CN105228557A

CN105228557A - For compositions and the method for dental tissue regeneration

Info

Publication number: CN105228557A
Application number: CN201480028994.6A
Authority: CN
Inventors: J·J·毛; Y·郑; G·孙; S·王
Original assignee: Columbia University in the City of New York
Current assignee: Columbia University in the City of New York
Priority date: 2013-03-21
Filing date: 2014-03-21
Publication date: 2016-01-06
Also published as: EP2976044A1; WO2014153548A1; EP2976044A4

Abstract

The invention provides a kind of method regenerating dental tissue, comprise and the support containing Wnt3a with BMP-7 and optional VEGF, bFGF or NGF of existing is contacted with dental tissue, promote the odontoblast differentiatio` of CFU-GM thus, promote that CFU-GM migrates in dental tissue, or regeneration dental tissue.Present invention also offers the compositions for regenerating dental tissue, it comprises support and Wnt3a and BMP-7, and optional VEGF, bFGF or NGF existed.

Description

For compositions and the method for dental tissue regeneration

The cross reference of related application

This application claims the rights and interests of the U.S. Provisional Application Ser numbers 61/804,138 of the U.S. Provisional Application Ser application in number on March 21st, 61/831,595 and 2013 of application on June 5th, 2013, each application is incorporated herein for referencial use in full.

About the research of federal funding or the statement of exploitation

The present invention completes under government-funded, described subsidy is the fund 5RC2DE020767 issued by NationalInstitutesofHealth/NationalInstituteofDentalandC raniofacialResearch, and the fund R01DE15391 issued by TheNationalInstitutesofHealth.Government has certain right in the present invention.

Introduce material for referencial use

Sequence table as a part of the present invention comprises computer-reader form, and it contains nucleotide of the present invention and/or aminoacid sequence.The theme of sequence table is incorporated herein for referencial use in full.

Background of invention

Biology tooth can survive, and this is mainly due to dental pulp.At present, dental pulp that is ill, disappearance or wound is by covering or displacement with inertia synthetic material.The most frequently used filler is gutta-percha, and this is the thermoplastic polymer of rubber mass.Removing ill, disappearance or wound natural dental pulp after, gutta-percha is melted and injects with root canal filling.Although dental pulp or the process of root pipe have been the routine techniquess of contemporary dental medical field, there have been some shortcomings (Salvietal.2007) of adverse effect the quality of life still had for patient.First, the tooth through root canal is tending towards frangible, and easily ruptures.Secondly, after root canal, usually there is tooth discoloration.Its patient that tooth discoloration occurs through root canal needs extra high price dental prosthetics program usually.3rd, the dental pulp that is ill, disappearance or that infect of the tooth (deciduous teeth) come off lacks therapeutic choice usually, and is usually unsuitable for root canal.Pulp necrosis occurs in the evulsed tooth and 70-100% intrusion tooth (intrudedteeth) of 85-96%.The tooth of untreated or bad process infects and can cause systemic infection (Shay2002; Brennanetal.2007).

Ideally, a kind of improvement for ill, disappearance or the therapeutic modality of wound dental pulp, biology living tissue is recovered.Tissue engineering technique is for developing the method and composition recovering cranium covering weave and bone.See such as AlhadlaqandMao, 2003; EdwardsandMason, 2006; Fongetal., 2005; GoldbergandSmith, 2004; HongandMao, 2004; Lovschalletal., 2001; Maoetal., 2006; Mathieuetal., 2006; Murrayetal., 2002; Murrayetal., 2007; NakashimaandAlamine, 2005; NakashimaandReddi, 2003; StosichandMao, 2007; Youngetal., 2002; U.S. Patent No. 5,885,829; And U.S. Patent Application Publication 20050079470.Most of technology comprises use timbering material, and it comprises mammalian cell as dental pulp stem cell or mescenchymal stem cell, and/or bioactive ingredients is as bone morphogenetic protein (BMP).The technology be inoculated in by cell on timbering material has the shortcoming being difficult to prepare and store, because living cells must be inoculated, cultivates and maintain on support.In addition, for the source of the cell in tissue regeneration and underproduce.

The key factor of CFU-GM being induced into odontoblast (Od) is not yet known.

Summary of the invention

In various aspects of the present invention, provide the compositions for regenerating dental tissue and method.

One aspect of the present invention provides the compositions for regenerating dental tissue.In some embodiments, compositions comprises Wnt3a polypeptide and the BMP-7 (BMP-7) of substrate or support (such as comprising substrate or the support of hydrogel) and treatment effective dose.In some embodiments, compositions comprises VEGF (VEGF), basic fibroblast growth factor (bFGF) or the nerve growth factor (NGF) for the treatment of effective dose.In some embodiments, compositions does not comprise living cells.In some embodiments, substrate or support comprise Wnt3a and BMP-7, and optional VEGF, bFGF or NGF existed.The further feature of described compositions is discussed in hereafter using method.Those skilled in the art understand this feature can equally for compositions self.

The present invention provides the method for regenerating dental tissue on the other hand.In some embodiments, described method comprises provides above-mentioned composition, and in the natural or artificial hole of compositions being inserted mammalian tooth or chamber.In some embodiments, described compositions promotes the odontoblast differentiatio` of CFU-GM, promotes that CFU-GM migrates in dental tissue, promotes that angiogenic, odontogenesis, fiber generation or neurogenic are grown, to regenerate dental tissue.In some embodiments, described compositions regeneration vessel pulp tissue or regenerate new dentin (neodentin).

In some embodiments, described support comprises further and is selected from following compound: platelet derived growth factor (PDGF), endothelial cell growth factor (ECGF) (ECGF), transforming growth factor-beta 1 (TGF-β 1), epidermal growth factor (EGF), hepatocyte growth factor (HGF), the factor-1 (SDF1) of stroma cell derivative, bone morphogenetic protein (BMP) except BMP-7, TGF-β, GDF (GDF), insulin-like growth factor-i (IGF1), dentin matrix protein, dentin sialoprotein, resorption lacunae, amelogenin, integrin, angiogenin (angiogenin), angiopoietin-1 (angiopoietin-1), del-1, follistatin, granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor/dispersion factor (HGF/SF), interleukin 8 (IL-8), leptin, Midkine, placental growth factor, thymidine phosphorylase/platelet-derived endothelial cell growth factor (PD-ECGF), PDGF-BB (PDGF-BB), many trophic factors (PTN), progranulin, proliferin, transforminggrowthfactor-α (TGF-α), transforming growth factor-β (TGF-β), tumor necrosis factor-alpha (TNF-α), matrix metalloproteinase (MMP), angiogenin 1 (ang1), ang2, delta-sample part 4 (DLL4), Connective Tissue Growth Factor (CTGF), brain derived neural factor (BDNF), NT-4 and NT-3.In some embodiments, support comprises antibiotic or analgesic further.

In some embodiments, Wnt3a polypeptide, BMP-7, VEGF, bFGF or NGF are injected in, are mixed in, are encapsulated in, are bound by (tetheredto) or are adsorbed in substrate or support.

In some embodiments, support has people's incisor, people's canine tooth, people's bicuspid (bicuspid) or people grinds one's teeth in sleep or the shape of wherein natural or artificial hole or chamber.

In some embodiments, substrate or support comprise natural polymer, are selected from collagen protein, gelatin, polysaccharide, chitosan, hydroxyapatite (HA) and polyhydroxyalkanoate.In some embodiments, substrate or support comprise the polymer of synthesis, are selected from aliphatic polyester and the Polyethylene Glycol of poly-('alpha '-hydroxy acids).In some embodiments, substrate or support comprise hydroxyapatite.In some embodiments, substrate or support comprise Alginate hydrogel.In some embodiments, substrate or support are biodegradable.

In some embodiments, support comprises diameter is (i) 50-500 μm; Or (ii) microchannel of about 200 μm.In some embodiments, Wnt3a and BMP-7 and optional VEGF, bFGF or NGF existed is embedded in the gel in support microchannel.In some embodiments, Wnt3a and BMP-7 and optional VEGF, bFGF or NGF existed is encapsulated in microsphere.

In some embodiments, described method comprises use computer-aided design (CAD) further and makes tooth or model cavity, and synthesizes support with biological drawing apparatus (bioplotter).

In some embodiments, Wnt3a polypeptide comprises aminoacid sequence shown in SEQIDNO:1, SEQIDNO:2, SEQIDNO:3 or SEQIDNO:4.In some embodiments, Wnt3a polypeptide comprises and has at least about 95% sequence thereto with SEQIDNO:1, SEQIDNO:2, SEQIDNO:3 or SEQIDNO:4 and the aminoacid sequence with Wnt3a activity.

In some embodiments, Wnt3a exists to the concentration of about 1,000mgWnt3a/ml solution with about 1ng, and wherein said solution is imported in substrate or support.In some embodiments, BMP-7 exists to the concentration of about 1,000mgBMP-7/ml solution with about 1ng, and wherein said solution is imported in substrate or support.In some embodiments, Wnt3a exists with the concentration of about 10ng/ml solution, and BMP-7 exists with the concentration of about 200ng/ml solution, and wherein said solution is imported in substrate or support.In some embodiments, VEGF exists to the concentration of about 1,000mgVEGF/ml solution with about 1ng.In some embodiments, bFGF exists to the concentration of about 1,000mgbFGF/ml solution with about 1ng.In some embodiments, NGF exists to the concentration of about 1,000mgNGF/ml solution with about 1ng.In some embodiments, above-mentioned solution is imported in substrate or support.

In some embodiments, described method comprises wound or ill pulp tissue in Exposed Pulp chamber or root pipe further; And cover or filling pulp cavity or root pipe at least partially by described compositions, wherein newborn after covering or filling vascularization dental pulp sample is organized in pulp cavity or root pipe and is formed.

In some embodiments, described method comprises further and from tooth, removes wound or ill pulp tissue, to produce the pulp cavity or root pipe that there is no wound or illing tissue.In some embodiments, described method comprises further remove substantially all pulp tissues from tooth.In some embodiments, described method comprises further with inert material filling pulp cavity at least partially.

Other object of the present invention and adjustment are later by obvious for part and point out.

Accompanying drawing describes

Those skilled in the art understand hereinafter described accompanying drawing and just illustrate the present invention.Accompanying drawing does not limit the present invention in any way the scope of instruction.

Fig. 1 is through the adult tooth photo of clinical same root pipe process.Through the root pipe of pulp treatment and pulp cavity with inactive composition (a figure), or only there is basic fibroblast bioactive ingredients (bFGF) (b figure), or both there is the collagen protein sponge filling that bFGF also has blood vessel endothelium bioactive ingredients (VEGF) (c figure).Tooth is maintained 2 weeks the mice of subcutaneous implantation immunodeficiency, in the root pipe and pulp cavity of pulp treatment, whether vascularization occurs to assess.Become divisional processing (only collagen protein sponge) (a) contrary with inactive, only bFGF (b) and VEGF and bFGF combination (c) process all illustrates and is inserting the vascularization in the collagen protein sponge in root pipe.

Fig. 2 illustrates the microphotograph of the adult tooth section processed as shown in Figure 1.A illustrates the root pipe of people's permanent incisor of the collagen protein sponge implanting inactive composition.In the mice of immunodeficiency after et al. Ke, there is not the inside growth of any host tissue from apical foramen.B illustrates the root pipe of people's permanent incisor with the collagen protein sponge that VEGF-loads, and illustrates to there is vascularization (arrow indication) and host tissue inwardly grows.The host tissue infiltrated is attached to dentin.C illustrates the root pipe of people's permanent incisor with the collagen protein sponge that bFGF-loads, and illustrates to there is vascularization (arrow indication) and host tissue inwardly grows.The host tissue infiltrated is attached to dentin.D illustrates the root pipe of people's permanent incisor with the collagen protein sponge that VEGF+bFGF-loads, and illustrates to there is vascularization (arrow indication) and host tissue inwardly grows.The host tissue infiltrated is attached to dentin.

Fig. 3 illustrates that TGF β 3 is from the release dynamics figure PLGA microsphere in 1%BSA solution.Detected by ELISA, TGF β 3 with sustained release fashion until 36 and 42 days discharge from the PLGA microsphere of the co-polymer ratio of 50:50 or 75:25 respectively.These two co-polymer ratios all observe initial outburst sample release, although 50:50PLA/PGA ratio produces rate of release faster than 75:25PLA/PGA ratio.

Fig. 4 is scanning electron microscopy, and manufacture and the degraded of PLGA microsphere are shown.A illustrates the representative SEM image being had the microsphere of TGF β 3 by poly-d-l-lactic-co-glycolic acid (PLGA) with the encapsulation of 50:50PLA/PGA ratio manufacture.B illustrates the representative SEM image of the expection degraded that encapsulation has the PLGA microsphere of TGF β 3 in PBS solution.

Fig. 5 illustrates the chart of BMP-7 from the cumulative mean release PLGA microsphere.

Fig. 6 illustrates the chart of NGF from the cumulative mean release PLGA microsphere.

Fig. 7 describes reporter gene in col1a1 (2.3kb)-EGFP mice to express and a series of images of odontoblast and osteoblastic microarray analysis, scatterplot and array of figure.Fig. 7 A is transgenic schematic diagram, and it contains the 2.3kbcol1a1 promoter driving EGFP to express.Fig. 7 B is the image of the jawbone being separated transgenic mice, and wherein GFP signal all can detect in bone and tooth.Fig. 7 C illustrates the cranium of the Col1a1 mice through dissecting, and observes transgene expression in suture and bone.The section that Fig. 7 D illustrates jawbone and grinds one's teeth in sleep.Fig. 7 E illustrates and observe GFP signal in alveolar bone, periodontal membrane and odontoblast.Fig. 7 F and Fig. 7 G illustrates the incisor of GFP positive mice, and GFP positive cell is in line along dentin surface, and GFP signal is arranged in odontoblast.Fig. 7 H illustrates that the positive odontoblast of GFP forms dentinal tubule.Fig. 7 I illustrates that the positive odontoblast of GFP moves out from Col1a1-EGFP mice dental pulp.Fig. 7 J illustrates 10,000 GFP positive cell (based on GFP sorting) from the dental pulp of subcutaneous vaccination in collagen gel (0.1ml).The X-line image inserted in Fig. 7 J left hand corner illustrates that 3 weeks rear mineralized tissues are formed.Fig. 7 K illustrates that mineralized tissue is the GFP positive.Fig. 7 L illustrates that the positive odontoblast of the GFP of conveying forms the tissue slice of the mineralized structures similar to disorderly dentin.Fig. 7 M is image (green, the GFP of the dentin sample tissue that the DSP positive is shown; Redness, DSP; Purple, DAPI).Fig. 7 N illustrates 10,000 GFP positive cell (based on GFP sorting) from the dental pulp be inoculated in renal capsule (0.1ml).The X-line image inserted in the left hand corner of Fig. 7 N illustrates that 3 weeks rear mineralized tissues are formed.Fig. 7 O illustrates that the H & E of mineralized tissue dyes.Fig. 7 P illustrates that cell forms the positive mineralized tissue of DSPP.Fig. 7 Q and Fig. 7 R illustrates that the osteocyte of cranium in Col1a1-EGFP mice and osteoblast are the GFP positives.Fig. 7 S illustrates and is separated cultured cells from cranium and in OIM, and wherein GFP cell formed tuberosity in one week.Fig. 7 T and Fig. 7 U illustrate the Agilent full-length genome microarray analysis of the mRNA of the GFP positive cell be separated from tooth (odontoblast) and cranium (osteoblast).The scatter diagram of signal intensity a little.Fig. 7 U illustrates the example (n=4) of array based assay data.The signal intensity of each feature represents with round dot.

Fig. 8 is a series of images of the gene that the differential expression detected by immunohistochemistry and in situ hybridization is shown.Carry out immunohistochemistry to detect the protein level of the gene selected.Fig. 2 A, Fig. 8 B, Fig. 8 C, Fig. 8 E illustrate at incisor, grind one's teeth in sleep and the expression of gene of selection that the odontoblast camber of cranium is expressed.Fig. 8 F and Fig. 8 G illustrates expression (scale=20 μm) (De: dentin of the gene of the selection expressed at osteoblast camber; P: dental pulp).Fig. 8 H, Fig. 8 I and Fig. 8 J illustrate the expression of the somatomedin detected by situ hybridization, because Wnts and BMP7 is the somatomedin of secretion, it can diffuse to surrounding tissue (scale: A-G, 50 μm; H, I, J, 250 μm).

Fig. 9 illustrates a series of bar diagram being confirmed microarray by RT-PCR.Fig. 9 A illustrates the gene of the selection expressed at odontoblast's camber.Fig. 9 B illustrates the gene of the selection expressed at osteoblast camber.The expression of the gene selected is confirmed by PCR in real time, uses to be separated to carry out (n=3) from the positive odontoblast of GFP and osteoblastic RNA.The gene selected is somatomedin or excreted factor and transcription factor.

Figure 10 is a series of bar diagram and image that illustrate that Wnt3a selective induction odontoblast moves.Figure 10 A is bar diagram, and the result that the Boyden room that the use PDL cell of the cell migration for studying response Wnt3a, BMP-7 carries out measures is shown.100k PDL cell is seeded in hole, and in migration inducement is added entering the room.After 12 hours, the cell of migration is counted through trypsinization.Then by plating cells and propagation to study further (n=4*p<0.05).Figure 10 B is the image of the cell of the migration broken up in Osteogenic Induction Medium, 3 weeks rear cma stainings (vonKossa).Figure 10 C and Figure 10 D is the bar diagram (n=3*p<0.05) illustrating that the marker gene detected by PCR is expressed.

Figure 11 is a pair image that the minipig dental pulp removed by standard pulp amputation is shown.Entrance is done at pulp cavity.After elimination pulp tissue, clean pulp cavity.The effective hand file of root is shaped, and diameter is 0.1-2.5mm, and uses salt water washing.With aseptic paper point dry root pipe.

Figure 12 illustrates that Wnt3a or BMP-7 regenerates dental pulp and Dentinal a series of images and bar diagram.Figure 12 A, Figure 12 B, Figure 12 C and Figure 12 D illustrate that the injectable collagen gel combined with somatomedin (tester, BMP-7 or Wnt3a) is injected in root pipe.Open access cavity (accesscavity) seals with base material and composite resin.Figure 12 E and Figure 12 F is the enlarged drawing of Figure 12 D part.Figure 12 H, Figure 12 I, Figure 12 J and Figure 12 K illustrate at the incisor gathered in the crops afterwards in 6-8 week and check with histology and miniature-CT.Figure 12 L and Figure 12 M illustrates the new dentin volume of formation and the quantity (p<0.05, n=4) of density.

Figure 13 is a series of figure that dental pulp decompression filling film is shown, wherein: 1 is film; 2 is marginal adhesion of composite; 3 is for filling out tooth cavity; 4 is adsorbable biomaterials; 5 is connections of microporous membrane and tooth cavity; And 6 is pulp tissues.Figure 13 A illustrates the top view of filling film, and it illustrates the core of film and peripheral part of composite.Figure 13 B illustrates the sectional view of filling film, and it illustrates the film carried in composite.Figure 13 C illustrates with the sectional view of the teeth cavity sealing device shown in diascopy.In teeth cavity, the exudate of inflammation can be absorbed immediately by alginate, ooze out subsequently, and the antibacterial in oral cavity is stoped by this film from film.Figure 13 D illustrates the sectional view of sealing device and tooth.

Figure 14 is the reaction schematic diagram that Alginate hydrogel is formed.

Figure 15 is swelling capacity (%) bar diagram of the Alginate hydrogel of physical crosslinking.

Figure 16 is the oral cavity photo of the minipig of anesthesia, wherein places rubber dam to isolate incisor.By with high-speed dental drill mechanicalness opening to touch pulp cavity.

Figure 17 is the tissue cross face figure of minipig incisor, and the dental pulp (r.p.) of the regeneration that 4 weeks are formed after the hydrogel with BMP-7 (200ng/ml) is injected into root pipe and pulp cavity, the blood vessel (b.v.) of regeneration, the dentin (r.d.) of regeneration and natural dentin (n.d.) are shown.

Figure 18 is the tissue cross face figure of minipig incisor, and the dental pulp (r.p.) of the regeneration that 4 weeks are formed after the hydrogel with Wnt protein (10ng/ml) is injected into root pipe and pulp cavity, the blood vessel (b.v.) of regeneration, the dentin (r.d.) of regeneration and natural dentin (n.d.) are shown.

Figure 19 is the tissue cross face figure of minipig incisor, and the dental pulp (r.p.) of the regeneration that 4 weeks are formed after the hydrogel with BMP-7 (200ng/ml) and Wnt protein (10ng/ml) is injected into root pipe and pulp cavity, the blood vessel (b.v.) of regeneration, the dentin (r.d.) of regeneration and natural dentin (n.d.) are shown.

Detailed Description Of The Invention

The present invention is at least partly based on finding that the dental pulp of ill, wound or disappearance can with the compositions displacement comprising substrate or support and one or more bioactive ingredients, and described compositions promotes that angiogenic, odontogenesis, fiber generation or neurogenic are grown.This compositions can promote that the generation of pulp cavity medium vessels, odontogenesis, fiber generation or neurogenic are grown, and keeps tooth vigor.

The invention provides compositions, it comprises for one or more bioactivator, to regenerate dental pulp or dentin.Various compositions can comprise bioactivator as Wnt3a, BMP-7, VEGF, bFGF, NGF or its combination.

Present invention also offers some biocompatible materials to keep the vigor of dental pulp in the recoverability pulpitis stage.As shown here, some can absorb again or can not resorbent biomaterial, comprise perforated membrane and hydrogel, not only allow blood, tissue fluid or water diffusion, and pathogen in oral cavity can be stoped to enter pulp tissue.Therefore, the dental pulp in recoverability pulpitis can be saved to maintain its vigor.

The present invention is at least partly based on having been found by overall gene expression pattern from CFU-GM as stem cell carries out the key factor of odontoblast's specialization.These key factors are in clinical front dental pulp in body and dentin model.As shown here, these key factors, comprise Wnt albumen, not only induce dental pulp but also induce Dentinal brute force regeneration.The conveying of these key factors can replace root tube material, teeth cavity charges or dentin.

In some embodiments, substrate or support (such as acellular matrix or support) is provided.Support can be mammalian tooth shape or moulding enter mammalian tooth with filling natural or artificial hole or teeth cavity in.Substrate can be suitable for natural or artificial hole or the chamber of insertion or part or all of filling mammalian tooth.Substrate or support can comprise bioactivator as herein described (such as chemotaxis, ostosis, dentin generation, enamel generation or cementum generation preparation) (such as Wnt3a, BMP-7, VEGF, bFGF or NGF) or its active analogue thereof.Substrate or support can with or do not implant together with the cell of exogenous application.Such as, comprise Wnt3a, BMP-7, VEGF, bFGF or NGF or encode its substrate of nucleic acid or support can import on tooth or wherein (such as in teeth cavity), to promote that the one-tenth dentin of CFU-GM breaks up, promote cell migration (such as CFU-GM migration), to regenerate pulp tissue (such as vascularization pulp tissue), or regenerate new dentin.

In some embodiments, compositions described herein is used for carrying out tooth, dental pulp or root pipe handling procedure to mammalian tooth in need.Described method can comprise wound or ill pulp tissue in Exposed Pulp chamber and/or root pipe; Cover or filling pulp cavity and/or root pipe at least partially by the compositions comprising bioactive ingredients.Described bioactive ingredients can promote that angiogenic, odontogenesis, fiber generation or neurogenic are grown.In some embodiments, described compositions does not comprise living cells during covering or filling.In some embodiments, described method comprises further and from tooth, removes wound or ill pulp tissue, to produce the pulp cavity and/or root pipe that there is no wound or illing tissue.In some embodiments, described compositions comprises substrate or support.

Bioactivator

Compositions described herein can comprise chemotaxis in doped matrix support, ostosis, dentin generation, enamel generation or cementum generation compound.As used herein, chemotaxis compound is the compound of luring cell.Ostosis compound is the compound promoting the synthesis of new bone.Dentin generation compound is the compound promoting the synthesis of new dentin.Enamel generation compound is the compound promoting enamel synthesis.Cementum generation compound is the compound promoting the synthesis of pressure cementum.

Compositions as herein described can comprise any bioactivator promoting that angiogenic, odontogenesis, fiber generation or neurogenic are grown.Compositions described herein can comprise more than a kind of bioactive ingredients, and such as 2,3,4 or more plant bioactive ingredients.

Bioactive ingredients can be that it is incorporated to herein incorporated by reference as described in U.S. Patent Publication No.2012/0282573.The example of nonrestrictive bioactivator comprises platelet derived growth factor (PDGF), endothelial cell growth factor (ECGF) (ECGF), transforming growth factor-beta 1 (TGF-β 1), epidermal growth factor (EGF), CXCL12 (SDF1), bone morphogenetic protein (BMP), GDF (GDF), insulin-like growth factor-i (IGF1), VEGF (VEGF), fibroblast growth factor (FGF), dentin matrix protein, dentin sialoprotein, resorption lacunae, amelogenin, integrin, VEGF (VEGF), basic fibroblast growth factor (bFGF), angiogenin, angiogenin (angiopoietin)-1, del-1, follistatin, granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor/dispersion factor (HGF/SF), interleukin 8 (IL-8), leptin, Midkine (midkine), placental growth factor, thymidine phosphorylase/platelet-derived endothelial cell growth factor (PD-ECGF), PDGF-BB (PDGF-BB), many trophic factors (PTN), progranulin, proliferin, transforminggrowthfactor-α (TGF-α), transforming growth factor-β (TGF-β), tumor necrosis factor-alpha (TNF-α), matrix metalloproteinase (MMP), angiogenin 1 (ang1), ang2, delta-sample part 4 (DLL4), Connective Tissue Growth Factor (CTGF), brain derived neural factor (BDNF), NT-4 and NT-3.

Bioactive ingredients can from any mammalian species.In some embodiments, bioactive ingredients is people's bioactive ingredients, particularly when the mammal treated is people.Bioactive ingredients can be restructuring bioactive ingredients.

Compositions described herein can comprise antibiotic or analgesic.Compositions described herein can comprise antibiotic.The antibiotic of citing is potassium v calcium, amoxicillin, Augmentin (augmentin), clindamycin or azithromycin.

Compositions as herein described can comprise analgesic.The analgesic of citing is acetaminophen, diclofenac, ketoprofen (ketoprofen), aspirin, naproxen, indomethacin (indomethacin), ketorolac (ketorolac), ibuprofen (ibuprofen), piroxicam (piroxicam), celecoxib (celecoxib), meloxicam (meloxicam), mefenamic acid (mefenemicacid), rofecoxib (rofecoxib), nimesulide (nimesulide) or prostaglandin.

The concentration of bioactivator described herein provides with the quality of per unit liquor capacity usually, and wherein solution can import in substrate or support.

Bioactivator can be encapsulated in various vector delivery system and give.The vector delivery system of citing comprises microsphere, hydrogel, polymer implants, intelligent polymer carrier and liposome and (usually sees UchegbuandSchatzlein, eds. (2006) PolymersinDrugDelivery, described in CRC, ISBN-10:0849325331).The system based on carrier of delivery of molecules or biomolecule formulations can: conveying in cell is provided; Adjustment biomolecule/preparation rate of release; Increase the ratio reaching the biomolecule of its site of action; Improvement medicine is to the transhipment of its site of action; Allow and other preparations or excipient local deposits altogether; Improved formulations stability in vivo; The time of staying of preparation at its site of action is extended by reducing clearance rate; Reduce the non-specific conveying of preparation for non-target tissue; Reduce the stimulation caused by preparation; The preparation institute reduced due to high predose is causing toxicity; Change the immunogenicity of preparation; Reduce administration frequency, improvement product taste; Or the storage period of improvement product.

Wnt3a

Regeneration dental pulp described herein or Dentinal compositions can comprise Wnt polypeptide (such as Wnt3a).This compositions can comprise other bioactivator of Wnt polypeptides in combination as VEGF, bFGF, BMP-7 or NGF.

Wnt3a is can exist to the concentration of about 1,000mgWnt3a/ml solution with about 0.1ng in compositions described herein.Such as, in compositions described herein, Wnt3a can exist to the concentration of about 100mgWnt3a/ml solution with about 0.1ng.Again such as, in compositions described herein, Wnt3a can exist to the concentration of about 10mgWnt3a/ml solution with about 0.1ng.Again such as, in compositions described herein, Wnt3a can exist to the concentration of about 1mgWnt3a/ml solution with about 0.1ng.Again such as, in compositions described herein, Wnt3a can exist to the concentration of about 0.1mgWnt3a/ml solution with about 0.1ng.Again such as, in compositions described herein, Wnt3a can exist to the concentration of about 100 μ gWnt3a/ml solution with about 0.1ng.Again such as, in compositions described herein, Wnt3a can exist to the concentration of about 10 μ gWnt3a/ml solution with about 0.1ng.Again such as, in compositions described herein, Wnt3a can exist to the concentration of about 1 μ gWnt3a/ml solution with about 0.1ng.Again such as, in compositions described herein, Wnt3a can exist to the concentration of about 100ngWnt3a/ml solution with about 0.1ng.Again such as, in compositions described herein, Wnt3a can exist to the concentration of about 10ngWnt3a/ml solution with about 0.1ng.Again such as, in compositions described herein, Wnt3a can exist with the concentration of about 10ngWnt3a/ml solution.

Solution containing above-mentioned concentration Wnt3a can to import in substrate or support or with its combination.

As shown here, canonical Wnt signal pathway for CFU-GM if odontoblast's specialization of mescenchymal stem cell or differentiation are crucial, and can come layout dental pulp or dentin regeneration as the transferable medical apparatus of such as microencapsulation, it can without the need to cell transplantation.This method can replace such as root tube material, teeth cavity charges or other dentistry charges (see embodiment 3).

As described herein, Wnt3a (without wing MMTV integration site family, member 3A) can promote that the one-tenth dentin of CFU-GM breaks up, promotes cell migration, regeneration vessel pulp tissue, or regenerates new dentin.

Wnt protein induces separately dental pulp and dentin regeneration.Wnt protein combination such as BMP-7 or PDGF can strengthen dental pulp and dentin regeneration further.Wnt protein can strengthen PDGF and BMP-7 signal pathway, and therefore can promote dental pulp or dentin regeneration further.

In some embodiments, Wnt3a can be used for promoting that CFU-GM is as the odontoblast differentiatio` from Level of Alveolar Bone marrow or pericemental mescenchymal stem cell.Wnt3a also can be used for promoting cell migration.In the body of Wnt3a conveying can in the root pipe of such as pulp treatment regeneration vessel pulp tissue.Be conducted through recruitment host endogenous cell in the body of Wnt3a and can regenerate the new dentin in natural tubulose dentin surface with the odontoblast of polarization.

In some embodiments, Wnt3a polypeptide or its nucleic acid of encoding can contact with tooth, bone, dental pulp or other dental tissue.As described herein, this contact can promote the odontoblast differentiatio` of CFU-GM, promotes cell migration (such as CFU-GM migration), regenerate pulp tissue (such as vascularization pulp tissue), or regenerates new dentin.Such as, Wnt3a polypeptide or its nucleic acid of encoding can to import on tooth or among (such as importing in teeth cavity), promoting the odontoblast differentiatio` of CFU-GM, promote cell migration (such as CFU-GM migration), regeneration pulp tissue (such as vascularization pulp tissue) or regenerate new dentin.

Wnt3a polypeptide should be understood by WNT3A gene code (usually seeing Saitohetal.2001BiochemBiophysResCommun284 (5), 1168 – 1175).The genomic constitution that WNT gene family is correlated with by the structure of the signal protein of secretion of encoding.These protein participates in tumor and is formed and some growth courses, is included between embryo's emergence period and regulates cell fate and pattern.This gene is the member of WNT gene family.Its coding and mice Wnt3A protein have 96% amino acid identity and the protein with another kind of WNT gene outcome-people WNT3 protein-have 84% homogeny.This gene and another family member WNT14 gene cluster in chromosome 1q42 region.

Wnt3a polypeptide can comprise aminoacid sequence shown in SEQIDNO:1, SEQIDNO:2, SEQIDNO:3 or SEQIDNO:4, or there is with it sequence of at least about 80% homogeny (such as at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%), it is active that wherein said polypeptide retains Wnt3a, or its function fragment.

Wnt3a polypeptide can be the aminoacid sequence relevant according to SwissProtIDP27467, and it is incorporated to herein incorporated by reference.Wnt3a polypeptide can be the aminoacid sequence relevant according to UniParcP56704, and it is incorporated to herein incorporated by reference.Wnt3a polypeptide can be the aminoacid sequence relevant according to UniParcQ3SY79, and it is incorporated to herein incorporated by reference.Wnt3a polypeptide can be the aminoacid sequence relevant according to UniprotaccessionQ9GTJ9 (hydra), and it is incorporated to herein incorporated by reference.Wnt3a polypeptide can be the aminoacid sequence relevant according to SwissProtIDP27467 (mice), and it is incorporated to herein incorporated by reference.

Wnta polypeptide can be the active fragment (the amino acid residue 250-350 of such as people Wnt3a) of total length Wnta peptide.

Wnt3a polypeptide and above-mentioned any sequence can have at least about 80% sequence thereto and retain Wnt3a activity.Such as, Wnta polypeptide and above-mentioned any sequence can have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence thereto, and it is active that wherein said polypeptide retains Wnta.

Compositions described herein, support or device can comprise the nucleic acid of Wnt3a polypeptide or coding Wnt3a polypeptide.Wnta nucleic acid can be the nucleic acid of above-mentioned any Wnta peptide sequence of encoding.Wnta nucleic acid can have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence thereto with the nucleic acid of the above-mentioned any sequence of coding, and it is active that the polypeptide of encoding thus retains Wnta.In the embodiment of nucleic acid coding Wnt3a polypeptide wherein, this nucleic acid can provide in carrier as described herein or construct.

Wnt3a polypeptide is commercially available from various source (such as ABCAM, Cambridge, MA).

Known Wnt3a is by TIKI1 and TIKI2 proteolysis, and it promotes larger disulfide bond oligomer oxidation and is formed, and causes WNT3A inactivation.Compositions described herein can comprise the inhibitor of TIKI1 and TIKI2.In some embodiments, Wnt3a polypeptide can be to such as by its derivant of TIKI1 or TIKI2 proteolysis resistance.

BMP-7

Regeneration dental pulp described herein or Dentinal compositions can comprise BMP-7 (BMP-7).This compositions can comprise BMP-7 and combine other bioactivator, as Wnt3a, VEGF, bFGF or NGF.

BMP-7 can exist to the concentration of about 1,000mgBMP-7/ml solution with about 0.1ng in compositions described herein.Such as, BMP-7 can exist to the concentration of about 100mgBMP-7/ml solution with about 0.1ng in compositions described herein.Again such as, BMP-7 can exist to the concentration of about 10mgBMP-7/ml solution with about 0.1ng in compositions described herein.Again such as, BMP-7 can exist to the concentration of about 1mgBMP-7/ml solution with about 0.1ng in compositions described herein.Again such as, BMP-7 can exist to the concentration of about 0.1mgBMP-7/ml solution with about 0.1ng in compositions described herein.Again such as, BMP-7 can exist to the concentration of about 100 μ gBMP-7/ml solution with about 0.1ng in compositions described herein.Again such as, BMP-7 can exist to the concentration of about 10 μ gBMP-7/ml solution with about 0.1ng in compositions described herein.Again such as, BMP-7 can exist to the concentration of about 1 μ gBMP-7/ml solution with about 0.1ng in compositions described herein.Again such as, BMP-7 can exist to the concentration of about 500ngBMP-7/ml solution with about 0.1ng in compositions described herein.Again such as, BMP-7 can exist to the concentration of about 200ngBMP-7/ml solution with about 0.1ng in compositions described herein.Again such as, BMP-7 can exist to the concentration of about 100ngBMP-7/ml solution with about 0.1ng in compositions described herein.Again such as, BMP-7 can exist to the concentration of about 10ngBMP-7/ml solution with about 0.1ng in compositions described herein.Again such as, BMP-7 can exist to the concentration of about 200ngBMP-7/ml solution with about 0.1ng in compositions described herein.

Solution containing above-mentioned concentration BMP-7 can to import in substrate or support or with its combination.

BMP-7 is commercially available.

VEGF

Regeneration dental pulp described herein or Dentinal compositions can comprise VEGF (VEGF).This compositions can comprise VEGF and combine other bioactivator, as Wnt3a, bFGF, BMP-7 or NGF.

VEGF can exist to the concentration of about 1,000mgVEGF/ml solution with about 0.1ng in compositions described herein.Such as, VEGF can exist to the concentration of about 100mgVEGF/ml solution with about 0.1ng in compositions described herein.Again such as, VEGF can exist to the concentration of about 10mgVEGF/ml solution with about 0.1ng in compositions described herein.Again such as, VEGF can exist to the concentration of about 1mgVEGF/ml solution with about 0.1ng in compositions described herein.Again such as, VEGF can exist to the concentration of about 0.1mgVEGF/ml solution with about 0.1ng in compositions described herein.Again such as, VEGF can exist to the concentration of about 100 μ gVEGF/ml solution with about 0.1ng in compositions described herein.Again such as, VEGF can exist to the concentration of about 10 μ gVEGF/ml solution with about 0.1ng in compositions described herein.Again such as, VEGF can exist to the concentration of about 1 μ gVEGF/ml solution with about 0.1ng in compositions described herein.Again such as, VEGF can exist to the concentration of about 500ngVEGF/ml solution with about 0.1ng in compositions described herein.Again such as, VEGF can exist to the concentration of about 200ngVEGF/ml solution with about 0.1ng in compositions described herein.Again such as, VEGF can exist to the concentration of about 100ngVEGF/ml solution with about 0.1ng in compositions described herein.Again such as, VEGF can exist to the concentration of about 10ngVEGF/ml solution with about 0.1ng in compositions described herein.Again such as, VEGF can exist with the concentration of about 33ngVEGF/ml solution in compositions described herein.

Solution containing above-mentioned concentration VEGF can to import in substrate or support or with its combination.

VEGF is commercially available.

bFGF

Regeneration dental pulp described herein or Dentinal compositions can comprise basic fibroblast growth factor (bFGF).This compositions can comprise bFGF and combine other bioactivator, as Wnt3a, VEGF, BMP-7 or NGF.

BFGF can exist to the concentration of about 1,000mgbFGF/ml solution with about 0.1ng in compositions described herein.Such as, bFGF can exist to the concentration of about 100mgbFGF/ml solution with about 0.1ng in compositions described herein.Again such as, bFGF can exist to the concentration of about 10mgbFGF/ml solution with about 0.1ng in compositions described herein.Again such as, bFGF can exist to the concentration of about 1mgbFGF/ml solution with about 0.1ng in compositions described herein.Again such as, bFGF can exist to the concentration of about 0.1mgbFGF/ml solution with about 0.1ng in compositions described herein.Again such as, bFGF can exist to the concentration of about 100 μ gbFGF/ml solution with about 0.1ng in compositions described herein.Again such as, bFGF can exist to the concentration of about 10 μ gbFGF/ml solution with about 0.1ng in compositions described herein.Again such as, bFGF can exist to the concentration of about 1 μ gbFGF/ml solution with about 0.1ng in compositions described herein.Again such as, bFGF can exist to the concentration of about 500ngbFGF/ml solution with about 0.1ng in compositions described herein.Again such as, bFGF can exist to the concentration of about 200ngbFGF/ml solution with about 0.1ng in compositions described herein.Again such as, bFGF can exist to the concentration of about 100ngbFGF/ml solution with about 0.1ng in compositions described herein.Again such as, bFGF can exist to the concentration of about 10ngbFGF/ml solution with about 0.1ng in compositions described herein.Again such as, bFGF can exist with the concentration of about 167ngbFGF/ml solution in compositions described herein.

When having been found that VEGF and bFGF adds collagen protein sponge and inserts in pulp cavity after the process of root pipe, it effectively can repair viability tissue (see such as embodiment 1) in pulp cavity and root pipe.

In some embodiments, wherein compositions comprises VEGF and bFGF, and described compositions comprises about 0.001ng to about 10,000 μ gVEGF and about 0.001ng to about 10,000 μ gbFGF/ml solution.In other embodiments, described compositions comprises about 0.01ng to about 1,000 μ gVEGF and about 0.02ng to about 2,000 μ gbFGF/ml solution.In further embodiment, described compositions comprises about 10ng to about 200ngVEGF and about 50ng to about 500ngbFGF/ml solution.In further embodiment, described compositions comprises about 33ngVEGF and about 167ngbFGF/ml solution.

Solution containing above-mentioned concentration bFGF can to import in substrate or support or with its combination.

BFGF is commercially available.

NGF

Regeneration dental pulp described herein or Dentinal compositions can comprise nerve growth factor (NGF).This compositions can comprise NGF and combine other bioactivator, as Wnt3a, VEGF, bFGF or BMP-7.

NGF can exist to the concentration of about 1,000mgNGF/ml solution with about 0.1ng in compositions described herein.Such as, NGF can exist to the concentration of about 100mgNGF/ml solution with about 0.1ng in compositions described herein.Again such as, NGF can exist to the concentration of about 10mgNGF/ml solution with about 0.1ng in compositions described herein.Again such as, NGF can exist to the concentration of about 1mgNGF/ml solution with about 0.1ng in compositions described herein.Again such as, NGF can exist to the concentration of about 0.1mgNGF/ml solution with about 0.1ng in compositions described herein.Again such as, NGF can exist to the concentration of about 100 μ gNGF/ml solution with about 0.1ng in compositions described herein.Again such as, NGF can exist to the concentration of about 10 μ gNGF/ml solution with about 0.1ng in compositions described herein.Again such as, NGF can exist to the concentration of about 1 μ gNGF/ml solution with about 0.1ng in compositions described herein.Again such as, NGF can exist to the concentration of about 500ngNGF/ml solution with about 0.1ng in compositions described herein.Again such as, NGF can exist to the concentration of about 200ngNGF/ml solution with about 0.1ng in compositions described herein.Again such as, NGF can exist to the concentration of about 100ngNGF/ml solution with about 0.1ng in compositions described herein.Again such as, NGF can exist to the concentration of about 10ngNGF/ml solution with about 0.1ng in compositions described herein.

NGF can exist to the concentration of about 500ngNGF/ml solution with about 0.2ng in compositions described herein.Such as, NGF can exist to the concentration of about 100ngNGF/ml solution with about 0.5ng in compositions described herein.Again such as, NGF can exist to the concentration of about 10ngNGF/ml solution with about 1ng in compositions described herein.

In some embodiments, wherein compositions comprises BMP-7 and NGF, and described compositions comprises about 0.2ng to 10,000ngBMP-7 and about 0.2ng to 500ngNGF/ml solution.In other embodiments, described compositions comprises about 1ng to 1000ngBMP-7 and about 0.5ng to 100ngNGF/ml solution.In further embodiment, described active component composition comprises about 5ng to 50ngBMP-7 and about 1ng to 10ngNGF/ml solution.

Solution containing above-mentioned concentration NGF can to import in substrate or support or with its combination.

NGF is commercially available.

Substrate or support

One aspect of the present invention provides and is suitable for inserting the substrate in pulp cavity or support, the compositions wherein comprising one or more bioactivator described herein to be included among described substrate or support or on.The substrate or the support that comprise the compositions of bioactivator can promote that when inserting in pulp cavity described substrate or support medium vessels organization formation or nerve are formed.In some embodiments, described substrate or support do not comprise living cells.This material can be used in tooth, dental pulp or root pipe processing method.

As used herein, " substrate " is impalpable structure, such as gel, and wherein can suspend one or more bioactive ingredients." support " is interpreted as having secondary or tertiary structure (such as column construction or loose structure, as in typical collagen protein sponge, such as have the on all four hole between about 250-400 μM, wherein one or more bioactive ingredients can permeate).The invention is not restricted to any specific substrate or support.Preferred described substrate or support are biodegradable.

In some embodiments, substrate or support comprise hydrogel.Hydrogel is interpreted as having polymer chain network, and it is hydrophilic, is colloidal gel sometimes, and wherein water is disperse medium.Hydrogel can be the polymer that highly absorbable (such as the above water of about 80%, 85%, 90%, 95%, 99% or 99.9%) is natural or synthesize.Hydrogel also can have the flexibility with natural tissues similarity degree due to its significant water content.Hydrogel can comprise such as polyvinyl alcohol, sodium polyacrylate, acrylate polymer or have the copolymer of hydrophilic radical.Natural hydrogel can comprise agarose, methylcellulose, hyaluronic acid or other natural derivative polymer.

The compositions comprising one or more bioactivator can by any mode known in the art and described substrate or holder combination.Such as, the compositions comprising one or more bioactivator can be injected in substrate or support.Again such as, the compositions comprising one or more bioactivator can mix with substrate or support.Again such as, the compositions comprising one or more bioactivator can be encapsulated in substrate or support by methods known in the art, or chemistry is bound by or is adsorbed in substrate or support.

Substrate or support can be placed in operation preparation teeth cavity on or insert removing pathological tissues, comprise in the pulp cavity after rotten enamel and dentin.For the substrate inserted in the teeth cavity of preparation or timbering material as Alginate hydrogel, blood, tissue fluid or water can be preferentially absorbed in biomaterial.Substrate or support be retained in original position or removing material after, pulp cavity can be closed or ill tooth can recover.

In some embodiments, biomedical devices can remove after about 24 hours.Such as, biomedical devices can remove after about 24 to about 48 hours.Again such as, biomedical devices can remove after about 48 hours.

Substrate or support can provide the substrate of Growth of Cells or organization formation.The beneficial property of substrate or support is the ability of porous, biocompatibility and biodegradable, sustenticular cell growth, or it is used as the ability (Murphy1999) of controlled gene and protein delivery carrier.The three-dimensional macromolecular structure provided by support can instruct the net shape (Murphy1999) of Bioengineered tissue.

Support described herein can have the shape of any mammalian tooth or hole wherein or chamber.Such as, support can have people's incisor, people's canine tooth, people's bicuspid or people grinds one's teeth in sleep or the shape of hole wherein or chamber.Substrate described herein can be applicable to any mammiferous natural or artificial hole or chamber.

Much research has investigated exogenous growth factor in the carrier to carry somatomedin to the importance and functions of implant site.Although perhaps carrier is not provided as the required any other factor of organization formation, it can be still the important component (Wozney1990) of growth course.One of function of carrier keeps the described factor at implant site and therefore increases its local concentration.Carrier also can as environment, and tissue can be formed wherein, and therefore contributes to limiting the region (Whang1998) that new organization can be formed.Collagen or synthetic vectors has been used as conveying carrier, and its physicochemical properties and its microenvironment produced work in generalise results (inductiveoutcome).Carrier can be solid xenogenesis (such as hydroxyapatite) (Kuboki1995, Murata1998), solid alloplastic (polyethylene polymer) material (Saito1998, or spontaneous gel (Sweeney1995 Isobe1999), Schwartz1998), allochthonous (Bax1999, or alloplastic source (Santos1998), and the combination of above-mentioned material (Alpaslan1996) Viljanen1997).

Substrate or support can comprise other bioactive molecule any further, such as antibiotic or chemotaxis somatomedin.In some embodiments, substrate or support strengthen, by adding such as human serum albumin (HSA), hetastarch, glucosan or its combination.For the suitable concn of these compounds in the present composition for it be known to those skilled in the art that or can being easy to without undo experimentation determine.

In substrate or support, the concentration of compound can change according to the treatment of the character of compound, its physiological role and hope or diagnosis effect.Treatment effective dose normally therapeutic agent show wish effect and without excessive toxicity enough concentration.

Compound can by any known method doped matrix or support.In some embodiments, compound is embedded in gel, such as, in collagen gel, in the hole of doped matrix or support, as be shown in the examples.

Or chemical modification method can be used for covalently bound for compound in substrate or rack surface.The surface functional group of substrate or support can, with the reactive functional groups coupling of compound to form covalent bond, use coupling agent well known in the art as acetaldehyde compound, carbodiimide etc.In addition, spacer molecule can be used for making the reactive group of surface reaction group and biomolecule to form breach, to make this molecule in stromal surface more flexible.Biomolecule and its content or outside other similarity method adhered to is made to be that those skilled in the art are known.

Compound or can by importing in substrate or on it based on system such as the encapsulation vehicle (vehicle) of carrier (carrier).This carrier can be used as slow releasing composition.Such as, compound can be microencapsulation with the time of delivery of the stability or prolongation that provide enhancing.Capsulation carrier includes but not limited to microgranule, liposome, microsphere etc., or the combination of any above-mentioned carrier, to provide the release mode of the hope of various ratio.Other method of Co ntrolled release conveying preparation is that those skilled in the art are known.In addition, these and other system can combine or adjust to optimize the integration/release of Medium Culture preparation.

Polymer that is that polymer microballoon can use natural generation or that synthesize produces, and it is the microparticulate systems of size 0.1-500 μm of scope.Polymer micelle and polymer vesicle (polymeromes) have the polymer transport carrier with microsphere similar characteristic, also can promote that the capsulation of compound described herein and substrate are integrated.Manufacture, the encapsulation of the microsphere of various payload and be stabilized in (see such as Varde & Pack (2004) ExpertOpin.Biol.4 (1) 35-51) within the scope of art technology.The rate of release of microsphere can by the type of polymer, polymer molecular weight, copolymer composition, add excipient in microsphere dosage forms and microsphere size and adjust.Polymeric material for the formation of microsphere comprises the DPPG (such as Solidose) of PLA, PLGA, PLGA, DPPC with DPPC bag quilt, DSPC, EVAc, gelatin, albumin, chitosan, glucosan, DL-PLG, SDLMs, PEG (such as ProMaxx), hyaluronate sodium, Diketopiperazine derivative (such as Technosphere), calcium phosphate-PEG microgranule and/or oligosaccharide derivatization.Capsulation can such as use water/oily single emulsification method, water-oil-water double emulsification process or lyophilization to realize.Some business capsulation technology be obtainable (such as alkerme).

Liposome also may be used for integration compound and substrate or support.The preparation carrying capacity of liposome and rate of release can be dependent on lipid components, size, electric charge, drug/lipid ratios and carrying method.Conventional liposome is made up of neutral or anion lipid (natural or synthesis).Conventional lipid is lecithin preparations, as phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, sphingomyelins, Phosphatidylserine, phosphatidyl glycerol and phosphatidylinositols.Liposomes enclose method is known in the art (Galovicetal. (2002) Eur.J.Pharm.Sci.15,441-448; Wagneretal. (2002) J.LiposomeRes.12,259-270).Target liposomes and reactive liposome also may be used for combining described preparation and substrate.Target liposomes has targeting part and is attached to its surface as monoclonal antibody or agglutinin, makes to interact with specific receptor and/or cell type.Reactive or polymorphism liposome comprises various liposome, and its total character is that it changes the tendency (such as pH-sensitive liposome) of its phase and structure based on specific interaction.See such as Lasic (1997) LiposomesinGeneDelivery, CRCPress, FL).

Any material manufacture that substrate or support can be approved by those skilled in the art.Suitable substrate or timbering material are at such as MaandElisseeff, ed. (2005) ScaffoldinginTissueEngineering, CRC, ISBN1574445219; Saltzman (2004) TissueEngineering:EngineeringPrinciplesfortheDesignofRep lacementOrgansandTissues, discusses in OxfordISBN019514130X.Limiting examples for all or part substrate or the potential available material of support comprises Polyethylene Glycol, polyactide, polyglycolic acid, PLGA, poly-(caprolactone), polyanhydride, polyglactin, Merlon, polyamide, polyanhydride, polyamino acid, polyorthoesters, polyacetals, polycyanoacrylate), polyphosphazene, degradable polyurethane, polyacrylate, the cellulose acetate of ethylene-vinyl acetate polymer and other acyl substituted and derivant thereof, polyurethane, polystyrene, polrvinyl chloride, polyvinyl fluoride, polyvinylpyrrolidone, poly-(vinyl imidazol), chlorosulfonated polyolefin, polyethylene glycol oxide, polyvinyl alcohol, , nylon, agarose, alginate (such as calcium alginate gel), fibrin, Fibrinogen, fibronectin, collagen protein (such as collagen gel), gelatin, hyaluronic acid, chitin, and other suitable polymer and biopolymer, or analog, mixture, combination, and the derivant of above-mentioned material.

In some embodiments, substrate or support comprise natural polymer.The natural polymer of citing is collagen protein, chitosan and polysaccharide.In other embodiments, substrate or support comprise synthetic polymer.The synthetic polymer of citing is aliphatic polyester and the Polyethylene Glycol of poly-('alpha '-hydroxy acids).The polymer of synthesis is in addition the mixture (PLGA) of polylactic acid (PLA), polyglycolic acid (PGA) and PLA and PGA.In some embodiments, synthetic polymer is the PLGA comprising about 50%PLA and 50%PGA.In other embodiments, substrate or support comprise collagen protein sponge or PLGA.

In some embodiments, substrate or support are by the compositions manufacture comprising bone guided material.The limiting examples of bone guided material is hydroxyapatite (HA).HA was used as bone substitute (Liao2006, Nebahat2006, Lijun2006, Wei2003) due to its good biocompatibility and high bioactivity over many years.

Although HA has good biological activity and bone guided, it is very frangible and have not good intrinsic tensility matter.Therefore, in some embodiments, HA and ε-polycaprolactone (PCL) combination.PCL is good bone matrix or timbering material because its in several years degradation in vivo and be biocompatible, relatively inexpensive and can greatly quantity obtain (Rich2002, Kim2004).PCL and HA combination (PCL-HA) provides the combination (Patcharaporn2005, Rezwan2006, Landis1995, Ziv1994) of the hope of biological activity, biodegradable and intensity.The PCL-HA material of compound is considered to have the biocompatibility of best substrate or support, cell adhesion, propagation and differentiation character (Zhao2008).In some embodiments, substrate or support comprise the mixture of about 80wt% polycaprolactone and about 20wt% hydroxyapatite.In other embodiments, substrate or support comprise about 60wt% polycaprolactone and about 40wt% hydroxyapatite extremely about 95wt% polycaprolactone and about 5wt% hydroxyapatite.Such as, substrate or support can comprise about 70wt% polycaprolactone and about 30wt% hydroxyapatite.Again such as, substrate or support can comprise about 90wt% polycaprolactone and about 10wt% hydroxyapatite.

In some embodiments, substrate or support have more highly porous.This loose structure provides the ingrown space of cell migration, adhesion and new bone tissue (Gazdag1995, Rezwan2006, Mano2004, Shin2003, Kim2001, Leong2003).

The hole of support and passage can be engineered to various diameter.Such as, the diameter in the hole of support can be that micron is to millimeter scope.In some embodiments, the hole of host material comprises microchannel.The average diameter of microchannel be about 0.1 μm to about 1,000 μm, such as about 50 μm to about 500 μm (such as about 100 μm, 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm or about 550 μm).The distribution that technical staff understands microchannel diameter can have any distribution mode, comprises normal distribution or improper distribution.In some embodiments, microchannel is the naturally occurring feature of host material.In other embodiments, microchannel is engineered to and exists in host material.

Present invention also offers the method for displacement mammalian mouth Tooth.In these embodiments, absence of tooth, and have alveolus at the dental area of disappearance in oral cavity.Described method is included in the acellular support implanted in alveolus and have the teeth patterning of disappearance.

Certain methods is for the manufacture of porous support, comprise particle leaching method, gas foaming method, electrostatic spinning, lyophilization, enamel foaming (foamingofceramicfromslurry) from pulp material, and organic foam forms (Mikos1994, Mooney1996, Qing2002, Sylvain2006).But, by the support using these methods to prepare, there is the structure of limiting holes and the shortcoming of being connected to each other property, can be limited it like this and be applied to Premeabilisation of cells aspect (Yeong2004, Tan2003) in organizational project.

In some embodiments, described method comprise further by computer-aided design (CAD) make missing tooth model and with biological drawing apparatus synthesis support.This method can provide has support that is more highly porous and being well connected to each other property.Propose Rapid Prototype Design (RP) method as fusion sediment modeling, selective laser sintering art, 3D printing, heterogeneous injection solidification and 3D drawing practice (Hutmacher2001, Moroni2006).

The key feature of Rapid Prototype Design is free entity shaping (SFF) method: 3D computer model is cut into series of thin layers, and it is for building composite laminate object.Thin layer is produced by the bonding of melting and solidification, layer light polymerization or microgranule, uses the sintering (selective laser sintering) of laser beam induction or special binding agent to carry out (Landers2002).In recent years, specialization rapid prototyping system (Bioplotter has been introduced ^tM, EnvisionTec, Germany), make it possible to the support that Design and manufacture has the anatomic shape of different internal structure, thus make it possible to accurate control hole size, porous, permeability and hardness (Landers2002; Landers2005).Use Bioplotter ^tMneed the 3D morphologic information of target tissue or tissue defects with the prototype method manufacturing tissue specificity PCL-HA support, this can pass through computerised tomography (CT) or nuclear magnetic resonance (MRI) obtains.When the tooth lacked has homologue at oral cavity opposite side, this homologue can be used as model to design the support of missing tooth.

Then the information of above-mentioned acquisition is used for by CAD design functionality support, and moves to Bioplotter ^tMsystem.Within the system, Bioplotter ^tMmechanical fusion also distributes timbering material (such as PCL-HA) with laminated on collecting board.Hole, such as microchannel, can produce as a part for design.The 3D support manufactured by RP system produces obvious Premeabilisation of cells, and therefore has desirable support character (Heo2007).These 3D supports can have clinical practice potentiality, by providing patient for nutrient transhipment and the tissue specificity anatomic shape of vascularization and the internal microstructure of optimization.More detailed discussion about these methods provides in the open WO2009/006558 of PCT, and it is incorporated herein by reference.

Invention additionally provides the method making tooth scaffold.Described method comprises the Acellular matrix of synthesis mammalian tooth shape and adds compound as herein described.In some embodiments of these methods, tooth is made into the shape the same with the tooth lacked in mammal, and described method comprises the tooth model being made disappearance by computer-aided design further, and synthesizes support with biological drawing apparatus.When the tooth lacked has corresponding tooth in the oral cavity, such as grind one's teeth in sleep, described method comprises further carries out such as in the similar CT scan of grinding one's teeth in sleep of oral cavity opposite side, wherein the CAD CT scan design data support that utilizes second to grind one's teeth in sleep.

In some embodiments of these methods, bioactivator described herein or its nucleic acid of encoding comprise in the bracket.

In some embodiments of these methods, the compound comprised in support can be Wnt3a polypeptide, platelet derived growth factor (PDGF), endothelial cell growth factor (ECGF) (ECGF), transforming growth factor-beta 1 (TGF-β 1), epidermal growth factor (EGF), hepatocyte growth factor (HGF), CXCL12 (SDF1), bone morphogenetic protein (BMP), TGF-β, GDF (GDF), insulin-like growth factor-i (IGF1), vascular endothelial cell growth factor (VEGF), fibroblast growth factor (FGF), dentin matrix protein, dentin sialoprotein, resorption lacunae, amelogenin or integrin.

In other embodiments, support is by the compositions manufacture comprising bone guided material.As discussed above, the example of available bone guided material is hydroxyapatite.Further example is the mixture as above-mentioned ε-polycaprolactone and hydroxyapatite.In the various embodiments of these methods, support comprises and has the microchannel that diameter is 50-500 μm.In further embodiment, support comprises atresia cap further.

CFU-GM

As used herein, CFU-GM (such as stem cell) is relatively undifferentiated cell, upgrades, also can be divided into the cell type of more specialization by cell mitogen self.As known in the art, stem cell comprises embryonic stem cell, it is all-round, the all cells type of the organism that it therefrom derives can be divided into, and adult stem cell, it is that multipotency (pluripotent) stem cell (can be divided into nearly all cell type, comprise the type of all three germinal layers), multipotency (multipotent) stem cell (can be divided into some cell types of cells into close associated families) or unipotent stem cell (can be divided into only a kind of cell type, but distinguish with non-stem cell the ability be by mitosis self renewal).

As used herein, tooth stem cell (DSC; Also referred to as the stem cell that tooth is derivative, TSC) be CFU-GM derived from vertebrates dental pulp.DSC can from tool any vertebrate any tooth dentulous.In some embodiments, tooth is stem cell-derived from deciduous teeth.In other embodiments, tooth stem cell-derived from premolars (premolar), grind one's teeth in sleep, incisor or canine tooth.DSC can be divided into the cell of all three germinal layers, is therefore pluripotent cell.

Dental tissue

As described herein, dental tissue can regenerate when contacting with Wnt3a polypeptide.Dental tissue can be dental pulp, coronal pulp (such as occlusal surface, central authorities, tip, buccal surface, lingual surface or bottom), radicular pulp, periapical tissue, the all connective tissues of cusp, periodontal tissue, Accessory canals, apical foramen of tooth, hole (foramina), Rinaggio district, Weil district, odontoblast layer, bone, gingiva, blood vessel, neural, Raschkow plexus nervorum, cementum, dentin, sclerosis dentin, compensating dentin, reactive dentin, reparative dentin, dentinal tubule, new dentin, or containing any ameloblast, fibroblast, odontoblast, histiocyte, macrophage, granulocyte, mastocyte or plasmacytic tissue.

Formulation

Preparation described herein and compositions can be prepared by any usual manner, use one or more materia medica acceptable carrier or excipient, as at Remington ' sPharmaceuticalSciences (A.R.Gennaro, Ed.), 21stedition, describe in ISBN:0781746736 (2005), be incorporated to herein incorporated by reference.This formulation contains the bioactivator described herein for the treatment of effective dose, and it can be purified form, and appropriate carrier is to provide the form correctly giving object.This formulation can among doped matrix or support or on.

Each preparation also can combine one or more other preparation or give together with other biological activity or biologically inert preparation.These biological activitys or inert formulation can be liquid, or lead to described pharmaceutical machine sexual intercourse, or are adhered to by ion, covalency, Van der Waals, hydrophobicity, hydrophilic or other physical force and described preparation.

(or sustained release) prepared product of Co ntrolled release can be prepared, to expand the activity of preparation and to reduce administration frequency.The prepared product of Co ntrolled release also can be used for influence time of origin or other characteristic, as the blood level of preparation, and therefore affects the generation of side effect.The prepared product of Co ntrolled release can be designed as the preparation that initial release produces the amount of the therapeutical effect of wishing, and progressively and constantly discharges other preparation measured with maintaining treatment exposure level within the time extended.In order to maintain the preparation close to constant level in body, described preparation can to replace being discharged from dosage form by metabolism or the speed of the amount of preparation of draining from body.The Co ntrolled release of preparation can be stimulated by various inducement, such as pH change, variations in temperature, enzyme, water or other physiological conditions or molecule.

Preparation described herein or compositions also may be used for combining other Therapeutic mode, as further discussed below.Therefore, except Therapeutic Method described herein, also known other Therapeutic Method useful for disease therapy, imbalance or disease can be provided for object.

Therapeutic Method

Present invention also offers a kind ofly is needing to give to treat to regenerate the method for dental tissue comprising in the substrate of Wnt3a, BMP-7, VEGF, bFGF or NGF or its combination or the object of support of effective dose, to promote the odontoblast differentiatio` of CFU-GM, promote cell migration, regeneration vessel pulp tissue, or regenerate new dentin.

Present invention also offers a kind of method regenerating dental tissue in object needing to give the Wnt3a polypeptide for the treatment of effective dose, to promote the odontoblast differentiatio` of CFU-GM, promoting cell migration, regeneration vessel pulp tissue, or regenerating new dentin.

As used herein, dental treating method can relate to any method of tooth.The dental treating method of citing comprises pulpal treatment, and it relates to dental pulp.The process of root pipe is a kind of dental treating method, and wherein whole dental pulps and root tubing are all removed, and replaces by inert material or compositions described herein, and it can recover living tissue in pulp cavity.

One aspect of the present invention provides the method for to carry out tooth, dental pulp or root canal in mammalian tooth in need.Described method can comprise wound or ill pulp tissue in Exposed Pulp chamber or root pipe; Cover by the compositions (such as comprising the support of the compositions of bioactivator) comprising bioactive ingredients or filling pulp cavity at least partially or root pipe.As described herein, described compositions can promote that angiogenic, odontogenesis, fiber generation or neurogenic are grown.In some embodiments, described compositions does not comprise living cells during covering or filling.In some embodiments, described method comprises further remove wound or ill pulp tissue from tooth, produces the pulp cavity or the root pipe that there is no wound or illing tissue.In some embodiments, substrate or support contain the compositions of bioactivator, and described substrate or support insert in teeth cavity or root pipe.

An application of the present invention is root canal, and wherein all pulp tissues all remove from tooth.Substrate described herein or support can partially or completely replace current dental pulp filler as the gutta-percha in those methods.Current method is not got rid of and is combinationally used described substrate or support and current material as gutta-percha.Therefore, in some embodiments, inert material such as gutta-percha also inserts in pulp cavity.

The dental pulp substituted can due to specify carry out tooth, dental pulp or the process of root pipe any disease caused by.Such as, pulp tissue can be bacteriological infection.Or pulp tissue destroys due to wound, or there is defect in pulp tissue.

Methods described herein are carried out object in need usually.Need the object of Therapeutic Method described herein can be suffer from, diagnose have, doubtful suffer from or be in there is the damage relevant to dental tissue, disease or disease as the object in tooth wound, pulpitis or dental caries equivalent risk.Such as, need the object of Therapeutic Method described herein can be suffer from, diagnose have, doubtful suffer from or be in there are medicable pulpitis or conventional needs and remove object in the Other diseases of healthy dental pulp or disease risk along with diseased pulp together.Determine whether to need treatment typical case to be evaluated by the medical history consistent with described disease or disease and physical examination.The various diagnosis by the medicable disease of methods described herein is in those skilled in the art's technical scope.Object can be animal, comprises mammal as horse, cattle, Canis familiaris L., cat, sheep, pig, mice, rat, monkey, hamster, Cavia porcellus and chicken, Yi Jiren.Such as, object can be people.

Normally, the safe and effective amount comprising Wnt3a, BMP-7, VEG, bFGF or NGF or its substrate combined or support is the therapeutical effect such as producing hope in object, simultaneously the amount of undesirable minimize side effects.In various embodiments, comprise Wnt3a, BMP-7, VEGF, bFGF or NGF or its substrate combined or support of effective dose promote the odontoblast differentiatio` of CFU-GM, promote cell migration, regeneration vessel pulp tissue or regenerate new dentin.

Normally, the safe and effective amount of Wnt3a polypeptide is the therapeutical effect such as producing hope in object, simultaneously the amount of undesirable minimize side effects.In various embodiments, the Wnt3a polypeptide of effective dose promotes the odontoblast differentiatio` of CFU-GM, promotes cell migration, regeneration vessel pulp tissue or regenerate new dentin.

When using in treatment described herein, Wnt3a, BMP-7, VEGF, bFGF or NGF for the treatment of effective dose can purified form application, and wherein this form exists with the acceptable salt form of materia medica, have or without the acceptable excipient of materia medica.Such as, reasonable benefit/risk-ratio that compound of the present invention all can be suitable for for any therapeutic treatment gives with q.s, to promote the odontoblast differentiatio` of CFU-GM, promote cell migration, regeneration vessel pulp tissue or regenerate new dentin.

The amount that can combine the compositions to produce single dose form from materia medica acceptable carrier is different according to the host treated and the AD HOC that gives.Those skilled in the art recognize each dosage form individually dosed in the unit content of preparation that comprises himself do not need composition treatment effective dose, because required treatment effective dose can be many individually dosed and reach by giving.

Toxicity and the treatment effect of compositions as herein described can determine LD by standard pharmaceutical procedures in cell culture or laboratory animal ₅₀(median lethal dose(LD 50)) and ED ₅₀(half treatment effective dose) and determine.Dosage rate between toxicity and therapeutical effect is therapeutic index, can be expressed as LD ₅₀/ ED ₅₀, wherein larger therapeutic index is best being commonly understood in the art to.

The specific treatment effective dose level dependant of any special object is in various factors, comprise the disease for the treatment of and the seriousness of disease, the activity of the specific compound of application, the particular composition of application, the age of object, body weight, general health situation, sex and diet, give the time, give approach, the drainage rate of the compositions of application, the treatment persistent period, the medicine combining with the specific compound of application or use simultaneously, and the known other factors of field of medicaments etc. is (see such as Koda-Kimbleetal. (2004) AppliedTherapeutics:TheClinicalUseofDrugs, LippincottWilliams & Wilkins, ISBN0781748453, Winter (2003) BasicClinicalPharmacokinetics, 4 ^thed., LippincottWilliams & Wilkins, ISBN0781741475, Sharqel (2004) AppliedBiopharmaceutics & Pharmacokinetics, McGraw-Hill/Appleton & Lange, ISBN0071375503).Such as, the initial dose that those skilled in the art know described compositions lower than the level needed for the therapeutical effect needing to reach hope, and progressively increases dosage until reach the effect of hope.If needed, effective dose can be divided into and repeatedly giving every day.Therefore, single dose compositions can be about quantity (submultiple) to form every daily dose containing this amount or its.But total dosage every day should understanding compound of the present invention and compositions can be determined within the scope of rational medical judgment by attending doctor.

Again, often kind of pathological state as herein described, disease, imbalance and disease and other disease all can have benefited from compositions as herein described and method.Normally, but treatment of pathological conditions, disease, imbalance or disease comprise and stop or postpone to get involved in mammal or tend to described pathological state, disease, imbalance or disease do not experience or show the appearance of clinical symptoms of clinical or inferior clinical symptom yet.Treatment also can comprise and suppress described pathological state, disease, imbalance or disease, such as, stop or reduce disease or its at least one generation that is clinical or inferior clinical symptom.In addition, treatment can comprise releasing disease, such as, cause described pathological state, disease, imbalance or disease or its at least one decline that is clinical or inferior clinical symptom.Benefit for treatment target can be statistically significant or be at least object or the perception of doctor institute.

Giving of compositions of the present invention can be once give or give in treatment time-histories.Such as, compositions can every day, weekly, every two weeks or monthly give once.For the treatment of acute disease, treatment time-histories normally at least several days.Some disease can extended treatment from several days to a few week.Such as, treatment can extend more than one week, two weeks or three weeks.For chronicer disease, treatment can from extending to some months even 1 year or the longer time a few week.

According to the treatment of the inventive method can before the conventional therapy pattern of the damage relevant to dental tissue, disease or disease, simultaneously or carry out afterwards.

Compositions described herein can with another preparation as antibiotic, antiinflammatory or another preparation simultaneously or in succession give.Such as, comprise the substrate of Wnt3a, BMP-7, VEGF, bFGF or NGF or support can with another preparation as among antibiotic or antiinflammatory or substrate or support or on other preparation that can comprise give simultaneously.Give can be undertaken by giving independent compositions, often kind of compositions contains one or more substrate or support, Wnt3a, BMP-7, VEGF, bFGF, NGF, antibiotic, antiinflammatory or another preparation above-mentioned simultaneously.The substrate or the support that comprise Wnt3a, BMP-7, VEGF, bFGF or NGF can give in succession with antibiotic, antiinflammatory or another preparation.Such as, the substrate or the support that comprise Wnt3a, BMP-7, VEGF, bFGF or NGF can give before or after giving antibiotic, antiinflammatory or another preparation.

List Wnt3a polypeptide above.Those skilled in the art's understanding is discussed above can in the same manner for the nucleic acid of Wnt3a polypeptide of encoding.

Molecular engineering

There is provided as given a definition with method to define the present invention better and to instruct those skilled in the art to implement the present invention.Unless otherwise indicated, in literary composition, term is the common usage according to various equivalent modifications.

As used herein, term " allogeneic dna sequence ", " foreign DNA section " or " heterologous nucleic acids " all refer to the sequence being derived from the source different from specific host cell, if or identical source, modified from its primitive form.Therefore, the heterologous gene in host cell comprises the endogenous gene still modified by such as using DNA reorganization of particular host cell.Multiple copies that the non-natural that this term also comprises naturally occurring DNA sequence exists.Therefore, this term refers to for cell it is the region of DNA section of external source or allos, or with cell-isogenic but in host cell nucleic acid the not position that usually finds of this element.Foreign DNA section is expressed and is produced allogenic polypeptide." homology " DNA sequence is relevant DNA sequence natural in the host cell among its importing.

Expression vector, expression construct, plasmid or recombinant dna construct are generally understood as the nucleic acid referring to and produced by manual intervention; comprise by recombination form or direct chemosynthesis mode, use a series of specific nucleic acid elements to allow specific nucleic acid in such as host cell transcription or translation.Expression vector can be a part for plasmid, virus or nucleic acid fragment.Typically, expression vector can comprise the transcribed nucleic acid be operably connected with promoter.

" promoter " is generally understood as nucleic acid control sequence, and it instructs transcribing of nucleic acid.It is the promoter of transcribing mediating the gene response particular stimulation operably connected that inducible promoter is generally understood as.Promoter can be included in the required nucleotide sequence of near transcriptional start sites, as the TATA element when polymerase Il type promoter.Promoter can optionally comprise end enhancer or repressor element, and it can be positioned at the position with transcriptional start site several thousand base pairs.

As used herein, " transcribed nucleic acid molecules " refers to any nucleic acid molecules that can be transcribed into RNA molecule.Known in a certain way by construct transfered cell, nucleic acid molecules transcribed is thus transcribed into functional mRNA molecule, and it is translated and is therefore expressed as protein.Construct also can be constructed as energy antisence RNA molecule, to suppress the translation of interested specific RNA molecule.For enforcement the present invention, conventional composition and preparation and use the method for construct and host cell to know (see such as SambrookandRussel (2006) CondensedProtocolsfromMolecularCloning:ALaboratoryManual for those skilled in the art, ColdSpringHarborLaboratoryPress, ISBN-10:0879697717; Ausubeletal. (2002) ShortProtocolsinMolecularBiology, 5thed., CurrentProtocols, ISBN-10:0471250929; SambrookandRussel (2001) MolecularCloning:ALaboratoryManual, 3ded., ColdSpringHarborLaboratoryPress, ISBN-10:0879695773; Elhai, J.andWolk, C.P.1988.MethodsinEnzymology167,747-754).

" transcriptional start site " or " initiation site " is the position around first nucleotide, and it is a part for transcription sequence, is also defined as position+1.About this site, other sequences all of gene and control zone thereof all can be numbered.Downstream sequence (i.e. the further protein coding sequence in 3 ' direction) can called after just, and upstream sequence (mainly 5 ' direction controlling district) called after bear.

" operably connection " or " functional connection " preferably refer to the associating of nucleotide sequence in single nucleic acid fragment, and its function interacts thus.Such as, if the position of two sequences is modulability DNA sequence affect mode that DNA sequences encoding expresses (namely coded sequence or functional r NA promoter transcribe control under), then modulability DNA sequence is " being operably connected " or " associating " with the DNA sequence of coding RNA or polypeptide.Coded sequence can operably be connected in sense or antisense direction with adjustment sequence.Two nucleic acid molecules can be parts for single continuous print nucleic acid molecules, and can be adjacent.Such as, record at transit cell if promoter regulates or mediates interested gene, then this promoter and interested gene are operably connected.

" construct " is generally understood as any recombinant nucleic acid molecules as plasmid, cosmid, virus, autonomous replicating nucleic acid molecule, phage, or linear or annular strand or double-stranded DNA or RNA nucleic acid molecules, derived from any source, can genome conformity or independently copy, comprise the nucleic acid molecules that wherein one or more nucleic acid molecules has operably connected.

Construct of the present invention can contain the promoter be operably connected with transcribed nucleic acid molecules, and described transcribed nucleic acid molecules is operably connected with 3 ' tanscription termination nucleic acid molecules.In addition, construct can including but not limited to the other adjustment nucleic acid molecules of such as 3 ' untranslated region (3'UTR).Construct can including but not limited to the 5 ' untranslated region (5'UTR) of mRNA nucleic acid molecules, and it can play an important role in translation initiation, and also can be the genetic constitution in expression construct.These other upstream and downstreams regulate nucleic acid molecules can derived from source that is natural with other element of existing on promoter construct or allos.

Term " conversion " refers to that nucleic acid fragment moves in host cell gene group, causes the heredity of inheritance stability.Host cell containing the nucleic acid fragment transformed is called " transgenic " cell, and the organism comprising transgenic cell is called " Transgenic Organisms ".

" conversion ", " genetically modified " and " restructuring " refer to the host cell or organism that have wherein imported exogenous nucleic acid molecule, as antibacterial, cyanobacteria, animal or plant.As known in the art and disclose, nucleic acid molecules can enter (Sambrook1989 in genome by stable integration; Innis1995; Gelfand1995; Innis & Gelfand1999).Known PCR method includes but not limited to the method using paired primer, primer set, single specific primer, degraded primer, gene-specific primer, vector-specific primers, part mismatched primers etc.Term " unconverted " refers to the normal cell not carrying out Transformation Program.

" wild type " refers to the virus without any known mutations or the organism of natural discovery.

There is the percent identities of above-mentioned requirements and retain the design of the Variant nucleotide of activity of protein requirements and the polypeptide of coding thereof of expressing, generation and detection within the scope of art technology.Such as, the orthogenesis of mutant and sharp separation can carry out according to the method described in list of references, include but not limited to Linketal. (2007) NatureReviews5 (9), 680-688; Sangeretal. (1991) Gene97 (1), 119-123; Ghadessyetal. (2001) ProcNatlAcadSciUSA98 (8) 4552-4557.Therefore, those skilled in the art can produce numerous nucleotide and/or polypeptide variants, and it has such as at least 95-99% homogeny compared with reference sequences described herein, and screen according to the phenotype of this area conventional method for hope.

Nucleotide and/or Amino acid sequence identity percentage ratio (%) are interpreted as being when contrast two sequences, nucleotide identical in candidate sequence compared with reference sequences or the percentage ratio of amino acid residue.In order to determine percent identities, sequence being compared, if needed, importing breach to realize largest percentage sequence thereto.Determine that the sequence alignment programme of percent identities knows for those skilled in the art.The computer software that usually can openly obtain such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used for aligned sequences.Those skilled in the art can determine that suitable parameter is to measure contrast, comprise the high specific of realization at the sequence of contrast to required algorithm.When aligned sequences, the percent sequence identities (can be described as certain percent sequence identities that specified sequence A has or comprises specified sequence B in addition) of specified sequence A and specified sequence B may be calculated: percent sequence identities=X/Y100, wherein X is the residue number being counted as identical match by the alignment programs of A and B or the comparison of algorithm, and Y is the total number of residues in B.If the length of sequence A is not equal to the length of sequence B, then the percent sequence identities of A and B is different from the percent sequence identities of B and A.

Normally, conservative replacement can be carried out in any position, as long as retain the activity needed.Can carry out so-called conservative substitution, wherein replaced aminoacid has and Original amino similar quality, and such as Glu is replaced by Asp, Gln is replaced by Asn, Val is replaced by Ile, Leu is replaced by Ile, and Ser is replaced by Thr.Disappearance is that aminoacid is replaced by Direct Bonding.The position of disappearance comprises the connection between the end of polypeptide and independent protein domain.Insertion imports aminoacid in polypeptide chain, and Direct Bonding is replaced by one or more aminoacid.Aminoacid sequence can adjust by means of computer simulator known in the art, and it can produce the polypeptide of the regulating action with activity or the change such as improved.Based on the peptide sequence of this artificial generation, using the specific cryptosystem of the host cell of wishing to use can this corresponding nucleic molecule of polypeptide through adjustment of composite coding in vitro.

" high stringency conditions " is defined as in 65 DEG C of hybridization in 6 × SSC buffer (i.e. 0.9M sodium chloride and 0.09M sodium citrate).In view of these conditions, by calculating the melting temperature (T of DNA duplex between two sequences _m) can determine to specify the sequence of series whether to hybridize.If the melting temperature of a specific duplex is lower than 65 DEG C under 6 × SSC salt condition, then two sequences will not be hybridized.On the other hand, if melting temperature under identical salt condition higher than 65 DEG C, then two sequences will be hybridized.Normally, the melting temperature of the DNA:DNA sequence of any hybridization can use following formula to determine: T _m=81.5 DEG C of+16.6 (log ₁₀[Na ⁺])+0.41 (fraction G/C content) – 0.63 (% Methanamide) – (600/l).In addition, nucleotide sequence identity often reduces by 1%, then the T of DNA:DNA crossbred _mreduce 1-1.5 DEG C (see such as SambrookandRussel, 2006).

Host cell can use various standard technique known in the art to transform (see such as SambrookandRussel (2006) CondensedProtocolsfromMolecularCloning:ALaboratoryManual, ColdSpringHarborLaboratoryPress, ISBN-10:0879697717; Ausubeletal. (2002) ShortProtocolsinMolecularBiology, 5thed., CurrentProtocols, ISBN-10:0471250929; SambrookandRussel (2001) MolecularCloning:ALaboratoryManual, 3ded., ColdSpringHarborLaboratoryPress, ISBN-10:0879695773; Elhai, J.andWolk, C.P.1988.MethodsinEnzymology167,747-754).This technology includes but not limited to the conveying, receptor-mediated picked-up, cell fusion, electroporation etc. of viral infection, calcium phosphate transfection, liposome-mediated transfection, microgranule mediation.Can select and the cell of breeding transfection to provide recombinant host cell, it comprises stable integration and enters expression vector in host cell gene group.

But the nucleic acid that can import in host cell comprises such as from DNA sequence or the gene of another species, or even comes from or to be present in same species to mix gene in acceptor cell or sequence by genetic engineering method.Term " external source " refers to that gene is improper and is present in the cell of conversion, or be not perhaps only with the existence such as form, structure that finds in the region of DNA section transformed or gene, or normal presence also need the gene of expressing as process LAN mode in the mode being different from natural expression pattern.Therefore, term " external source " gene or DNA refer to and are imported into any gene in recipient cell or region of DNA section, no matter whether there is similar gene in this cell.The DNA type that foreign DNA comprises can comprise the DNA in Already in cell, from the DNA of identical type another individuality organic, from the organic DNA of difference, or the outside DNA produced, as the DNA sequence containing antisense gene information, or the DNA sequence of the gene forms of coding synthesis or modification.

Can by many known way assessments (see such as Studier (2005) ProteinExprPurif.41 (1), 207 – 234 according to the host strain that methods described herein produce; Gellissen, ed. (2005) ProductionofRecombinantProteins:NovelMicrobialandEukaryo ticExpressionSystems, Wiley-VCH, ISBN-10:3527310363; Baneyx (2004) ProteinExpressionTechnologies, Taylor & Francis, ISBN-10:0954523253).

The method of downward or silent gene is known in the art.Such as, the protein active of expressing can use antisense oligonucleotide, protein aptamers, nucleotide aptamers and RNA to disturb (RNAi), and (such as siRNA s (siRNA), short hairpin RNA (shRNA) and ceramic micro-nanocomposite (miRNA) etc. are lowered or are eliminated (see such as FanningandSymonds (2006) HandbExpPharmacol.173,289-303G, describes hammerhead ribozyme and children purpura nephritis; Helene, C., etal. (1992) Ann.N.Y.Acad.Sci.660,27-36; Maher (1992) Bioassays14 (12): 807-15, describe targeting deoxyribonucleotide sequence; Leeetal. (2006) CurrOpinChemBiol.10,1-8, describes aptamers; Reynoldsetal. (2004) NatureBiotechnology22 (3), 326-330, describes RNAi; PushparajandMelendez (2006) ClinicalandExperimentalPharmacologyandPhysiology33 (5-6), 504-510, describes RNAi; Dillonetal. (2005) AnnualReviewofPhysiology67,147-173, describes RNAi; DykxhoornandLieberman (2005) AnnualReviewofMedicine56,401-423, describe RNAi).RNAi molecule is commercially available from multiple source (such as Ambion, TX; SigmaAldrich, MO; Invitrogen).Some siRNA molecule of the various algorithm of use known in the art are designed program (see such as Cenixalgorithm, Ambion; BLOCK-iT ^tMrNAiDesigner, Invitrogen; SiRNAWhiteheadInstituteDesignTools, Bioinofrmatics & ResearchComputing).The character affecting the definition of best siRNA sequence is included in the G/C content of siRNA end, the Tm in the specific internal territory of siRNA, siRNA length, the position of target sequence in CDS (coding region), and the nucleotide content of 3 ' jag.

Test kit

Present invention also offers test kit.This test kit can comprise preparation described herein or compositions, and also comprises in some embodiments and use description.This test kit can be convenient to implement methods described herein.When providing with test kit, the heterogeneity of compositions can be packed in a separate container, at once mixes before the use.Composition components includes but not limited to substrate or support and Wnt3a polypeptide or polynucleotide, BMP-7, VEGF, bFGF or NGF, antibiotic, antiinflammatory, or as another preparation above-mentioned.If needed, the composition of this independent packaging may reside in a packaging or distributor, and it can containing one or more unit dosage form with described compositions.Packaging such as can comprise metal or plastic tab is packed as blister package.The composition of this independent packaging also can allow long storage periods in some cases and not lose the activity of described composition.

Test kit also can be included in the reagent in independent container, and such as sterilized water or saline have in the active component of the lyophilizing of independent packaging to be added.The glass ampule such as sealed can contain lyophilizing composition, and has in independent ampoule at neutral non-reactive gas as sterilized water, the Sterile Saline or without fungus packed under nitrogen.Ampoule can be made up of any suitable material, if glass, organic polymer are if Merlon, polystyrene, pottery, metal or typical case are for holding other material any of reagent.Other example of suitable container comprises bottle, and it can be made by with ampoule similar substance, and bag, and it can by paillon foil inwall as aluminum or alloy foil sheet form.Other container comprises test tube, bottle, flask, bottle, syringe etc.Container can have sterile access port, as having the bottle of the stopper that can be pierced through by subcutaneous injection injection needle.Other container can have two compartments separated by the film being easy to extract, and when being removed by film, composition mixes.Removable film can be glass, plastics, rubber etc.

In some embodiments, test kit can provide description.Description can be printed on paper or in other substrate, and/or can with electronically readable medium, and such as floppy disk, mini-CD-ROM, CD-ROM, DVD-ROM, Zip dish, video-tape, audiotape etc. provide.Detailed description book is not perhaps together with test kit; Replacing user can the internet site that specifies of the distributor of directed Producer or test kit.

Following patent is open to be incorporated to for referencial use with its full content: U.S. Patent Publication No.2011/0171607, BIOPULP; U.S. Patent Publication No.2013/0022989, DENTALSTEMCELLPROGRAMMING; U.S. Patent Publication No.2011/0236977, DENTALSTEMCELLDIFFERENTIATION; U.S. Patent Publication No.2012/0282573, TOOTHSCAFFOLDS; U.S. Patent Publication No.20110171607, BIOPULP; WO2012/045097, PRODUCTIONOFDENTIN, CEMENTUMANDENAMELBYCELLS.

The molecular biology method that compositions as herein described and method utilize can based on various standard technique known in the art (see such as SambrookandRussel (2006) CondensedProtocolsfromMolecularCloning:ALaboratoryManual, ColdSpringHarborLaboratoryPress, ISBN-10:0879697717; Ausubeletal. (2002) ShortProtocolsinMolecularBiology, 5thed., CurrentProtocols, ISBN-10:0471250929; SambrookandRussel (2001) MolecularCloning:ALaboratoryManual, 3ded., ColdSpringHarborLaboratoryPress, ISBN-10:0879695773; Elhai, J.andWolk, C.P.1988.MethodsinEnzymology167,747-754; Studier (2005) ProteinExprPurif.41 (1), 207 – 234; Gellissen, ed. (2005) ProductionofRecombinantProteins:NovelMicrobialandEukaryo ticExpressionSystems, Wiley-VCH, ISBN-10:3527310363; Baneyx (2004) ProteinExpressionTechnologies, Taylor & Francis, ISBN-10:0954523253).

Definition as herein described and method is provided to be to define the present invention better and instructing those skilled in the art to implement the present invention.Unless otherwise indicated, term uses according to various equivalent modifications convention.

In some embodiments, for describing and require the quantity of the expression composition of embodiment of the present invention, the numeral of character as molecular weight, reaction condition etc. be interpreted as being revised by term " approximately " in some cases.In some embodiments, term " approximately " is for representing that numerical value comprises the standard deviation of the meansigma methods for the device or method determining numerical value.In some embodiments, the numerical parameter described in description and claims is approximation, can change according to the character of the expectation obtained by particular.In some embodiments, described numerical parameter and should be applied usual approximation technique and explain according to the restricted numeral of report.Although the numerical range and the parameter that describe the wide region of some embodiments of the present invention are approximations, the numerical value in specific embodiment is accurately reported strictly according to the facts.The numerical value existed in some embodiments of the present invention can comprise some errors certainly led to by the standard deviation found in its separately thermometrically.Herein quoting of logarithm value is only method for simplifying as referring to each independent numerical value fallen within the scope of this one by one.Unless particularly pointed out in this article, each numerical value is all incorporated in this description, just as it is quoted in this article one by one.

In some embodiments, the term " " in the context (particularly at more following claim contexts) describing particular and " described " can be understood to include odd number and plural number, except as otherwise noted.In some embodiments, comprise claim, as used herein, term " or " refer to "and/or", unless clearly shown only to refer to options, or options is mutually exclusive.

Term " comprises ", " having " and " comprising " is open connection verb.Any form of these verbs or tense are also open.Such as, any method of " comprising ", " having " or " comprising " one or more step is not limited to only have those one or more steps, also can cover other unlisted steps.Similarly, any being combined as of " comprising ", " having " or " comprising " one or more feature is not limited to only have those one or more features, also can cover other unlisted features.

Methods described herein can be carried out with any proper order, unless otherwise indicated herein or the obvious contradiction of context.Use any and whole embodiment or citing language (such as " such as ") to be only better describe the present invention for some embodiments herein, do not limit the scope of the present invention.Language in description should not be considered to the element implementing any failed call protection of the present invention.

The set of optional element of the present invention described herein or embodiment should not be interpreted as restriction.Each set member can refer to or claimed one by one, or with other members of this set or other element combination in any of finding herein.One set one or more member can for convenience of or patentability and be included in or from one set delete.When any this comprise or delete occur time, description of the present invention is regarded as comprising the set through adjustment, meets the written description of all Ma Kushi set used in following claims thus.

Should not think to admit that it is prior art of the present invention to quoting of list of references herein.

The present invention is described in detail, under the prerequisite not departing from the scope of the invention limited in appended claims, can modifies to the present invention, change and the embodiment of equivalence.In addition, should recognize that all embodiments provided in the present invention are all non-limiting examples.

Embodiment

There is provided following non-limiting example with further illustration the present invention.Those skilled in the art should realize the technology disclosed in subsequent embodiment and represent the method that inventor has confirmed to have good function in enforcement of the present invention, and therefore can think that composition implements instance mode of the present invention.But according to the present invention, those skilled in the art should recognize that can carry out some to the embodiment of citing under prerequisite without departing from the spirit and scope of the present invention changes, and still can obtain same or analogous result.

Embodiment 1

People's incisor of choosing is carried out the process of root pipe.Then will to have or collagen protein sponge without bFGF and/or VEGF is implanted in root pipe.Then by the mice of subcutaneous for this incisor implantation immunodeficiency.Remove tooth after 2 weeks, evaluate the vascularization in pulp cavity and root pipe.

By perusal, to occur without obvious blood vessel with the tooth of the collagen protein sponge process without any bioactive ingredients that (Fig. 1 a).But, show vascularization (Fig. 1 b and 1c) with the tooth of the collagen protein sponge process with bFGF or bFGF and VEGF combination inserting in the collagen protein sponge in root pipe.

The root pipe of the tooth of above-mentioned process is assessed further by microscope.Show in root pipe with the tooth of the collagen protein sponge process without any bioactive ingredients and show inorganization growth (Fig. 2 A), and illustrate that vascularization and host tissue inwardly grow (Fig. 2 B-D) with the tooth of the collagen protein sponge process with bFGF or VEGF or bFGF+VEGF combination.The host tissue infiltrated in these process is attached to dentin.

Embodiment 2

Following embodiment confirms dentin generation bioactive ingredients, bone morphogenesis protein-7 (BMP-7), and neurogenic bioactive ingredients, the capsulation of neural bioactive ingredients (NGF) in biocompatible poly-d-l-lactic-co-glycolic acid (PLGA) microsphere and Co ntrolled release.PLGA is made up of 50:50 (PLA:PGA), slowly degrades.When PLGA microsphere was degraded along with the time, BMP-7 and NGF discharges gradually.After 4-6 week, the release mode of BMP-7 and NGF is determined by ELISA, and confirms the release concentration curve of accumulation.These find to provide to apply BMP-7 and NGF to regenerate the evidence of the viewpoint of dental pulp in vivo in biocompatible microsphere.

Poly-d-l-lactic-co-glycolic acid (PLGA, Sigma, St.Louis, MO) microsphere (Moiolietal., 2006 of 50:50PLA/PGA ratio are selected due to the open discovery in cumulative release pattern; 2007a, b; Clarketal., 2007) (Fig. 3).

First the 0.1%PVA of 100mL is prepared, and before importing other composition any, 450rpm prolonged agitation 30 minutes.Use double emulsion technique (W/O/W) (Moiolietal., 2006; 2007a, b; Clarketal., 50:50 ratio 2007) is prepared.The PLGA of 0.25g to be altogether dissolved in completely in 1mL dichloromethane and with the 2.5 μ g recombinant human B MP-7 diluted in 50 μ L solution or NGF emulsifying (maximum turn speed) 1 minute (Water-In-Oil).Then by 1% polyvinyl alcohol (PVA, 30,000 – 70, the 000MW) turn 1 minute (W/O/W) of elementary emulsion and 2mL.Then this mixture to be added in the 0.1%PVA in stirring and to stir 1 minute.In final emulsion, add 2% isopropyl alcohol of common 100mL, in chemical hood, prolonged agitation 2 hours is with except desolventizing.PLGA microsphere containing cytokine is separated by filtration (2 μm of filter membranes), freezing 30 minutes and lyophilizing 48 hours with distilled water wash and in liquid nitrogen.Before the use by the PLGA microsphere of lyophilizing-20 DEG C of storages.

The PLGA microsphere two groups of dentin generation and neurogenic cytokine encapsulation all distributes 4 samples (n=4).Often group has the cytokine of the 10mg encapsulation in the 1%BSA solution of 1mL, in 37 DEG C of prolonged agitations on shaking table.The acquisition number strong point by the supernatant in 4-6 all collections weekly whole amount.The 1%BSA solution of 1mL is changed after each collection.The amount of BMP-7 and NGF carries out measures of quantization by using BMP-7ELISA test kit and NGFELISA test kit for each sample.

Result illustrates the PLA/PGA using 50:50 ratio, BMP-7 and NGF microsphere discharges in vitro until 30-44 days.Between the period 1, find the release of outburst sample, compared with TGF β 3 Co ntrolled release result previously, similar release mode (Fig. 3) is shown.These two kinds of release modes all illustrate that 50:50PLGA can encapsulation BMP-7 and NGF, and have similar degradation rate to the bioactive ingredients of other previous encapsulation.

Table 1:BMP-7 is along with the release-ELISA data of time

Table 2:NGFELISA data, to illustrate from PLGA microsphere weekly and cumulative release

Embodiment 3

Primary odontoblast (Od) is separated the dental pulp from birth Col1a1 (the 2.3kb)-GFP mouse incisor of latter 14 days, and is separated with non-odontoblast by GFP sorting.Primary osteoblast cells (Ob) is separated the cranium from same mouse.Overall gene expression pattern is by Alilgent-mouse-genome-oligo microarray analysis.Use >5 doubly and the target gene different between odontoblast (Od) from osteoblast (Ob) of p<0.01 (adjustment) selection, and by the confirmation of qRT-PCR, immunohistochemistry and in situ hybridization.Wnt3a and BMP7 encoded by the corresponding gene from microarray data is encapsulated in collagen gel, and is delivered to minipig lower incisor in vivo in the root pipe of pulp treatment (N=8).3 months later evaluation dental pulp regeneration situations.

Table 3: the Select gene of differential expression.Runic: the gene of expressing at odontoblast's camber.Non-runic: the gene of expressing at osteoblast camber.

gene	acquisition number	multiple	the function describing and estimate
				wnt 10a	nM_009518	126	activate classical Wnt/β-catenin signal, be positioned at DSP upstream
alx3	nM_007441	51	mesoderm is formed between the period of development
				pax9	nM_011041	40	work when there is not wisdom teeth in some crowds
dlx1	nM_010053	33	the growth of veutro forebrain.Can work in cranium face is formed and form occurs.
				bMP7	nM_007557	30	promote that bone/tooth generates
foxq1	nM_008239	28	fetal development, Cycle Regulation, tissue-specific gene is expressed
				tinagl1	nM_023476	28	relating to adrenal cortex divides band to grow
lhx8	nM_010713	25	the differentiation of some neuron and mesenchymal cell
				sox11	nM_009234	17	may be important in nervous system development
gDF10	nM_145741	200	the positive regulator of osteoblast differentiation
				wnt 16	nM_053116	97	relevant to bone mineral density
rANKL	nM_011613	25	bone remodeling
				sox 6	nM_011445	18	promote osteoblast differentiation

Result illustrates that Col1a1 (2.3kb) promoter specificity drives the expression of GFP in Od and Ob.Od accounts for about 2% of total pulp cells.Microarray analysis illustrates that the brute force of 341 genes in Od is expressed, and comprises wnt family gene, bmp7, dsp, dlx and pax9,196 gene expressions in Ob.Wnt3a illustrate be promote from alveolar bone bone marrow and pericemental mesenchymal stem/progenitor cells odontoblast differentiatio` institute must and enough.Wnt3a also illustrates promotion cell migration.Be conducted through in the body of Wnt3a and recruit host's endogenous cell, not only regenerate the pulp tissue of the root pipe medium vessels of the pulp treatment of minipig mandibular incisors, and regeneration has the new dentin being polarized to dentin cell in natural tubulose dentin surface.

Therefore confirm canonical Wnt signal pathway for CFU-GM if odontoblast's specialization of mesenchymal cell or differentiation are crucial, and come layout dental pulp or dentin regeneration as the transferable medical treatment device of the microencapsulation without the need to cell transplantation.

Embodiment 4: biomaterial dental pulp is protected

Biomaterial (such as Alginate hydrogel) is placed at removing pathological tissues comprise rotten enamel and dentin after the tooth cavity prepared of surgical operation in or insert pulp cavity (see such as Figure 13 A-D).For the biomaterial of inserting in the tooth cavity of preparation as Alginate hydrogel, absorption enters in this biomaterial by blood, tissue fluid and water.Biomedical devices can remove after 24-48 hour, restored ill tooth with rear enclosed pulp cavity.

In crosslinked period, calcium and alginate form stable compound to form network (see such as Figure 14).Alginate hydrogel can retain the water (see such as Figure 15) of 60 ~ 75 times of its original weight.

Embodiment 5: the pulp treatment in the minipig of tooth maturation

Following embodiment illustrates the pulp treatment in the minipig of tooth maturation.Illustrate especially BMP7 or Wnt3a as a supplement thing hydrogel mediation dental pulp and dentin regeneration.

Under anaesthesia, place rubber dam to isolate the incisor of minipig, this is the nursing standard (see such as Figure 16) of dental pulp disease patient.By with high-speed dental drill mechanicalness opening to touch pulp cavity.Eradicate corona and tip of a root dental pulp, use instrument process root pipe dentin subsequently, process as in dental pulp disease patient.After sterilization and the drying of paper using point, will to have or hydrogel without BMP7 (200ng/ml) or Wnt3a (10ng/ml) is injected in root pipe and pulp cavity.Cavit and composite resin is used to recover corona opening, as in dental pulp disease patient.

The result of the hydrogel after 4 weeks containing BMP7 (200ng/ml) illustrates dental pulp and dentin regeneration (see such as Figure 17).The result of the hydrogel after 4 weeks containing Wnt3a protein (10ng/ml) illustrates dental pulp and dentin regeneration (see such as Figure 18).The result containing the hydrogel of BMP7 (200ng/ml) and Wnt3a protein (10ng/ml) after 4 weeks illustrates dental pulp and dentin regeneration (see such as Figure 19).

List of references

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U.S. Patent Application Publication No. 20050079470

Sequence

SEQIDNO:1

Wnt3a polypeptide (people)

UniParcP56704

SEQIDNO:2

Wnt3a polypeptide (people)

UniParcQ3SY79

SEQIDNO:3

Wnt3a polypeptide (mice)

From the recombinant full-lenght mice Wnt3a albumen of cell culture.SwissProtID＝P27467

SLAVGPQYSSLSTQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGVKAGIQECQHQFRGRRWNCTTVSNSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGSAAICGCSSRLQGSPGEGWKWGGCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCEVKTCWWSQPDFRTIGDFLKDKYDSASEMVVEKHRESRGWVETLRPRYTYFKVPTERDLVYYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARTERRREKCHCVFHWCCYVSCQECTRVYDVHTCK

SEQIDNO:4

Wnt3a polypeptide (hydra)

At the recombinant full-lenght Wnt3a albumen (UniProtAccessionQ9GTJ9) from hydra of expression in escherichia coli

MQYKLALNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTELVPRGSQLWMALGTQTSAIESRPRSSINKNLCRALYLHHYQRTVCLNYTDLMLSVAEGIRLGIDECQVQFKHRKWNCTINEHGTSVFGPIITTASRESAFISGIISAGVAFSVTESCAEGKSVHCRCDNSVRGQTDEGWRWGGCNRPITYGIWFSQLFIDQVEKIVKKRKDPRKIMNLHNNKAGREVIKNLLQTECKCHGTSGNCNLKTCWRSQPHFSEIGKILKEKYDSAHEMEFLYKVKANGERKIKDLIPKYKEYLPPSSLDFIYYEESPNYCVKNETLGIAGTKGRSCNITSSGVDGCELMCCQRGYNVNIVQKTHSCECKFVWCCKVSCNSCIKMTPEYTCKLVPRGSLEHHHHHH

Claims

1., for regenerating a compositions for dental tissue, it comprises:

Comprise substrate or the support of hydrogel; And

The Wnt3a polypeptide for the treatment of effective dose; And

The BMP-7 (BMP-7) for the treatment of effective dose; And

Optionally, the VEGF (VEGF) of effective dose, basic fibroblast growth factor (bFGF) or nerve growth factor (NGF) is treated;

Wherein

Described compositions does not comprise living cells, and

Substrate or support comprise Wnt3a and BMP-7, and optional VEGF, bFGF or NGF existed.

2., for regenerating a method for dental tissue, comprising:

The compositions of claim 1 is provided; And

By in the natural or artificial hole of described compositions insertion mammalian tooth or chamber;

Wherein said compositions promotes the odontoblast differentiatio` of CFU-GM, promotes that CFU-GM migrates in dental tissue, promotes that angiogenic, odontogenesis, fiber generation or neurogenic are grown, to regenerate dental tissue.

3. the method for claim 2, wherein said compositions regeneration vessel pulp tissue or regenerate new dentin.

4. the method for any one of claim 2-3, wherein said support comprises further and is selected from following compound: platelet derived growth factor (PDGF), endothelial cell growth factor (ECGF) (ECGF), transforming growth factor-beta 1 (TGF-β 1), epidermal growth factor (EGF), hepatocyte growth factor (HGF), CXCL12 (SDF1), bone morphogenetic protein (BMP) except BMP-7, TGF-β, GDF (GDF), insulin-like growth factor-i (IGF1), dentin matrix protein, dentin sialoprotein, resorption lacunae, amelogenin, integrin, angiogenin, angiopoietin-1, del-1, follistatin, granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor/dispersion factor (HGF/SF), interleukin 8 (IL-8), leptin, Midkine, placental growth factor, thymidine phosphorylase/platelet-derived endothelial cell growth factor (PD-ECGF), PDGF-BB (PDGF-BB), many trophic factors (PTN), progranulin, proliferin, transforminggrowthfactor-α (TGF-α), transforming growth factor-β (TGF-β), tumor necrosis factor-alpha (TNF-α), matrix metalloproteinase (MMP), angiogenin 1 (ang1), ang2, delta-sample part 4 (DLL4), Connective Tissue Growth Factor (CTGF), brain derived neural factor (BDNF), NT-4 and NT-3.

5. the method for any one of claim 2-4, wherein said support comprises antibiotic or analgesic further.

6. the method for any one of claim 2-5, is wherein injected in Wnt3a polypeptide, BMP-7, VEGF, bFGF or NGF, is mixed in, is encapsulated in, is bound by or is adsorbed in substrate or support.

7. the method for any one of claim 2-4, wherein said support has the shape that people's incisor, people's canine tooth, people's bicuspid or people grind one's teeth in sleep, or the shape of wherein natural or artificial hole or chamber.

8. the method for any one of claim 2-7, wherein said substrate or support comprise:

Natural polymer, is selected from collagen protein, gelatin, polysaccharide, chitosan, hydroxyapatite (HA) and polyhydroxyalkanoate;

The polymer of synthesis, is selected from aliphatic polyester and the Polyethylene Glycol of poly-('alpha '-hydroxy acids);

Hydroxyapatite; Or

Alginate hydrogel.

9. the method for any one of claim 2-8, wherein said support comprises the microchannel that diameter is (i) 50-500 μm or (ii) about 200 μm.

10. the method for claim 9, wherein Wnt3a and BMP-7 and optional VEGF, bFGF or NGF existed is embedded in the gel in support microchannel.

The method of 11. claim 9, wherein Wnt3a and BMP-7 and optional VEGF, bFGF or NGF existed is encapsulated in microsphere.

The method of 12. any one of claim 2-11, comprises the model using computer-aided design (CAD) to make tooth or tooth cavity further, and synthesizes support with biological drawing apparatus.

The method of 13. any one of claim 2-12, wherein Wnt3a polypeptide comprises:

Aminoacid sequence shown in (i) SEQIDNO:1, SEQIDNO:2, SEQIDNO:3 or SEQIDNO:4; Or

(ii) there is at least about 95% sequence thereto with SEQIDNO:1, SEQIDNO:2, SEQIDNO:3 or SEQIDNO:4 and there is the aminoacid sequence of Wnt3a activity.

The method of 14. any one of claim 2-13, wherein said substrate or support are biodegradable.

The method of 15. any one of claim 2-14, wherein Wnt3a exists to the concentration of about 1,000mgWnt3a/ml solution with about 1ng, and wherein said solution is imported in substrate or support.

The method of 16. any one of claim 2-15, wherein BMP-7 exists to the concentration of about 1,000mgBMP-7/ml solution with about 1ng, and wherein said solution is imported in substrate or support.

The method of 17. any one of claim 2-16, wherein Wnt3a exists with the concentration of about 10ng/ml solution, and BMP-7 exists with the concentration of about 200ng/ml solution, and wherein said solution is imported in substrate or support.

The method of 18. any one of claim 2-17, wherein:

VEGF exists to the concentration of about 1,000mgVEGF/ml solution with about 1ng;

BFGF exists to the concentration of about 1,000mgbFGF/ml solution with about 1ng; Or

NGF exists to the concentration of about 1,000mgNGF/ml solution with about 1ng;

Wherein said solution is imported in substrate or support.

The method of 19. any one of claim 2-18, comprises further:

Wound or ill pulp tissue in Exposed Pulp chamber or root pipe; And

Cover by described compositions or filling pulp cavity at least partially or root pipe;

Wherein newborn after covering or filling vascularization dental pulp sample is organized in pulp cavity or root pipe and is formed.

The method of 20. any one of claim 2-19, comprises further:

Wound or ill pulp tissue is removed, to produce the pulp cavity or root pipe that there is no wound or illing tissue from tooth;

Substantially all pulp tissues are removed from tooth; Or

With inert material filling pulp cavity at least partially.