CN101743014A - oligopeptide for enhancing osteointegration and osteogenesis - Google Patents
oligopeptide for enhancing osteointegration and osteogenesis Download PDFInfo
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Abstract
The present invention relates to an oligopeptide for enhancing osteointegration and osteogenesis. More specifically, it relates to bioactive oligopeptide compounds having the general structural formula (C or K)-(I, K or P)-(I, P or K)-(K or P)-(K, P or S)-(P or S)-(A or S)-(A, P or S)-(A, P or T)-(E, P or T)-(E, L or T)-(E, L or S)-(A, L or S)-(A, I or S)-(A, I or S)-(I, M or S)-(L, M or S)-(C, L or M). The oligopeptide of the present invention is effective in bone filler replacement, vertical treatment and horizontal treatment. By using this invention, the continuous action of the oligopeptide on the surface of an implant treated with a thin film of the same can reduce the time of initial osteointegration. Additionally, an increase in the success rate of surgical operations on osteopenia patients can be achieved by inducing an increase in osteoblasts and function. It is also possible to achieve bone formation when bone mass is insufficient, without bone replacement, leading to a reduction in surgical operation costs.
Description
Technical field
The present invention relates to a kind of be used for promoting general implantation body synosteosis and osteoplastic oligopeptide.
Background technology
The present invention relates to a kind of be used for promoting general implantation body synosteosis and osteoplastic oligopeptide.Particularly, the present invention concentrates on the oligopeptide that is used for Dental Implant (dental implant).
1. the shape of implantation body's assembly and term
Dental Implant has the shape shown in Fig. 1, and has comprised implantation body or fixed body (fixture), the corona of using as artificial tooth root (being the artificial tooth) and will plant the abutment that body is connected to corona.Implantation body is made of with the implant surface that directly contacts alveolar bone the tooth joint that is connected to abutment.Because implant surface is determined the function of synestotic length and Dental Implant, so it is the key element of Dental Implant.
2. the function of implantation body's assembly and material
Dental Implant is meant and comprises the assembly that is placed in the artificial titanium root of the tooth in the alveolar bone that it can provide sensation and the function identical with original tooth, and can not damage other contiguous tooth.When Dental Implant being placed in the alveolar bone back one period scheduled time, Dental Implant experience synosteosis, and abutment is connected with it successively with corona.Normal function such as for example chew because corona can be carried out, and can after corona is connected to Dental Implant, use 15 to 20 years, so the synosteosis of implantation body should be able to keep semi-durablely.For this reason, implantation body generally can reach 5,000,000 cycle by the pre-fatigue life test of experience (pre-fatigue life test) under the load of 250 Ns (N).
A key of Dental Implant performance is: shorten the synosteosis phase.That is to say that if can't realize synosteosis in a short time, the treatment time limit of implantation body will increase so, make thus to be difficult to form Dental Implant market, and implantation body can not carry out normal masticatory function.Therefore, need to shorten initial bone in conjunction with the phase.
For addressing this problem, in implantation body settles, with the somatomedin that promotes osteoblastic proliferation and differentiation (for example, transforming growth factor-beta (transforming growth factor-β, TGF-β) and insulin like growth factor-1 (insulin-like growth factor-1, IGF-1)) or bioactive materials (for example, bone morphogenetic protein(BMP) (bone morphogenetic protein, BMP)) directly be coated on implant surface, or be deposited on around the operative site.Yet, before settling implantation body, bioactive materials directly being coated on the method for implant surface and impracticable, this is because this method needs a large amount of bioactive materials, and can't guarantee after arrangement effectively bioactive material retains.In addition, the sedimentary organism active material can only be carried out in extremely limited zone around operative site, can't carry out effective synosteosis and osteanagenesis on whole implant surface thus, and the operation cost is increased.In addition, the standardization of this operation method still has to be waited to set up, and the valid function of the method and case control can not be guaranteed, so that in fact can active adoption sedimentary organism active material around operative site.
3. the technology trend of Dental Implant
The Dental Implant technology generally is divided into designing technique and resurfacing technology.The resurfacing technology is to determine the initial bone of implantation body in conjunction with the key factor in phase and life-span, and can be divided into for 4 generations according to the development of this technology.
First generation implantation body is the machining implantation body of nineteen sixty-five by Branemark (Sweden (Sweden)) exploitation.First generation implantation body needed 4 to 8 months to realize synosteosis, brought various problems thus.Therefore, develop the second filial generation implantation body that long-pending increase of implant surface and synosteosis time shorten.Second filial generation implantation body relates to the method for for example acid treatment, sandblast, sandblast reconstruction such as acid treatment, sintering subsequently implant surface, and it makes the synosteosis time shorten to 3 to 6 months.Third generation implantation body relates to hydroxyapatite (hydroxyapatite, the surface treatment of HA) coating, anodic oxidation, interpolation fluorine (fluorineaddition surface treatment), the hydrophilic surface treatment of enhancing etc.Although according to the age of exploitation, the HA coating belongs to second filial generation technology, and according to the technical development classification, it belongs to third generation technology.Anodic oxidation is to be designed to improve synosteosis by forming porous surface and thick oxide layer.The surface treatment of the interpolation fluorine of being developed is (for example, from Sweden ASTRA Tech, the OsseoSpeed of Inc
TM) strengthen synosteosis by adding fluoride to implant surface.The hydrophilic surface treatment of the enhancing of being developed is (for example, from the SLActive of Switzerland (Switzerland) ITI company
TM) shorten the synosteosis time by strengthening surface energy and increasing hydrophilic.Third generation implantation body uses and adds for example method of bone formation materials such as fluorine, calcium and phosphorus, and the method for making porous surface and formation thick oxide layer, so that the synosteosis time is shortened to 2 to 4 months.Yet except that prior art, third generation technology also will spend at least 3 months and repair dental defect.In addition, although third generation implantation body is more successful than second filial generation implantation body, its success rate still can not be satisfactory for insecure alveolar bone, and limited the application in the unstable individuality of alveolar bone (for example, gerontal patient).Therefore, be lower than 3 months restriction, still need to develop the 4th generation implantation body (table 1, Dental Implant surface-treated type and feature) because third generation resurfacing has the synosteosis time shortened to.
Table 1
| The surface-treated type | The synosteosis time (moon) | Feature | |
| The first generation | Mechanical processing process | ??4-8 | Initial implantation body only passes through machining processes |
| The second filial generation | Increase surface area via sandblast and etching | ??3-6 | Give surface roughness by the implantation body that machining is crossed and strengthen synosteosis |
| The third generation | HA coating, Osseospeed, SLActive, anodic oxidation | ??2-4 | Strengthen synosteosis by chemical surface treatment |
| The surface-treated type | The synosteosis time (moon) | Feature | |
| The 4th generation | Be coated with bioactive materials | ??1.5-2 | Strengthen synosteosis by the biochemistry surface treatment, and even under weak bone mass situation, increase plantation program possibility of success |
Disclose about osteoplastic multinomial research, the bone morphogenetic protein(BMP) (BMP) that belongs to TGF group plays an important role in alveolar bone forms.Because first part of disclosed result of study is about BMP, many scientists have utilized animal to experimentize, and deliver experimental result, that is, BMP not only can both induce the bone reparation and strengthen bone structure in animal but also in the mankind.Yet when BMP directly being thrown with individuality, its can be decomposed rapidly by intravital enzyme, and loses its function in several minutes.In other words, for effectively inducing new bone formation, must during the plantation program, throw BMP rapidly with suitable high concentration.Therefore, when using BMP to promote bone formation, there are various problems in stability, pharmacodynamics and cost aspect.
Protein is to constitute to thousands of aminoacid by hundreds of.The report of the research of protein involved 26S Proteasome Structure and Function claims that epi-position (epitope) is present in the proteinic amino acid whose sequence of formation.According to this result, carrying out a research, can carry out the technology that can simulate described proteinic function with the minimum aminoacid sequence of target protein identical functions so that find.
Recently, developed the technology of using peptide synthesizer to make amino acid needed sequence rapidly.In general, by the target protein gene is introduced in microorganism or the zooblast gene, large-scale culture and purification are made recombiant protein subsequently.The introducing that produces the gene of target protein relates generally to viral vector, cell culture medium, non-virus particle carrier, liposome transfection carrier (lipofection) etc.Use viral vector to have multiple advantage, but also can cause problem: genetically modified stability and expression degree obviously reduce in cell and the humoral immune reaction.
On the other hand, the method of using peptide synthesizer to make oligopeptide does not need series of genes to modify, and its effective part is in fact: can use the method exploitation and synthetic a kind of oligopeptide, described oligopeptide does not provide the complete amino acid sequence of native protein, and provide the one partial amino-acid series, especially can represent the pass key sequence of described proteinic function.When synthetic finishing, (high performance liquid chromatography HPLC) comes the described oligopeptide of purification, and this relates to comparatively simply handles to carry out high performance liquid chromatography, and produce the higher product of purity, compared with the target protein of purification by the genetic modification acquisition.In addition, because the use synthesizer allows the aminoacid sequence of (for example, acetylation, amidatioon, the phosphorylation etc.) oligopeptide of part modification optionally, therefore possibility composite part aminoacid sequence is different from the oligopeptide of the manually modified mistake of native protein.Particularly, if aminoacid sequence is obviously short than described protein, so with the exploitation via genetic modification (polymerase chain reaction (polymerase chainreaction, PCR), gene clone, colony screening etc.) stable produce the described cell line of cell line, large-scale culture of target protein and the method for separation and purification target material is compared, the organic synthesis that uses synthesizer to carry out can shorten the time that oligopeptide is made, and reduces its cost.
In recent years, fully research participates in osteoplastic BMP and extracellular matrix, to find to carry out in the complete amino acid sequence aminoacid sequence of key function.In addition, also to use synthesizer make these aminoacid sequences with and measures of effectiveness study.
In addition, also at will be for example enhancing such as protein or oligopeptide synosteosis and osteoplastic bioactive materials be coated on implant surface and carry out multinomial research.
Following patent be applied for about the exploitation of bioactive materials such as for example oligopeptide and the patent of introducing described material in implant surface.
The open case of korean patent application discloses a kind of Dental Implant and a kind of method that is coated with described material that is coated with bioactive materials 2006-0110190 number.Described method comprises: the peripheral shape in Dental Implant becomes active film; On described active film, form the intermediate reaction layer; And use the host material that connects via chemical covalent bond and bioactive materials or with the polymer-coated described intermediate reaction layer of bond to bioactive materials, make thus even after sterilization, still keep biological activity.In addition, this Dental Implant not only allows to distribute expediently and store, but also makes the adhesion strength between bioactive materials and the implantation body be enough to shorten the synestotic time in the implant operation.
The oligopeptide that the open case of korean patent application discloses a kind of for the surface treatment Dental Implant 2006-0110189 number.Described oligopeptide comprises the peptide with the copolymer bond, and directly is coated on the Dental Implant surface, with enhance bone growth and shorten the synosteosis time.Particularly, described oligopeptide has formula R 1-(A-B-C) n-R2-(A-B-C) m-L, and wherein R1 is hydrogen (H), amino acid residue, fatty acid residue or the copolymer chain with biodegradable characteristics, and R2 is basic at interval, and L is for connecting base.
The open case of korean patent application 2006-0101019 number (patent No. 0676945) discloses a kind of bone graft that is coated with the peptide that strengthens osseous tissue formation, and the transformed support of a kind of organizational project.The surface coated of described bone graft has the adherent peptide of inducing cell and/or is derived from the peptide of tissue growth factor.Particularly, described patent discloses a kind of peptide that is derived from bone morphogenetic protein(BMP) (BMP-2,4,6).2006-0082060 number (patent No. 0630903) a kind of macromole screened film of announcement of the open case of korean patent application and a kind of peptide that strengthens osseous tissue formation that utilizes are fixed in its lip-deep implantation body.The surface coated of described macromole screened film and implantation body has the adherent peptide of inducing cell or is derived from the combination of the peptide and the cross-linking agent of tissue growth factor.Particularly, described patent discloses a kind of peptide that is derived from bone morphogenetic protein(BMP) (BMP-2).
Yet, above-mentioned peptide still need improvement with remarkable shortening initial bone in conjunction with the phase, and especially need to increase the lower patient's of bone mass success rate of operation.
The present inventor goes out to strengthen synestotic ability and native protein quite or be better than the new amino acid sequence of native protein via the orderly modular design of amino acid whose minimum, and its effect is analyzed.And the present inventor selects the oligopeptide that represents the optkmal characteristics that are applicable to implant surface, and obtains to have the oligopeptide of particular sequence, and as disclosed herein, described oligopeptide with particular sequence can strengthen initial bone combination and bone formation.
Summary of the invention
[technical problem]
The present invention relates to address the above problem, and embodiment comprise provide with BMP-2 quite or be better than the oligopeptide of the effect of BMP-2, described oligopeptide generally is used to promote osteoblastic proliferation and differentiation, also strengthen bone formation simultaneously, and its stability is higher than bioactive materials far away.
Another embodiment comprises a kind of Dental Implant allocation method, described method comprises the surface of oligopeptide being introduced Dental Implant with chemical mode, to promote the initial bone combination, shorten the high success rate of operation that implantation body settles the phase and especially guarantees the patient of the lower and bone quantity not sufficient of bone mass thus.
Another embodiment comprises a kind of Dental Implant, with low-cost uniform deposition oligopeptide is arranged on its whole surface, thereby represent and similar osteanagenesis of BMP and synosteosis efficient, and the extent of stability identical, reduce the extra duty of Dental Implant manufacturing, distribution and storage thus with inorganic coating material.
[technical solution]
According to an aspect of the present invention, strengthen synosteosis and osteoplastic oligopeptide and have following structural formula: (C or K)-(I, K or P)-(I, P or K)-(K or P)-(K, P or S)-(P or S)-(A or S)-(A, P or S)-(A, P or T)-(E, P or T)-(E, L or T)-(E, L or S)-(A, L or S)-(A, I or S)-(A, I or S)-(I, M or S)-(L, M or S)-(C, L or M).
The present invention relates to be applicable to the oligopeptide of general implantation body.In this article, detailed description will concentrate on the oligopeptide that is used for Dental Implant.
The oligopeptide that is applicable to Dental Implant can further comprise the amino acid residue with terminal SH group, with activation described oligopeptide is stablized in the titanium of introducing on the implant surface (Ti).Particularly, described oligopeptide may be for having residue (C, L or Y)
nThe peptide of (wherein n is 1 or 2), and more preferably comprise the peptide of cysteine.
Oligopeptide can be introduced with substrate, with according to the distance between the osteoblastic size Control oligopeptide, and adjusts the relative localization of oligopeptide.The titanium surface experience of implantation body is utilized and is connected the pretreatment that base carries out.For with the functional group's bond that is connected base, oligopeptide can have at N or C-terminal and have-amino acid residue of SH group, and preferred cysteine.
In fact, oligopeptide has free amine group or cysteine at N-terminal for example, easily described oligopeptide is introduced in the implantation body via connecting base with activation.In addition, according to an embodiment, can utilize silane-connection base-peptide bond that oligopeptide is introduced implant surface, thereby make the titanium (Ti) on oligopeptide and the implant surface be in steady statue.
In addition, be applicable to that the oligopeptide of Dental Implant can be for being selected from the oligopeptide by the following group that constitutes: PEP111 (SEQ ID 1), PEP121 (SEQ ID 2), PEP131 (SEQ ID 3), PEP112 (SEQ ID 4), PEP122 (SEQ ID 5), PEP132 (SEQ ID 6) and PEP133 (SEQ ID7).Oligopeptide more preferably can be the oligopeptide that is selected from PEP111, PEP112 and PEP122.Even more preferably, oligopeptide can be PEP111 or PEP122.Most preferably, oligopeptide can be PEP111.
When using peptide synthesizer to prepare oligopeptide with screening enhancing synosteosis and osteoplastic oligopeptide sequence, generated time shortens, and the time of the effective oligopeptide sequence of screening is also shortened.
Oligopeptide according to the embodiment of the invention can be easy to by the known method of one of ordinary skill in the art synthetic.In example of the present invention, use the oligopeptide that obtains from the Peptron company limited.
Because oligopeptide has the structural stability that is better than native protein or recombinant protein, so think that described oligopeptide can be guaranteed to sterilize and the stability of memory period.Therefore, when will represent with native protein quite or be better than native protein be used to strengthen synestotic active according to an embodiment of the invention when oligopeptide is coated on implant surface, expect that described oligopeptide will play crucial effects aspect initial adherence, osteoblastic proliferation and the differentiation, and cause the obvious shortening of synosteosis persistent period.Particularly, think that described oligopeptide is specially adapted to because of the lower patient that can't settle implantation body of bone mass.
According to an embodiment, when oligopeptide is coated on screened film or implant surface, described oligopeptide can the per unit surface area 0.1 to 5.0 milligram amount apply.More preferably described oligopeptide can contain 10 to 21 aminoacid, and can apply with the amount of 0.1 to 3.0 milligram of per unit surface area.
According to conventional methods (for example, above-mentioned document) or according to an embodiment of the invention method will be coated on implant surface according to the oligopeptide of the embodiment of the invention.Can use substrate this moment or connect base.A kind of method of rebuilding implant surface of above-mentioned document illustration; The use of cross-linking agent or substrate; Silane-connection base-peptide bond and with oligopeptide coating etc.
According to one embodiment of the invention, use oligopeptide with following sequence:
SEQ?ID?1：R1-CKIPKPSSAPTELSAISMLYL-R2；
SEQ?ID?2：R1-CIPKPSSAPTELSAISMLYL-R2；
SEQ?ID?3：R1-CPKPSSAPTELSAISMLL-R2；
SEQ?ID?4：R1-KIPKPSSAPTELSAISMLYLC-R2；
SEQ?ID?5：R1-KIPKPSSAPTELSAISMLYC-R2；
SEQ?ID?6：R1-KIPKPSSAPTELSAISMLC-R2；
SEQ?ID?7：R1-KIPKPSSAPTELSAISMC-R2。
Wherein, cysteine residues can be introduced the N or the C-terminal of oligopeptide, promote bond pretreated connection base on titanium (Ti) surface of implantation body thus.
Above represented single-letter be encoded to used always in this technology and be explained as follows:
| The single-letter coding | Abbreviation | Full name |
| ??A | ??Ala | Alanine |
| ??R | ??Arg | Arginine |
| ??N | ??Asn | Agedoite |
| ??D | ??Asp | Aspartic acid |
| ??C | ??Cys | Cysteine |
| ??Q | ??Gln | Glutamine |
| ??E | ??Glu | Glutamic acid |
| ??G | ??Gly | Glycine |
| ??H | ??His | Histidine |
| ??I | ??Ile | Isoleucine |
| ??L | ??Leu | Leucine |
| ??K | ??Lys | Lysine |
| ??M | ??Met | Methionine |
| ??F | ??Phe | Phenylalanine |
| ??P | ??Pro | Proline |
| ??S | ??Ser | Serine |
| ??T | ??Thr | Threonine |
| The single-letter coding | Abbreviation | Full name |
| ??W | ??Trp | Tryptophan |
| ??Y | ??Tyr | Tyrosine |
| ??V | ??Val | Valine |
[beneficial effect]
Can be used as the succedaneum of bone filler according to the oligopeptide of the embodiment of the invention, and provide and strengthen osteoplastic vertical therapeutical effect in vertical direction and strengthen osteoplastic horizontal stretcher effect in the horizontal direction.When with oligopeptide coating Dental Implant, the continuous action of oligopeptide makes initial bone shorten in conjunction with the phase induced osteogenesis cell proliferation and increased functionality thus; The lower patient's of bone mass success rate of operation is increased; Even in the bone lacks situation, when no bone filler, increase the bone formation probability; And reduction operation cost.
Description of drawings
In conjunction with the specific embodiment of alterations, will more clearly understand above-mentioned and others, feature and advantage of the present invention according to hereinafter.
Fig. 1 is the sketch map of Dental Implant.
Fig. 2 is the figure that the cell adhesion of oligopeptide material is analyzed.
Fig. 3 is the figure that the MTS of oligopeptide material analyzes.
Fig. 4 is alkali phosphatase (alkaline phosphatase, ALP) figure of Fen Xiing of oligopeptide material.
Fig. 5 is the figure of the damaged model of bone.
Fig. 6 is for describing the figure of the bone formation performance in 2 weeks and the damaged model of 4 week back bones.
Fig. 7 is in the damaged model of bone, and 2 whens week after processing respectively, coating BMP-2 plants body (impregnation drying method (dip ﹠amp; And the figure of the tissue sample of oligopeptide material processed group (impregnation drying method) dry)).
Fig. 8 is the figure of the comparison analyzed of the ALP of oligopeptide PEP111 and existing RGD sequence according to an embodiment of the invention.
The specific embodiment
Next, will the present invention be described in more detail with reference to following example.Yet one of ordinary skill in the art will be apparent, the invention is not restricted to these examples and can carry out various modifications, replacement, change and its equivalence variation under the situation that does not depart from scope of the present invention.
Example 1: preparation titanium dioxide (TiO
2) sull and substrate
Develop TiO by anodic oxidation
2Behind the sull, the present inventor produces a kind of anodized surface and handles product GSII CellNest.Described surface treatment is identical with the processing of the well-known Straumann Ltd. in affiliated field.
Particularly, carry out anodic oxidation (a kind of method that on titanium (Ti) surface, forms sull) with electrochemical method, on the Ti surface, to form the porous oxide thin film in hole, provide 0.8 to 1.2 the surface roughness Ra and the surface area of increase thus with tiny crater shape.The thickness of sull is adjusted to 2 to 8 microns (μ m),, increase corrosion resistance simultaneously so that stability to be provided to suppress metal ion from the Ti surface emitting.In this example, anodized surface is handled product GSIICellNest as TiO
2Sull.
Example 2: strengthen the design and the preparation of synosteosis and osteoplastic oligopeptide
The present inventor modifies the aminoacid sequence that serves as the pass key sequence of osteoblastic proliferation and differentiation in bone morphogenetic protein(BMP)-2 (BMP-2), fiber adhesion albumen (fibronectin) be connected albumen (vitronectin) with glass the complete amino acid sequence by part, uses peptide synthesizer to synthesize subsequently and prepares oligopeptide.After synthetic, with product purification to 95% or higher purity, utilize nuclear magnetic resonance, NMR (Nuclear Magnetic Resonance, NMR) determining molecular weight subsequently by HPLC.
＜2-1〉design of candidate material
Research participates in osteoplastic protein (for example somatomedin), extracellular matrix etc. on the material that participates in initial adherence and osteoblastic proliferation and differentiation, and is analyzed to obtain the pass key sequence in the described proteinic whole aminoacid sequence.BMP-2 has the specific binding site of osteoblast (bone formation cell), and ECM has a part of sequence that participates in cell adhesion.Given this, on the basis of these sequences, design the reorganization oligopeptide, it has makes that osteoblast can be protein and the aminoacid sequence of inducing described oligopeptide adhesion, propagation and differentiation with the oligopeptide sequence recognition.In example 3, will assess the effect of prepared oligopeptide pair cell.
＜2-2〉preparation of oligopeptide
By being to obtain from the Peptron company limited based on the synthetic oligopeptide of the synthetic method of Fmoc/tBu.After synthetic, with product purification to 95% or high-purity more, utilize the NMR determining molecular weight subsequently by HPLC.
The oligopeptide that is screened has following sequence:
1.PEP111(SEQ?ID?1)：R1-CKIPKPSSAPTELSAISMLYL-R2；
2.PEP122(SEQ?ID?5)：R1-KIPKPSSAPTELSAISMLYC-R2。
The activity of following each peptide of measurement.
Example 3: oligopeptide is to osteoblastic effect: in vitro test
For analyzing, use the cell line MG63 of analogy osteoblast, the cytoactive on the culture plate of the oligopeptide of assessment coating in vitro by cysteine residues being introduced the terminal prepared effect of oligopeptide for the product that is coated with.
＜3-1〉the oligopeptide processing method
The oligopeptide solution of 1 micro-molar concentration (μ M) of 1 milliliter (ml) (is introduced in each hole of 24 well culture plates in desalted water (DemineralizedWater, D.W.) in), and under 37 ℃, at carbon dioxide (CO
2) cultivated 3 hours in the calorstat, after this, oligopeptide is taken out from culture plate.Then, (phosphate buffer solution, PBS) the washing culture plate is 1 time, subsequently air drying on the cleaning platform to use phosphate buffered solution.
＜3-2〉cell adhesion: cresyl violet stains (cresyl violet stain)
With 1 * 10
5Individual MG63 cell is distributed in each hole of 24 well culture plates that are coated with oligopeptide, and under 37 ℃, cultivates 2 hours in carbon dioxide incubator.BMP-2 is as positive control.
For cell adhesion, use the DMEM (Dulbecco ' s Modified Eagle Medium, DMEM) (serum-free medium) that do not have 10% hyclone (fetal bovine serum).After 2 hours, use the not adherent cell on PBS buffer (pH 7.2) the removal culture plate, add 1 milliliter of 10% formalin (in the PBS buffer, pH 7.2) subsequently with fixed cell.For mensuration adheres to the amount of the cell on each hole of culture plate, 300 milliliter of 0.04% cresol-purple (in 20% methanol) is added in each hole, and at room temperature reacted 30 minutes, will adhere to the nucleic acid staining of the cell on each hole.By using D.W. will coil washing 3 times, non-reacted staining solution is removed from culture plate, add the citric acid (in 50% ethanol) of 300 milliliter of 0.1 molar concentration subsequently, the staining solution of eluting bond to the nucleic acid thus, thus measure cell adhesion (Fig. 2).
＜3-3〉propagation: the MTS analysis
With 1 * 10
5Individual MG63 cell is distributed in each hole of 24 well culture plates that are coated with oligopeptide, and under 37 ℃, cultivates 2 hours in carbon dioxide incubator.BMP-2 is as positive control.For cell adhesion, use the DMEM (serum-free medium) that does not have 10% hyclone.After 2 hours, use the not adherent cell on PBS buffer (pH 7.2) the removal culture plate.Then the DMEM that will contain 10% hyclone puts on the culture plate, and under 37 ℃, cultivates 5 days in carbon dioxide incubator.Cultivate the back every 2 days, changes culture medium with fresh culture, and after cultivation 1,3 and 5 day, CellTiter 96 used
TMAqueous non-radioactive cell proliferation assay kit (Non-Radioactive CellProliferation Assay Kit) (available from Promega Co., USA) is measured propagation.
＜3-4〉differentiation: the ALP analysis
By measuring early stage labelling alkali phosphatase (ALP) activity of osteoblast differentiation, observe the effect of oligopeptide in the bone formation cell differentiation procedure.For measuring the ALP activity, with 1 * 10
5Individual MG63 cell is distributed in each hole of 24 well culture plates that are coated with oligopeptide, and under 37 ℃, cultivates in carbon dioxide incubator.BMP-2 is as positive control.Cultivate the back every 2 days, changes culture medium, and measure the ALP activity (Fig. 4) when cultivating back 3 days and 8 days with fresh culture.
Example 4: oligopeptide is to osteoblastic effect: in vivo test
Physical Absorption (impregnation drying method) at the implant surface oligopeptide is carried out animal experiment.Be the preparation sample, each implantation body immersed in the oligopeptide solution of 100 micro-molar concentrations of 200 microlitres (μ l), and under 37 ℃, in calorstat, cultivated 3 hours.After the reaction, on the cleaning platform, surplus solution is coated on implant surface, air drying subsequently equably.
The uncoated body (impregnation drying method) of planting of planting body and coating BMP-2 is used as positive control, and with regard to bone formation the described body of planting is compared with the implantation body with oligopeptide.
＜4-1〉formation of the damaged model of bone: diagram
The damaged model of bone is illustrated among Fig. 5.
＜4-2〉osteoplastic comparison in the miniature pig of mandibular defect
Extract tooth from the mandibular bone both sides of 8 miniature pigs, these pigs were cured 3 months.It is damaged to form identical artificial bone near the cortical bone of healing, and the implantation body's (impregnation drying method) and the contrast that will be coated with oligopeptide (PEP111, PEP122) are subsequently put into wherein.After 2 weeks and 4 weeks, with sacrifice of animal, and measuring in the defective region the new surface of bone that forms by the histology, long-pending (bone area BA) calculates surface of bone and amasss ratio (%).
Particularly, in the damaged model of bone, oligopeptide processed group and the untreated fish group and the BMP-2 processed group that provide as negative control and positive control are respectively compared with regard to bone regeneration capability.Therefore, as shown in Figure 6, observe, compare with the untreated fish group that provides as negative control, the bone formation ability of oligopeptide processed group is in 2 weeks and 4 obviously enhancings of week back.(Fig. 6 and Fig. 7)
This result shows that oligopeptide PEP111 represents good osteanagenesis effect.
Example 5: with the comparison of other group
RGD (Arg-Gly-Asp) sequence that is present in fiber adhesion albumen and the collagen protein plays the cell attachment effect.PEP111 and RGD sequence are compared with regard to cell adhesion and ALP activity.Described relatively is to carry out described in example 3.The result shows that the PEP111 sequence is better than the RGD sequence, especially aspect the ALP activity (Fig. 8) like this.
Sequence table
＜110〉limited company of the safe implantation body of tooth difficult to understand
＜120〉strengthen synosteosis and osteoplastic oligopeptide
<150>KR10-2008-0054730
<151>2008-06-11
<160>7
<170>KopatentIn?1.71
<210>1
<211>21
<212>PRT
＜213〉artificial sequence
<220>
<223>PEP111
<400>1
Cys?Lys?Ile?Pro?Lys?Pro?Ser?Ser?Ala?Pro?Thr?Glu?Leu?Ser?Ala?Ile
1???????????????5???????????????????10?????????????????15
Ser?Met?Leu?Tyr?Leu
20
<210>2
<211>20
<212>PRT
＜213〉artificial sequence
<220>
<223>PEP121
<400>2
Cys?Ile?Pro?Lys?Pro?Ser?Ser?Ala?Pro?Thr?Glu?Leu?Ser?Ala?Ile?Ser
1???????????????5??????????????????10??????????????????15
Met?Leu?Tyr?Leu
20
<210>3
<211>18
<212>PRT
＜213〉artificial sequence
<220>
<223>PEP131
<400>3
Cys?Pro?Lys?Pro?Ser?Ser?Ala?Pro?Thr?Glu?Leu?Ser?Ala?Ile?Ser?Met
1???????????????5??????????????????10??????????????????15
Leu?Leu
<210>4
<211>21
<212>PRT
＜213〉artificial sequence
<220>
<223>PEP112
<400>4
Lys?Ile?Pro?Lys?Pro?Ser?Ser?Ala?Pro?Thr?Glu?Leu?Ser?Ala?Ile?Ser
1???????????????5???????????????????10??????????????????15
Met?Leu?Tyr?Leu?Cys
20
<210>5
<211>20
<212>PRT
＜213〉artificial sequence
<220>
<223>PEP122
<400>5
Lys?Ile?Pro?Lys?Pro?Ser?Ser?Ala?Pro?Thr?Glu?Leu?Ser?Ala?Ile?Ser
1????????????????5??????????????????10??????????????????15
Met?Leu?Tyr?Cys
20
<210>6
<211>19
<212>PRT
＜213〉artificial sequence
<220>
<223>PEP132
<400>6
Lys?Ile?Pro?Lys?Pro?Ser?Ser?Ala?Pro?Thr?Glu?Leu?Ser?Ala?Ile?Ser
1????????????????5??????????????????10??????????????????15
Met?Leu?Cys
<210>7
<211>18
<212>PRT
＜213〉artificial sequence
<220>
<223>PEP133
<400>7
Lys?Ile?Pro?Lys?Pro?Ser?Ser?Ala?Pro?Thr?Glu?Leu?Ser?Ala?Ile?Ser
1????????????????5??????????????????10??????????????????15
Met?Cys
Claims (6)
1. one kind strengthens synosteosis and osteoplastic oligopeptide, and it has following structural formula:
(C or K)-(I, K or P)-(I, P or K)-(K or P)-(K, P or S)-(P or S)-(A or S)-(A, P or S)-(A, P or T)-(E, P or T)-(E, L or T)-(E, L or S)-(A, L or S)-(A, I or S)-(A, I or S)-(I, M or S)-(L, M or S)-(C, L or M).
2. oligopeptide according to claim 1, it further comprises:
C or N-terminal at described oligopeptide have-amino acid residue of SH group, and described amino acid residue has (C, L or Y)
nStructure, wherein n is 1 or 2.
3. oligopeptide according to claim 2, wherein said amino acid residue are the cysteine residues that adds the C-terminal of described oligopeptide to.
4. according to the described oligopeptide of arbitrary claim in the claim 1 to 3, wherein said oligopeptide is the peptide that is selected from by the following group that constitutes: PEP111 (SEQ ID 1), PEP121 (SEQ ID 2), PEP131 (SEQ ID 3), PEP112 (SEQ ID 4), PEP122 (SEQ ID 5), PEP132 (SEQ ID 6) and PEP133 (SEQ ID 7).
5. oligopeptide according to claim 4, wherein said peptide are the peptide that is selected from PEP111 and PEP122.
6. oligopeptide according to claim 5, wherein said peptide are PEP111.
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| KR1020080054730A KR100879704B1 (en) | 2008-06-11 | 2008-06-11 | Oligopeptide for enhancing osteointegration and bone formation |
| PCT/KR2009/003141 WO2009151288A2 (en) | 2008-06-11 | 2009-06-11 | Oligopeptide for enhancing osteointegration and osteogenesis |
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| EP3505529A4 (en) * | 2016-08-26 | 2020-05-13 | Sewon Biotechnology Inc. | Peptide for promoting osteoanagenesis or osteogenesis and use of same |
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| JP3895792B2 (en) * | 1995-12-08 | 2007-03-22 | プロスケリア・エス・ア・エス | Bone formation promoter |
| WO2003072593A2 (en) * | 2002-02-21 | 2003-09-04 | University Of Virginia Patent Foundation | Bone targeting peptides |
| WO2005072403A2 (en) * | 2004-01-28 | 2005-08-11 | The Regents Of The University Of California | Bone morphogenic protein binding peptide |
| KR100757241B1 (en) * | 2006-09-13 | 2007-09-10 | 재단법인서울대학교산학협력재단 | Bone graft containing osteogenesis enhancing peptides |
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| CN110551201A (en) * | 2019-08-26 | 2019-12-10 | 杭州彗搏科技有限公司 | Novel cyclic peptide derived from bone morphogenetic protein 2, preparation method and application thereof |
| CN110551201B (en) * | 2019-08-26 | 2020-04-28 | 杭州彗搏科技有限公司 | Novel cyclic peptide derived from bone morphogenetic protein 2, preparation method and application thereof |
| WO2021036063A1 (en) * | 2019-08-26 | 2021-03-04 | 杭州彗搏科技有限公司 | Cyclic peptide from novel bone morphogenetic protein 2, preparation method therefor and application thereof |
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