule polypeptide |
| CN106890095A (en) * | 2016-12-07 | 2017-06-27 | 中日友好医院 | A kind of dental prosthetic material and its application for promoting dentine remineralization |
| CN106890095B (en) * | 2016-12-07 | 2019-11-12 | 中日友好医院 | A kind of dental prosthetic material and its application for promoting dentine remineralization |
| US11617711B2 (en) | 2018-08-30 | 2023-04-04 | University Of Washington | Compositions and methods for dental care |
| CN113527425A (en) * | 2021-08-17 | 2021-10-22 | 清华大学 | Polypeptide derivative, medicine and application thereof |
| CN114832155A (en) * | 2022-04-29 | 2022-08-02 | 大连理工大学 | Preparation and application of biomineralization-based hydroxyapatite with controllable hydrophilicity and hydrophobicity |
| CN114832155B (en) * | 2022-04-29 | 2022-11-15 | 大连理工大学 | Preparation and application of biomineralization-based hydroxyapatite with controllable hydrophilicity and hydrophobicity |
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Application publication date: 20111116 |