CN102639081B - Tooth scaffold - Google Patents

Abstract

The present invention provides a kind of acellular mammalian tooth shape support, and it comprises chemotaxis, osteogenic, one-tenth dentine, becomes enamel or become cementum compound.Also provide for a kind of method replacing tooth in the mouth of mammal, wherein absence of tooth and in mouth position at disappearance tooth there is alveolus.Described method is included in alveolus implants the Acellular matrix with the shape losing tooth.Further it is provided that a kind of method preparing tooth scaffold.Described method includes the Acellular matrix synthesizing mammalian tooth shape and adds at least one chemotaxis, osteogenic, one-tenth dentine, become enamel or become cementum compound.

Description

Tooth scaffold

Cross-Reference to Related Applications

This application claims the U.S. Provisional Application No. 61/187,875 and on June 11st, 2010 submitted on June 17th, 2009 The rights and interests of the U.S. Provisional Application No. 61/354,164 submitted to；They are incorporated herein by reference.

Background of invention

The present invention relates to tissue engineering bracket.

Biomedical engineering and regeneration of tooth.Fall tooth generally to be caused by various oral diseases and physiology cause, including dental caries, Periodontal disease, damage, heredopathia and aging (Amar 2003, Philstrom2005, Kim 2006).Fall tooth may result in health and The misery of spirit, its self-respect that can reduce individuality and quality of the life (Amar 2003, Philstrom 2005, Kim 2006).Permitted Multi-form odontopathy and some medical condition the most uncontrolled diabetes raising fall tooth risk.For the treatment of anodontia, mesh Front selection is confined to use tooth transplantation thing and/or the fixed or movable prosthese of routine.

Recently, appearance and the development of biomedical engineering instrument has caused in the new patient care scope of medical domain.Example As, preliminary people's clinical experiment has been reported in the child of osteogenesis imperfecta at total body perfusion bone marrow stroma stem cell (BMSSC) or bone After myelocyte, bone formation level improves (Horwitz 2001, Horwitz 2002).Dental tissue engineering, material science and The progress proposition that stem cell biology field is recent, regeneration of tooth will be possible (Duailibi 2006).Additionally, there are in The recent qualification of the different mescenchymal stem cell (MSC) of tooth or cranium covering weave extends and is helping dental tissue (such as Dentine, cementum and periodontal ligament (PDL)) potential clinical benefit scope (Shi 2005) in regeneration.It is present in deciduous teeth with solid Dental tissue progenitor cell in the myeloid tissue of tooth can be used for making dentine and Regeneration of Alveolar Bone (Shi 2005；Zhang 2005). Additionally, the cell separated by both rat and pig tooth bud can be used for biological engineering anatomy correction corona, but have limited Predictability (Duailibi 2004, Honda 2005, Young 2002, Young 2005).

The most individual organ of tooth/periodontal complex.Although it is believed that this organ is relatively small, but its structure and growth Complexity is well recognized as.Tooth structure is made up of the organization type (enamel, dentine and cementum and dental pulp) of three kinds of calcifications. Dentine occupies the main body of tooth, and enamel and cementum are covered each by corona and tip portion.Periodontal tissue has tooth Supporting function, and be made up of cementum, periodontal ligament, alveolar bone and gingiva.Periodontal ligament is for passing through perforating fiber (Sharpey ' s fiber) makes the connection tissue that cementum is connected with alveolar bone.Periodontal ligament energy perception and buffering chew period Mechanical force.

Although tooth structure is complicated, but the progress of biomedical engineering technology has produced the tooth of two kinds of current employings more Generation method.First method is based on organizational project, and target is to make regeneration of tooth by inoculation stem cell in support biomaterial (Young 2002, Duailibi 2004, Honda 2005).This technology has shown the result likely of paradenlal tissue regeneration (Nakahara2006).Second method attempts to replicate or imitate the growth course (Nakahara 2006) that embryo's tooth is formed. The method utilizes the embryonal tissue's (tooth epithelium and tooth mesenchyme) gathered by mouse fetal and it should be understood that adjusts in embryo The principle (Ohazama2004, Hu 2006, Nakao 2007) of joint tooth development in early days.Use these methods, in many research In, Bioengineered dental gram is transplanted in the health of animal reservoir, described animal reservoir is usually rodent, Qi Zhongcun Necessary nutrient and oxygen is provided, so that organizing the formation of optimal (Nakahara 2006) at enough blood flows.

Stem cell and the challenge run into is used in tissue regeneration.Stem cell is be present in normal structure static Cell mass, it presents asymmetric fissional different characteristic, forms the progenitor cell of two kinds of daughter cells-new/dry thin Born of the same parents and the another kind of daughter cell (Hawkins 1998, Lin 1998) that the tissue broken up can be formed.Mesenchyme remote ancestor is thin for tooth Born of the same parents are the most identified and are characterized as dental pulp (Gronthos 2000, the Mooney 1996, Shi of people's deciduous teeth and teeth consolidating 2005).As previously mentioned, these the posteriori epitheliums being present in immature tooth bud and mesenchyme tooth steam cell/remote CFU-GM is proved to produce Bioengineered and anatomical correction, but makes containing enamel, dentine, marrow and alveolar bone Corona size miniaturization (Shi 2005；Zhang 2005).

Their regeneration potential of known Periodontal Ligament Cells, to cause formation lamina propria, cementum, bone and periodontal ligament (Melcher 1985, McCulloh 1985).For the subgroup of the cell by pericemental main outer planting syntaxy, demonstrate,prove Bright periodontal ligament stem cell in vitro forms the ability (Arceo 1991, Cho 1992) of the deposit of mineralising.Believe periodontal ligament Stem cell need suitable support to come in inductor to be formed bone, dentine and cementum (Gronthos 2000, Krebsbach1998).When periodontal ligament stem cell is incorporated in hydroxyapatite/tricalcium phosphate support and at mouse back Subcutaneous area in heterotopic transplantation time, form typical cementum/periodontal ligament spline structure (Seo 2004).Additionally, be proved I-type collagen in graft-positive periodontal ligament sample tissue is connected with the cementum being newly formed, it morphologically with pass through Wear fiber similar (Seo2004).

The recent development of tooth steam cell biotechnology and cell-mediated Muridae regeneration of tooth has encouraged researcher spy Study carefully the potential (Duailibi2004, Ohazama 2004, Shi 2005) of the regeneration of tooth of the work with suitable functional character.Can Use many different stem cell to make Muridae regeneration of tooth, with cooperatively form in vivo tooth structure (Duailibi 2004, Ohazama 2004, Young 2005).Additionally, when in the mice being transplanted to immunocompromised host, pass through type I collagen respectively And periodontal ligament stem cell (PDLSC) makes dentine/myeloid tissue and cementum/periodontal complex regeneration (Gronthos (DPSC) 2000, Seo 2004).But, due to people's dental growth and the complexity of growth, with being currently available that regeneration biological technology, whole Individual tooth structure (including enamel, dentine/marrow complex and periodontal tissue) is one as the regeneration of the functional entity of people and chooses War (Sonomaya 2006).

Research previously it has been reported the challenge (Duailibi using stem cell in the regeneration of dental tissue 2004, Young 2002, Young 2005).Have been accepted as, although in Bioengineered tooth formation, form multiple Minitype tooth Hat is possible, but the regeneration of the whole tooth of actual size runs into many challenges.These challenges are attributed to answering of tooth development equally Polygamy matter (Duailibi 2006, Tummers 2003).

The concept of cell playback.It has been proposed that the purpose of the conventional method of stem cell-inoculation is to imitate cell knot in support Structure and regenerate functional organization's equivalent in vitro or in vivo.Cell-derived self terminal organ or more undifferentiated source ratio Such as bone marrow (Schantz 2007).These methods are limited by problem, such as from the donor site sickness rate of cell collection With the heterogeneous quality of the transplantation site constructed in cell-scaffold organize the formation of (Schantz 2007).Therefore, cell playbacks Concept causes more concern at present.Cell playback purpose is to induce desired cell to playback to filling at specific anatomical site The support (Schantz 2007) of full cytokine.The method attempts tissue regeneration in vivo, and without cell-inoculation.Therefore, carefully Born of the same parents' playback can be that organizational project provides the enhancing of cellular processes and provides novel minimally invasive selection for tissue regeneration (Schantz2007)。

Based on discussed above, need the further exploitation of tooth scaffold.The present invention solves this demand.

General introduction

In some embodiments, it is provided that a kind of acellular mammalian tooth shape support.Described support comprises chemotactic Property, osteogenic, one-tenth dentine, become enamel or become cementum compound.

In other embodiments, it is provided that a kind of method replacing tooth in the mouth of mammal.Implement at these In scheme, absence of tooth and in mouth disappearance tooth position there is alveolus.Described method is included in alveolus implantation tool There is the Acellular matrix of the shape losing tooth.

Further it is provided that the method for preparation tooth scaffold.Described method includes that the nothing synthesizing mammalian tooth shape is thin Born of the same parents' support also adds at least one chemotaxis, osteogenic, one-tenth dentine, becomes enamel or become cementum compound.

Other purpose and characteristic are apparent from, and part is indicated below.

Accompanying drawing is sketched

It will be appreciated by those skilled in the art that the purpose that figures described below is merely to illustrate.These accompanying drawings are not intended to appoint Where formula limits the scope of this teaching.

Fig. 1 is the flow chart of the design of the research that display describes in an embodiment.

Fig. 2 uses 3D print system (Bioplotter for display^TM) support manufacture photo.Figure A shows and is used for producing The Bioplotter of support^TM；Rat Mandibular central incisor root (left) of figure B display manufacture and people's molar shape PCL-HA support (right)；Figure C shows the ethylene oxide sterilizer for making support sterilizing；Figure D display somatomedin (SDF1 and BMP-7) processes Support；Figure E shows before transplantation for the support of the incubation of collagen cross-linking in the bracket；Figure F shows the grown factor Support with collagen gel load；Figure G shows at the support extracting groove and position, back is transplanted in rats.

Fig. 3 is the photo of people's mandibular molar shape support of the manufacture for transplanting at rat back.Hat and root are separately fabricated And merge later.Microchannel is obvious.

Fig. 4 is to extract lower jaw central incisor then to transplant the photo of root shape support in groove.The retraction of figure A display lower lip； The incision of figure B display gingival flap and reflection；The hurtless measure of figure C display lower-left jaw central incisor is extracted, the bone of the preservation of display channel Wall；Figure D shows the front tooth extracted and the comparison of root shape support；Figure E shows the support transplanted in extracting groove；Figure F display is sewed up And the main gingival flap closed.

Fig. 5 is the photo of people's mandibular molar shape support of subcutaneous transplantation in rat back position.The 2-that figure A display manufactures Cm otch；Figure B display produces subcutaneous pocket；Figure C shows in structural transplantation to bag；Figure D shows main Guan Bi.

Fig. 6 is the overall photo gathered of display lower jaw central incisor support.Figure A showed before gathering the most complete Wound healing；Figure B is displayed in proximity to the otch that support manufactures；The entirety of figure C display support (right) and adjacent front tooth (left) is adopted Collection.

Fig. 7 is the photo of the program showing the support collection for transplanting at position, back.Figure A showed before gathering complete Full wound healing；Figure B is displayed in proximity to the otch that support manufactures；The fascia encapsulating of figure C display support；What figure D display was reclaimed props up Frame.

Fig. 8 is the microphotograph that display selects the holder part of the dyeing for the region of histologic analysis.Figure A shows Three regions selected in lower jaw central incisor support；Figure B shows four regions selected in people's mandibular molar support.

Fig. 9 is the aobvious of the holder part of the dyeing of the tissue-mount interface of display door dental root shaped support in extracting groove Micro-photo (test and matched group).Figure A shows the microscope slide being described in the ingrown Feng Kesashi of Interface Bone (VK)-dyeing； Figure B display hematoxylin and the part of eosin (H&E)-dyeing, the intimate of cell wall is adapted to and integrates by its display support；Figure C shows Prove obvious angiogenesis and the higher enlarged drawing of soft tissue ingrowth between PCL-HA line.

Figure 10 is the cradle portion of the dyeing of the tissue-mount interface showing the people's mandibular molar shape support from position, back The microphotograph (test and matched group) divided.Figure A show online around and online between the angiogenesis of tissue and inwardly life Long；The higher enlarged drawing of figure B display explanation integration between support and encapsulating tissue.

Figure 11 is the microphotograph of the holder part of dyeing, and its display is from the representative diagram of the support extracting groove, and it shows Show the cell density difference of test bracket (figure A) and the crt bracket (figure B) without somatomedin.

Figure 12 is the microphotograph of the holder part of dyeing, and it shows the representative diagram of the support from position, back, its Display test bracket (figure A) and the cell density difference of the crt bracket (figure B) without somatomedin.

Figure 13 is the figure of display cell density difference between experimental group and transplantation site.GF+: test；GF-: comparison. " * ": p ＜ 0.05.

Figure 14 is the microphotograph of the holder part of dyeing, and its display is from the representative diagram of the support extracting groove, and it shows Show the vessel density difference of test bracket (figure A) and the crt bracket (figure B) without somatomedin.

Figure 15 is the microphotograph of the holder part of dyeing, and it shows the representative diagram of the support from position, back, its Display test bracket (figure A) and the cell density difference of the crt bracket (figure B) without somatomedin.

Figure 16 is the figure of display vessel density difference between experimental group and transplantation site.GF+: test；GF-: comparison. " * ": p ＜ 0.05.

Figure 17 is the microphotograph of the holder part of dyeing, and its display explanation is from extracting groove (figure A) and position, back (figure The representative diagram of the blood vessel diameter difference of support B).

Figure 18 is the figure of display blood vessel diameter difference between experimental group and transplantation site.GF+: test；GF-: comparison. " * ": p ＜ 0.05.

Figure 19 is the microphotograph of the holder part of VK-dyeing, and its display is from the representative of the test pack support extracting groove Property figure, it shows mineralising.

Figure 20 is the microphotograph of the holder part of VK-dyeing, and its display is from the generation of the test pack support at position, back Table figure, it shows mineralising.

Describe in detail

The present invention is based partially on following being found surprisingly that, when being transplanted in alveolus, attraction shifting is grown by tooth tooth shape support The cell of support is to provide the tooth lived, even if do not have to provide cell for support exogenously.

Therefore, in some embodiments, it is provided that a kind of acellular mammalian tooth shape support.Described support comprises Chemotaxis, osteogenic, one-tenth dentine, one-tenth enamel or one-tenth cementum compound.Therefore transplant these supports, and there is no external source The cell that property is used.As set up in an embodiment, by migrating into the local cell of support, the shifting of the support of transplanting is grown and is filled Divide and carry out.By the chemotaxis being incorporated in support, osteogenic, one-tenth dentine, become enamel or become cementum compound, moving Grow and encouraged further.

Described compound can have any structure, includes but not limited to that albumen, oligopeptide, little organic molecule (that is, are less than About 2000mw or about 1000mw or about 500mw), the molecule of metal ion, carbohydrate or lipid.Chemotactic used herein Property compound be attract cell compound.Osteogenic compound is the compound encouraging the synthesis of new bone.Become dentine compound For encouraging the compound of new dentine synthesis.Becoming enamel compound is the compound encouraging tooth enamel synthesis.Become cementum Property compound be encourage cementum synthesis compound.

" support " used herein is for providing the structure of substrate to the growth of cell and/or being formed of tissue.Having of support Character be porous, biocompatibility and biological degradability, can support cell growth and as controlled gene-and egg The purposes (Murphy1999) of in vain-delivery vehicle.The three-dimensional macromolecular structure that support provides guides Bioengineered tissue Net shape (Murphy 1999).

The support of these embodiments can have the shape of any mammalian tooth.In some of these embodiments In, support has people's front tooth, people's canine tooth, people's bicuspid or the shape of people's molar.

Compound in these embodiments can be chemotaxis, osteogenic, one-tenth dentine, becomes enamel or become cementum Any compound of property.Limiting examples includes the somatomedin (PDGF) of platelet-derivative, endothelial cell growth factor (ECGF) (ECGF), conversion growth factor-β 1 (TGF-β 1), epidermal growth factor (EGF), hepatocyte growth factor (HGF), substrate are thin The factor-1 (SDF1) of born of the same parents-derivative, bone morphogenetic protein (BMP), TGF-β, GDF (GDF), Insulin-Like Growth factor-1 (IGF1), VEGF (VEGF), fibroblast growth factor (FGF), dentine matrix albumen, Dentine salivary gland albumen, bone salivary gland albumen, amelogenin or integrin.

In some embodiments, described compound is the SDF1 with chemotaxis character.SDF1 is chemotactic factor, it is believed that It supplements in (recruitment) stem cell and progenitor cell for tissue repair after wound is necessary (Kollet 2003).SDF1 also can the migration (Kim 1998) in chemotaxis indoor of the induction of hematopoiesis progenitor cell.Additionally, SDF1 for It is important that marrow stromal cell migrates to bone marrow, and the dose-dependency stimulated such as mescenchymal stem cell (MSC) response SDF1 moves Move shown (Win 2004).SDF1 also has anti-apoptotic character, and directly protection hematopoietic stem cell is from other somatomedin In the presence of the apoptosis effect (Herodin 2003) of γ-radiation.Additionally, the bootable mescenchymal stem cell derived from bone marrow (MSC) (Schantz 2007) is migrated towards SDF1.

In other embodiments, described compound is BMP.BMP2,6 and 9 are clearly the most effective of MSC osteogenic differentiation Reagent, and remaining BMP in terms of promoting to pay the osteoclast precursor of (commit) and osteoblastic terminal differentiation more Effectively (Cheng 2003).

In in some aspects of these embodiments, described BMP is BMP-7.Mesenchymal cell is being converted into bone by BMP-7 With the effect playing key in cartilage.BMP-7 process be enough to induce all bases of osteoblast differentiation in various cell types Because of label (Chen 2004).Noticing, BMP-7 has obtained the approval of Food and Drug Administration (FDA) to be made for people's clinic With.

Many researchs have investigated exogenous growth factor by the work in the carrier of delivery of growth factor to transplantation site With and behavior.Although carrier may not contribute the required any other factor that organizes the formation of, but still can be growth course Important component (Wozney 1990).One of function vector is to maintain the factor of transplantation site, and therefore improves its localized rich Degree.Carrier also serves as and wherein can form tissue and therefore help to limit the environment (Whang that wherein can form neoblastic region 1998).Collagen protein or synthetic vectors have been used as delivery vehicle, and their physicochemical properties and the microcosmic of generation Environment works in the result of induction.Carrier can be solid heterogeneity (such as, hydroxyapatite) (Kuboki 1995, Murata 1998), solid alloplasm (polyethylene polymer) material (Saito 1998, Isobe 1999) or spontaneous solidifying Glue (Sweeney 1995, Schwartz 1998), heterologous (Bax 1999, Viljanen 1997) or alloplasm source (Santos 1998) and combinations of the above (Alpaslan 1996).

One of function vector is to keep the factor at transplantation site and therefore provide its local concentration.Collagen matrices is just Remain up to the BMP of 65% during phase dipping, and in two stages it discharged: in the initial stage transplanted in a few hours and Depend on the second stage (Uludag 1999) of support and geometrical property thereof.Believe that BMP is not combined (Uludag with carrier 1999), but Physical entrapment is in its structure, and this makes some design be more beneficial for self-bone grafting (Tsuruga compared with other 1997).In the case of collagen protein sponge carrier, substantial amounts of collagen cross-linking and sterilizing methods affect BMP and precipitate, and with The resistance (Friess1999) of the sponge degraded of rear collagenase.Compared with polymeric matrices, collagen carrier can also result in regeneration Bone density improves (Cochran 1997).

BMP-7 plays the effect of key being converted into by mesenchymal cell in bone and cartilage.BMP-7 process be enough to induce All gene markers (Chen 2004) of osteoblast differentiation in various cell types.It is also noteworthy that arrive, BMP-7 obtains Approval to Food and Drug Administration (FDA) is used for people's Clinical practice.

In some of these supports, there is chemotaxis somatomedin and osteogenic, one-tenth dentine, become enamel or one-tenth Cementum somatomedin.In a specific embodiment, described chemotaxis somatomedin is SDF1, and described skeletonization Property, become dentine, become enamel or become cementum somatomedin be BMP-7.

The support of these embodiments can further include other bioactive molecule any, such as antibiotic or other Chemotaxis somatomedin or another kind of osteogenic, one-tenth dentine, one-tenth enamel or one-tenth cementum somatomedin.Implement at some In scheme, by adding such as human serum albumin (HSA), hetastarch, glucosan or combinations thereof, support is added By force.Known to those skilled in the art for the suitable concentration of these compounds of the application compositions, or without mistake Degree experiment is readily determined.

In support the concentration of compound according to character, its physiological action and desired treatment or the diagnosis effect of compound and Become.Therapeutically effective amount be usually therapeutic agent display intended effect and without the enough concentration of excessive toxicity.Some embodiment party In case, support comprise the BMP-7 of about 10ng/g-1000 μ g/g support in the bracket with about 10ng/g-1000 μ g/g support SDF1 is in the bracket.In a more particular embodiment, with about 100 μ g/g supports in the bracket, and SDF1 is with 100 μ for BMP-7 G/g support is in the bracket.

By any of method, compound can be mixed in support.In some embodiments, compound is embedded Gel (such as, the collagen gel being incorporated in the hole of support), as described embodiments.

Or, chemical modification method can be used for covalently bound for compound on the surface of support.Use known in this field Coupling agent (such as aldehyde compound, carbodiimide etc.), can be by the reactive functional groups of the surface functional group of support Yu compound Coupling, to form covalent bond.Additionally, spacer molecule can be used for the reactive base of displaced surface reactive group and biomolecule Group so that these molecules are the most more flexible.Other class being interiorly or exteriorly connected by biomolecule with substrate It is known to a person skilled in the art like method.

Or by system based on carrier (such as encapsulating vehicle), compound can be incorporated in Medium Culture or substrate. These vehicles can be used as slow releasing composition.Such as, can be by micro-for somatomedin encapsulating, to provide the stability strengthened and/or to prolong Long delivery.Encapsulating vehicle includes but not limited to microgranule, liposome, microsphere etc., or any of above combination, to provide not Desired release profile in proportion.Other method of the controlled-release delivery of reagent is that professional and technical personnel is known.Additionally, Can be by these and other system in combination and/or amendment, so that integration/release that reagent is in the substrate is optimal.

Polymeric microspheres can use the polymer production naturally occurring or synthesizing, and is that size is at 0.1-500 μ m Interior particle system.Polymeric micelle and polymer waterfloocling (polymeromes) are passed with being polymerized of microsphere similar characteristics for having Send vehicle, and also can promote that the encapsulating of compound described herein and substrate are integrated.Microsphere for various payload The manufacture of body, encapsulate and stablize within the skill of the art (for example, see, Varde&Pack (2004) ExpertOpin.Bio1.4(1)35-51).By the type of polymer, polymer molecular weight, copolymer form, join micro- The excipient of spheroid preparation and sized micro-spheres, can adjust the rate of release of microsphere.Can be used for being formed the polymer of microsphere Material includes PLA, PLGA, scribbles the PLGA of DPPC, DPPC, DSPC, EVAc, gelatin, albumin, chitosan, glucosan, DL- PLG, SDLM, PEG (such as, ProMaxx), hyaluronate sodium, Diketopiperazine derivative (such as, Technosphere), phosphoric acid Calcium-PEG granule and/or oligosaccharide derivatization DPPG (such as, Solidose).Can such as use the single emulsion process of water/oil, water-oil- The double emulsion process of water or lyophilizing realize encapsulating.Available several industry wrapper technology is (such as,Alkerme)。

Liposome can be additionally used in be integrated compound with support.The reagent carrying capacity of liposome and rate of release can depend on In lipid composition, size, electric charge, drug/lipid ratios and delivering method.Conventional liposome is by neutral or anion lipid (naturally occurring or synthetic) forms.Conventional lipid is lecithin, such as phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, sphingomyelins, phosphatidyl Serine, phosphatidyl glycerol and phosphatidylinositols.Liposomal encapsulated method is (Galovic et al. commonly known in the art (2002) Eur.J.Pharm.Sci.15,441-448；Wagner et al. (2002) J.Liposome Res.12,259-270). The liposome of targeting and reactive liposome also can use with reagent and substrate combination.The liposome of targeting has targeting ligand, The monoclonal antibody being such as connected with their surface or lectin so that with specific receptor and/or cell type phase Interaction.Reactive or polymorphic liposome includes the liposome of wide scope, and their common property is when specific phase interaction Change their phase and structure (such as, pH-sensitive liposome) with tending to afterwards.For example, see Lasic (1997) Liposomes in Gene Delivery (liposome in gene delivery), CRC Press, FL).

Any material that the support of these embodiments can use professional and technical personnel there have been manufactures.Suitably base Material is such as in following discussion: Ma and Elisseeff edits (2005) Scaffolding in Tissue Engineering (at organizational project medium-height trestle), CRC, ISBN 1574445219；Saltzman (2004) Tissue Engineering: EngineeringPrinciples for the Design of Replacement Organs and Tissues (tissue work Journey: replace the engineering philosophy of the design of organ and tissue), Oxford ISBN 019514130X.For all or part of support The limiting examples of the material come in handy include Polyethylene Glycol, polylactide, polyglycolic acid, lactide coglycolide Copolymer, poly-(caprolactone), condensing model, polyglactin, Merlon, polyamide, condensing model, polyamino acid, poly-ortho acid Ester, poly-acetal, polybutylcyanoacrylate, polyphosphazene, degradable polyurethane, polyacrylate, ethane-acetic acid ethyenyl ester are poly- Compound and other acyl group substituted cellulose acetate salt and their derivant, polyurethane, polystyrene, polrvinyl chloride, poly-fluorine Ethylene, polyvinyl pyrrolidone, poly-(vinyl imidazole), chlorosulfonated polyolefin, poly(ethylene oxide), polyvinyl alcohol, TeflonNylon, agarose, alginate (such as, calcium alginate gel), fibrin, Fibrinogen, fibronectin, glue Former albumen (such as, collagen gel), gelatin, hyaluronic acid, chitin and other suitable polymer and biopolymer, Or above analog, mixture, combination and derivant.

In some embodiments, support is by the compositions manufacture comprising bone conduction (osteoconductive) material.Bone The limiting examples of conduction material is hydroxyapatite (HA).The biocompatibility excellent due to HA and high bioactivity, it Have been used as bone substitute many years (Liao2006, Nebahat 2006, Lijun 2006, Wei 2003).

Although HA has good biological activity and bone conductibility, but it is highly brittle and intrinsic tensile property is poor.Cause This, in some embodiments, combine HA Yu ε-polycaprolactone (PCL).Owing to PCL needs several years just degradation in vivo also And for biocompatibility, relatively inexpensive and can obtain in a large number, it is good bone holder material (Rich 2002, Kim 2004). The combination (PCL-HA) of PCL and HA provides the desired combination (Patcharaporn of biological activity, biological degradability and intensity 2005, Rezwan 2006, Landis 1995, Ziv 1994).Think the material of complex PCL-HA have biocompatibility, Cell-bond, the optimal support character (Zhao 2008) bred and break up.In some embodiments, support comprises about 80% Weight polycaprolactone and the mixture of about 20% weight hydroxyapatite.In other embodiments, support comprises about 60% weight Amount polycaprolactone and about 40% weight hydroxyapatite to about 95% weight polycaprolactone and about 5% weight hydroxyapatite times What quantity.Such as, support can include about 70% weight polycaprolactone and about 30% weight hydroxyapatite.Real as another Example, support can include about 90% weight polycaprolactone and about 10% weight hydroxyapatite.

In some embodiments, support has high porosity.This loose structure be new osseous tissue cell migration, Bonding and growth provide space (Gazdag 1995, Rezwan2006, Mano 2004, Shin 2003, Kim 2001, Leong 2003)。

The hole of support and passage can be engineered, to have various diameter.Such as, the hole of support can have a few micrometers to number The diameter range of millimeter.In some embodiments, the hole of host material includes microchannel.The usual average diameter in microchannel is about 0.1 μm-about 1,000 μm, such as, about 50 μm-about 500 μm (e.g., from about 100 μm, 150 μm, about 200 μm, about 250 μm, about 300 μ M, about 350 μm, about 400 μm, about 450 μm, about 500 μm or about 550 μm).It will be appreciated by those skilled in the art that microchannel diameter Distribution can have any distribution, including normal distribution or nonnormal distribution.In some embodiments, microchannel is host material Naturally occurring feature.In other embodiments, by microchannel through engineering approaches, to be present in host material.

In some embodiments, described compound is embedded in the gel in microchannel.Any gel can be used for this mesh 's.In some embodiments, described gel is collagen gel.

In various embodiments, support comprises atresia corona (cap) further.This corona provides further for support Intensity and prevent infect.Described atresia corona may simply be the material identical with the remainder of support, except not having hole. Or, described atresia corona can be different material, such as, typical tooth corona material, the most ceramic or golden.

The method that in the mouth of mammal replace tooth is also provided herein.In these embodiments, absence of tooth And there is alveolus in the position at disappearance tooth in mouth.Described method is included in alveolus to implant has the shape losing tooth Acellular matrix.

Use multiple method to manufacture porous support, including granule leaching, gas foaming, electricity spin, lyophilization, by starching Material makes ceramic foam and forms polymeric sponges (Mikos 1994, Mooney 1996, Qing 2002, Sylvain 2006).So And, by the support using these methods to prepare, there are in terms of the structure and interconnectivity of control hole some shortcomings, this can limit They application (Yeong 2004, Tan 2003) in terms of Premeabilisation of cells in organizational project.

In some embodiments, described method farther includes by computer-aided design (CAD) preparation disappearance The model of tooth, and use bioplotter to synthesize described support.This method can provide has high porosity with the most mutual The support of connection property.As described by an embodiment, can prepare and there is controlled and reproducible porosity and good limit Three-dimensional (3D) support of 3D microstructure.Have pointed out rapid prototype manufacturing (RP) method, such as fused deposition modelling, selectivity Laser sintered, 3D prints, multistage injection solidifies and 3D draws (Hutmacher 2001, Moroni 2006).

The key feature of rapid prototype manufacturing is that solid free form manufactures (SFF) method: cut by 3D computer model The sequence of stratification, it constructs complicated object for successively.Induced by the solidification of melt, layer photopolymerization or use laser beam Sintering one of (selective laser sintering) or particular adhesive make granule bonding payzone in next life (Landers 2002).Recently, Introduce the RPM Systems (Bioplotter of specialization^TM, EnvisionTec, Germany) so that can design and manufacture tool There is the support with anatomic shape of different internal structure, it is thus possible to accurately control hole size, porosity, permeability and rigidity (Landers 2002；Landers 2005).Use Bioplotter^TMFor manufacturing the former of tissue-specificity PCL-HA support Type manufacture method needs the 3D shape information of destination organization or tissue defects, and these can pass through computerized tomography roentgenography Or nuclear magnetic resonance (MRI) obtains (CT).When lacking tooth and having homologue at the opposite side of mouth, this homologue can be used as mould Type carrys out the tooth design support for losing.

Information derived above is used subsequently to by CAD design functional support, and transfers to Bioplotter^TMSystem. Within the system, Bioplotter^TMMachine-melt timbering material, and on collecting board, successively distribute timbering material (such as, PCL- HA).As a part for design, hole, such as microchannel can be produced.The 3D support manufactured by RP system causes significant cell Infiltration, and therefore there is the character (Heo 2007) of ideal stent.By for patient provide tissue-specific anatomical shape with And for nutrient transport and angiopoietic optimized internal microstructure, what these 3D supports can have a clinical practice can Energy.Other details of these methods provides in PCT Publication WO2009006558, and this announcement is incorporated by reference.

Further it is provided that a kind of method preparing tooth scaffold.Described method includes synthesizing mammalian tooth shape Acellular matrix also adds at least one chemotaxis, osteogenic, one-tenth dentine, becomes enamel or become cementum compound.? In some embodiments of these methods, the tooth that the shape picture of described tooth lacks in mammal, and described method Farther include the model by computer-aided design (CAD) preparation disappearance tooth and with described in bioplotter synthesis Support.When the tooth of disappearance has homologue (such as, molar) in mouth, described method farther includes the mortar carrying out being similar to The CT scan of tooth, such as, at the opposite side of mouth, wherein CAD utilizes the CT scan data of second molar to design described support.

In some embodiments of these methods, described compound is the somatomedin (PDGF) of platelet-derivative, interior Skin cell growth factor (ECGF), conversion growth factor-β l (TGF-β 1), epidermal growth factor (EGF), hepatocyte growth factor (HGF), the factor-1 (SDF1) of stromal cell-derivative, bone morphogenetic protein (BMP), TGF-β, GDF (GDF), insulin-like growth factor-i (IGF1), VEGF (VEGF), fibroblast growth factor (FGF), dentine matrix albumen, dentine salivary gland albumen, bone salivary gland albumen, amelogenin or integrin.

In other embodiments, described support is by the compositions manufacture comprising bone conduction material.As discussed above, The example of available bone conduction material is hydroxyapatite.Other example is ε-polycaprolactone as discussed above and hydroxyl phosphorus The mixture of lime stone.In the various embodiments of these methods, support comprises diameter microchannel between 50-500 μm.? In other embodiments, support comprises atresia corona further.

In some embodiments, for the expression composition of certain embodiments of the present invention is described and claimed as Amount, the numeral of character such as molecular weight, reaction condition etc. are interpreted as being modified by term " about " in some cases.Real at some Executing in scheme, described term " about " is for illustrating that this value includes the standard deviation of the meansigma methods for the device or method measuring this value Difference.In some embodiments, the numerical parameter described in printed instructions and claims is approximation, and it can root Become according to seeking the desired character that obtained by specific embodiments.In some embodiments, be considered as being reported has Imitate the numeral of number and apply the common technology that rounds up to explain described numerical parameter.Although describing some enforcements of the present invention The numerical range of the wide scope of scheme and parameter are approximation, but the numerical value described in a particular embodiment essence as far as possible cuts ground Report.The numerical value presented in some embodiments of the present invention can comprise by the mark found during test is measured accordingly at them Some error that quasi-deviation necessarily causes.Quoting of numerical range is provided merely as individually mentioning and falls into the every of this scope herein The method for simplifying of individual independent value.Unless otherwise specified herein, otherwise each single value is incorporated in description, just looks like herein In be individually recited as.

In some embodiments, unless otherwise indicated, otherwise describe specific embodiments context in (particularly In the context of some claim) term " " that uses may be interpreted as covering odd number and plural number with " being somebody's turn to do " and similar quoting Both.In some embodiments, the term "or" (including claim) herein and using is for referring to "and/or", unless clearly Explanation is only to refer to that substitute or substitute are mutually exclusive.

Term " comprises ", " having " and " including " is open connection verb.Any shape of these verbs one or more Formula or tense, such as " comprise ", " comprising ", " having ", " having ", " including " and " including " are also open.Such as, " bag Contain ", any method of " having " or " including " one or more steps be not limited to that only there are those one or more steps, and And also can cover other step do not enumerated.Similarly, " comprise ", any group of " having " or " including " one or more feature Compound or device are not limited to only have those one or more features, and can cover other feature do not enumerated.

All of method described herein can use any suitable order to carry out, unless herein additionally explanation or on Negate the most clearly.Any and all embodiment of the pass offer of some embodiment in this article or exemplary language are (such as " such as ") use is only intended to preferably illustrate the present invention, and the scope of the present invention requiring in addition that protection is not produced limit System.Language is not had to should be interpreted that the key element implementing necessary any failed call protection of the explanation present invention in the description.

The confession of present invention disclosed herein selects the packet of key element or embodiment to should not be construed as restriction.Each group membership can Relate separately to and claim or carry out any combination with other member of this group or other key element of finding herein.For side Just or the reason of patentability, the one or more members in group may be included in a group or delete from a group.When going out Incumbent what is the most this when including or delete, and description can be considered the group comprising amendment in this article, thus realize will in appended right Seek the written description that all marlcush group of middle use are closed.

All of publication, patent, patent application and other list of references quoted in this application are for all purposes It is incorporated herein by reference, and as each single publication, patent, patent application or other list of references tool Body with illustrate individually identical by quoting in full the degree of combination for all purposes.Quoting of reference herein should not It is construed as an admission that the prior art that this list of references is the present invention.

Describe in detail the present invention, it is obvious that without departing from the present invention defined in the appended claims In the case of scope, revise, change and equivalent embodiments is possible.Furthermore, it is to be understood that all embodiments in the disclosure There is provided as non-limiting example.

Embodiment

There is provided following non-limiting example to further illustrate the present invention.It will be understood by those skilled in the art that following Technology disclosed in embodiment represents the method that inventor finds good running in an embodiment of the present invention, therefore can recognize For constituting the embodiment of its embodiment.But, in view of the disclosure, it will be understood by those skilled in the art that without departing from the present invention In the case of spirit and scope, disclosed specific embodiments can carry out many changes and still obtain similar or similar Result.

Embodiment 1: by the regeneration of the anatomy correction tooth that cell playbacks

One of odontogical basic mission is for recovering tooth structure that is ill, loss and that lose.At present, to falling tooth Conventional treatment includes that the prosthodontia using or not making the tooth transplantation thing surgically placed is treated.Although having reported tooth The high success rate of tooth graft, but it is not without complication, such as loose, infection and bone loss.Recently, in doctor and science Exist in Jia and biomedical engineering prompting (cue) can be used to make single tooth structure or the common aspiration of whole regeneration of tooth. But, this emerging field encounters several obstacle, and maximum of which is the complexity of regeneration and tooth structure form.This research carries Go out to use Bioengineered tooth scaffold and by delivery is known, the somatomedin that tooth development is important set up whole tooth The regeneration of tooth organ.Use Bioplotter^TMMachine, by the coalition of ε-polycaprolactone and hydroxyapatite (PCL-HA) The rapid prototype manufacturing of layer deposition, first manufactures full-scale people's tooth scaffold.Concurrently, also Sprague Dawley is built with 3D The support of the lower jaw central incisor root shape of rat.This support is subsequently with the factor-1 (SDF1) and the Bones morphology of stroma cell derivative There is albumen 7 (BMP-7) perfusion.There are 22 Spraque Dawley rats, in test group and matched group each 11.Big at every In Mus, at position, back subcutaneous transplantation people's mandibular molar shape support, and surgical operation extract two lower jaw central incisor it After one in extracting groove transplanting rat lower jaw door dental root shaped support.Test pack support SDF1 and BMP-7 dipping, and matched group props up Frame does not contains somatomedin.After the transfer 9 weeks time gather the support of all of transplanting, and Histological evaluation's tissue ingrowth, thin Born of the same parents' infiltration, angiogenesis and mineralising.

Material and method.Before starting this research, obtain Columbia University's association's animal care and use committee (IACUC) approval.This research uses the lower jaw central incisor of 12 week old male Sprague Dawley Rats to extract groove and subcutaneous Position, back (Charles River, NY).All of position accepts PCL-HA support.Have 22 SD rats, they are random It is divided into two groups: test group and matched group, each group in two groups has 11 rats.As it is shown in figure 1, respectively every rat is given Give identifier-#1 to #11 and #12 to #22-test group and matched group.After point of purchase, during one week laundering period, rat is become To being maintained in 12 hours light/dark cycles, and before the surgical procedure of structural transplantation, random feeding rat chow (Rodent Laboratory Chow 5001, Ormond Veterinary Supply, Ontario, Canada) and water.Table 1 Summarise the number of holders of seminar and transplanting.

Table 1. seminar and the number of holders of transplanting

E=extracts groove location and transplants；The subcutaneous back of D=is transplanted.

Each Test sites (lower jaw central incisor extracts groove and subcutaneous back) of every rat is accepted one by surgical operation Individual PCL-HA support.Extract groove location and accept the support of the root as lower jaw central incisor.Position, back acceptor's mandibular molar shape The support of shape.Support SDF1 and the BMP-7 dipping transplanted in test group rat, and matched group accepts only have collagen protein The support of gel.All rats keep 9 weeks at surgical site infections, gather support subsequently for lab analysis with quantitative.

Prepared by support.Use 3D print system (Bioplotter^TM, Envision TEC, Gladbeck, Germany), pass through Layer by layer deposition manufactures the PCL-HA support (Fig. 2 A, B) of Rat Mandibular central incisor root shape and people's mandibular molar shape.This is multiple Compound is by 80% weight polycaprolactone (PCL) (Mw～65,000, Sigma, St.Louis, MO) and the hydroxy-apatite of 20% weight Stone (HA) (Sigma, St.Louis, MO) forms.PCL-HA is melted in room at 120 DEG C, and by 27-gauge metal pin (DL technology, Haverhill, MA) distributes, to produce the line and the microchannel (Fig. 3) of interconnection being clipped in the middle.Owing to making It is one piece of difficulty run in it manufactures continuously, individually produces hat and the root architecture of people's mandibular molar shape support, and melt subsequently Close to form whole tooth (Fig. 3).All of support includes what the interlock of the PCL-HA line by a diameter of 200 μm produced The microchannel (Fig. 3) of a diameter of 200 μm.The macromole 3D structure of support is intended to the formalness promoting to form final result, and The target of the internal structure with microchannel is to provide and occupies the space with tissue ingrowth for cell.Propping up of all manufactures Frame is sterilizing 24 hours in ethylene oxide sterilizer, subsequently with the collagen gel (test group) containing SDF1 and BMP-7 and Collagen gel (matched group) without somatomedin processes (Fig. 2 C).Use aseptic experiment room technology, at laminar flow hood In carry out the process of support.

For test group, by 100ng/ml stromal cell-derivative factor-1 (SDF1) (R&Dsystems, MN) and 100ng/ml bone morphogenetic protein-7 (BMP-7) (R&D systems, MN) 2mg/ml neutrality cattle I-type collagen (R&D Systems, Minneapolis, MN) middle combination.Subsequently somatomedin-collagen solution is filled into In the microchannel of PCL-HA support, and cross-link 1 hour in moist cultivating container at 37 DEG C.At PBS, 10X PBS, NaOH In mixture, the collagen gel of preparation load SDF1 and BMP-7, is summarized in table 2.SDF1 is selected based on work above Dosage with BMP-7.Chemotaxis measures and shows, mescenchymal stem cell grows towards SDF1 stimulus object, at 100ng/ml There is under concentration maximum chemical chemotaxis (Schantz 2007).The BMP-7 concentration of 100ng/ml has shown and has effectively facilitated skeletonization Cell grows (Laflamme 2008) in collagen carrier.The most unsupported any somatomedin of matched group support. But, use identical mode described with test group, at surgery operation consent, empty collagen gel is filled in microchannel.

Table 2. is delivered to test the details of the growth factor solution in pack support.

Inclusions

Amount (ml)

10X PBS	1
		1N NaOH	0.14
PBS	4.86
		Collagen protein I	2(5mg/ml)
BMP7	1(100ng/ml)
		SDF1	1(100ng/ml)
Cumulative volume	10

Surgical procedure.Buy 12 week old Sprague Dawley rat (CharlesRiver, NY), and allow them fit Should about 1 week.The all of rat mixture of KET (80mg/kg, IP) and xylazine (5mg/kg, IP) is anaesthetized.In journey Carry out the degree of depth of Monitored anesthesia by pinching toe (toe-pinch) during sequence；And when needed, individually give KET (all The 1/3:25mg/kg of dosage, IP), with on-demand holding depth of anesthesia.Pulse-oximeter device is used at surgery intra-operative Monitoring pulse rate and oxygen saturation level.

Surgery operating technology between the two groups is identical (Fig. 4,5).Carry out careful hurtless measure surgical operation and extract lower jaw Central incisor, immediately followed by transplanting root shape support (Fig. 4 C, D, E) in extracting groove.Use periosteotome (Periotome) as far as possible Carry out extracting program, the bone wall (Fig. 4 C) of careful retention groove hurtless measure.During transplanting structure, the careful quilt by this structure Move and be suitable for keeping lip wall (Fig. 4 E).After the transfer, propose (advance) limb (flap) and close for main, and each groove (polyglylactin suture material (Vicryl is used by one or two single (single-interrupt) sewn closed of interrupting 5-0, Johnson and Johnson, NJ)) (Fig. 4 F).

At the position, back of identical rat, the subcutaneous transplantation (Fig. 5) of the people's mandibular molar shape support being prepared.Place The long horizontal cut of 2-cm, and use sharp scissors for surgery make subcutaneous area relax and become bag (Fig. 5 B).In subcutaneous generation Transplanted Human mandibular molar shape support (Fig. 5 C) in Dai.This position with polyglylactin suture material (Vicryl 5-0, Johnson and Johnson, NJ) close, it is ensured that the bubble (Fig. 5 D) not retained before Guan Bi.Place breaking joint in multiple list Close, for main Guan Bi.

After completing transplanting program, give fourth the third promise coffee (0.05mg/kg, IP) and be used for pain relieving, relocate to subsequently move Thing intensive care unit.During the convalescent period of 3-5 days, veterinary technician closely monitor rat, and light/dark at 12 hours Cycle is maintained in the single cage taken, and random feeding rat chow (Rodent Laboratory Chow 5001, OrmondVeterinary Supply, Ontario, Canada) and water 9 weeks, euthanasia subsequently.During these 9 weeks, in routine On the basis of closely any infection of monitoring rat or disease indication.When observing this sign, carry out suitable treatment.Monitoring The continued growth of residue front tooth, to avoid malocclusion and the malnutrition caused.When producing instruction, clamp tooth, to hold Easily chew.When surgical site infections 9 weeks, by every rat humanity euthanasia, the pentobarbital of overdose is immediately used to note Penetrate IP to be acquired.

Gather and laboratory procedure.Before gathering, it should be apparent that keep lower jaw central incisor to extract the gingiva group on groove Knit, and do not expose support (Fig. 6 A).Position, back displays that its optimal wound healing (Fig. 7 A).

Monoblock (en bloc) gathers the support in lower jaw, including remaining adjacent center front tooth and alveolar bone (Fig. 6).Return Receive back bracket, around fascia encapsulating support (Fig. 7).The structure of all collections is stored in 10% formalin, transports subsequently Transport to histology experiment room, prepare the hematoxylin with each sample and eosin (H&E) and Feng Kesashi for 5 μm-thickness microscope slide (VK) dyeing.

Cell playback, vascularization and mineralising quantitative.Cell playback, vascularization and mineralising quantitative based on seminar And cell density between transplantation site, angiogenesis (blood vessel number and diameter) and existing mineralising is observed any Difference.

By do not know which rat belong to which group blind trier carry out quant program.Checking each support Microscope slide before, determine which region contributes to histological data analysis.Agree to check the support of preparation on microscope slide Corona 1/3rd, middle 1/3rd and the zone line of most advanced and sophisticated 1/3rd, as shown in Figure 8.Therefore, by extracting groove collection Support have three evaluate regions (Fig. 8 A), and the support gathered by position, back due to exist two roots and there are four The region (Fig. 8 B) evaluated.Numeral research microscope (LeicaDM6000, Leica, Switzerland) is used fully to examine under 100X amplifies Test each microscope slide.Microscope slide is carried out Digital photographic.Use the software program Leica that microscope provides ApplicationSuite (LAS) carries out the quantitative of cell and blood vessel in the region agreed to.Later counting was converted into quantity/ mm².Computer software programs (Photoshop CS) are used to measure blood vessel diameter and be converted into millimeter (mm).Also evaluate whether to deposit In mineralising.

Statistical analysis.Computer program (Microsoft Office Excel 2007) is used to carry out all of statistical Analysis.For foregoing each variable, calculate average and standard deviation value.Carry out t-inspection, to measure at two experimental grouies Between and significance level between transplantation site.P-value ＜ 0.05 is considered significant.

Result

Organizational integration with PCL-HA support.As in Fig. 9 and Figure 10 it will be apparent that, from two transplantation sites of two groups All of support shows, tissue-mount interface region shows the tissue of relative close and adapts to support.Do not appear in two groups it Between there is any significant difference, this is unrelated with transplantation site.But, the micro-characteristic of integration seems at two transplantation sites Between different.For the support in extracting groove, interface is characterised by that significant bone adapts to support, may serve as a contrast containing fiber In.The bone growth (Fig. 9 A, B) that some interfaces are presented between the line of support.It addition, deposit in the interface of support and cell wall Positive evidence (Fig. 9 C) at angiogenesis and soft tissue ingrowth.Seem to be organized in around PCL-HA line and at PCL-HA (Fig. 9 C) is grown between line.Tissue is also along bone cell wall (Fig. 9 C).Position, back display interface has well in support Soft tissue ingrowth (Figure 10 A, B) in region, portion.

Premeabilisation of cells and density.The previous research utilizing PCL-HA support is proved to comprise around the line of PCL-HA support Premeabilisation of cells and propagation (Heo 2007).As shown in FIG. 11 and 12, between test group and the matched group of each transplantation site There is significant cell density (cell/mm²) difference of level.Figure 11 describes by the representative district extracting the support that groove reclaims Territory, and Figure 12 represents the region from position, back support, test group respectively and matched group.By test pack support observe thin Born of the same parents' density, much larger than the cell density-p=0.049 observed by matched group and p=0.001, extracts groove location and back respectively Position.By the support extracting groove recovery, there is the cell mass-p=0.016 more more dense than the support reclaimed by position, back and p= 0.002, test group respectively and matched group (Figure 13).

Angiogenesis.In the support from all collections of two experimental grouies, angiogenesis is substantially (Figure 14,15).Logical Often, compared with matched group, the degree (blood vessel/mm of the angiogenesis observed in test pack support²) bigger, this and transplanting portion Position unrelated-p=0.011 and p=0.002, extracts groove and position, back (Figure 16) respectively.But, from the support extracting groove The density observed seems overall bigger, regardless of its statistics non-limiting-p=0.222 between group and p=0.095, surveys respectively Examination group and matched group (Figure 16).

Blood vessel diameter (μm) in the transplantation site of back is more than-p=0.028 and p=0.022 in extracting groove, respectively Test group and matched group (Figure 18).But, between experimental group, do not add up difference-p=0.426 and p=0.732, pull out respectively Go out groove and position, back (Figure 18).The representative photo of microscope slide shows, and by compared with extracting the support of groove collection, it appears that by The blood vessel diameter much bigger (Figure 17 A, B) of the support that position, back gathers.As shown in figure 18, in back bracket, at matched group Inside there is significantly larger blood vessel diameter meansigma methods, but this difference is not up to statistical significance (p=0.732).

Mineralising.Only in test pack support, observe mineralised zones (Figure 19, Figure 20).Carry out self-test group and extract groove and test The support of group back transplantation site shows mineralised zones in Feng Kesashi (VK) microscope slide.At two kinds of transplantation sites, well See that mineralising enters support, and at tissue with mount interface region without mineralising.

Discuss

Cell transplantation is acquiescence (default) strategy for the treatment of based on cell, and it includes transplanting or injection culture-swollen Swollen or modified tissue remote ancestor or the tissue (Mooney 1996) formed completely.But, the adult cell of culture-expansion Therapeutic engraftment has several critical obstacle, life-span limited during being included in the culture period of prolongation, slowly propagation and Lost cell phenotype (Muschler et al. 2004).Therefore, especially for substantive in vitro (ex vivo) cell manipulation of needs Those, suspected technology and the economic feasibility (Muschler et al. 2004) of cell delivery delivery method.Recently, more and more Pay close attention to and playbacked by cell, then make tissue regeneration (Stosich etc. frequently by the celliferous form in elaborately planned sky occurs People 2007).Cell playback is defined as actively being added to by endogenous cell in predetermined anatomy compartment, and represents at tissue again Also in the method (Quesenberry 1998) of research in life.Similarly, this strategy of Schantz is called " cell playback ", and will It is defined as " playback of position-guiding that induced tissue is formed in the support of cytokine-load of local stem cell " (Schantz 2007).In many adult tissues, stem cell is playbacked and migrates the replacement for ongoing mature cell Regeneration with damaged cell is critical (Laird et al. 2008).

This result shows, the interconnecting microchannel (diameter 200 μm) produced by layer by layer deposition PCL-HA (diameter 200 μm) The tissue that internal structure contributes to interface zone is intimate adapts to support, is followed by host cell and successfully playbacks inside with blood vessel Grow into extensive (～17mm) support.Support containing SDF1 and BMP-7 promotes host cell infiltration.

The factor-1 (SDF-1) of the stromal cell secreted in bone marrow microenvironment by stromal cell-derivative is by expressing it The progenitor cell of homoreceptor Gro-beta-T receptor 4 (CXCR4) supplement and promote cell playback in play necessity work With (Vandervelde 2005).CXCR4+ positive cell includes CD34+ hematopoietic stem cell (HSC) and mescenchymal stem cell (MSC) (Brenner et al. 2004 in bone marrow；Wynn et al. 2004；Honczarenko et al. 2006；Cheng et al. 2008).Owing to these cells are necessary for vascularization and osteanagenesis, it is presumed that the SDF-1 being incorporated in 3D support is not Only supplement local cell but also supplement hematopoietic stem cell and MSC.

In addition to SDF1, support also delivers BMP-7.Owing to mesenchymal cell is being converted in osteoblast by BMP-7 Play central role, thus it is speculated that the dystopy observed in our the most celliferous support or original position mineralising are by being supplemented by SDF1 Stem cell/progenitor cell BMP-7-mediation osteogenic differentiation and realize.But, result shows, containing SDF1 and In the support of BMP-7, at two transplantation sites, there is little and inconsistent mineralising evidence.Suboptimum osteogenesis be probably by In the quick release of BMP-7, because collagen protein fast degradation in vivo.

It is interesting that compared with back, observe less mineralised zones in extracting groove transplantation site, although blood and Medullary cell enriches.This is likely due to removing the healing extracting groove after lower jaw central incisor and be likely to be due to the transplanting of support And postpone.The lip wall that the surgical operation persistent period extended can also result in sensitive paper the thinnest is easier to postoperative re-absorption.Due to tooth Tooth and surrounding tissue are the most extremely brittle weak, it is known that the hurtless measure of Rat Mandibular front tooth extracts the great technical difficulty of program.Under little rat The process of jaw also makes this program the most complicated.

In this study, Histological evaluation confirms that many grooves have lost their lipstick plate.This is likely due to remaining Lip wall is very thin.During lip re-absorption and this process of reinventing with time slot, we improve the possibility that may increase broken bone active Property.The agglutination that previous built vertical rat molar extracts groove is divided into three phases: commitment (1-5 days), period clot Organize and groove has been covered by epithelium part；The bone formation stage (5-20 days)；With bone remodeling stage (20-60 days), now children Osseous maturation and alveolar ridge reinvent (Pietrokovski 1967).Histomorphometric shows, due to after extracting at most 112 days, height and width all reduced, and anodontia lower jaw experiences significant size and reduces (Elsubeihi 2004).Consider to gather journey The fact that sequence is carried out after the transfer on the 9th week, may reinvent and shrink the actively generation when gathering.

Generally speaking, this discovery proves in live body rat model, born cell migration can be induced to enter and have blood simultaneously Pipe generates and in angiopoietic PCL-HA support.This research is emphasized, compared with in the support of matched group, with SDF1 and In the PCL-HA support of BMP-7 dipping, Premeabilisation of cells and angiogenesis increase.Verified, compared with position, subcutaneous back, pulling out Go out the degree of cell ingrowth and angiogenesis in groove location bigger.These find to confirm in extracting groove location with SDF1 and The support of BMP-7 dipping will present the hypothesis of maximum proliferation potential.Therefore, pulled out in the presence of SDF1 and BMP-7 by proof Go out cell and the vessel density of the enrichment observed in groove, it is shown that diving of the tooth in-situ regeneration of use cell playback technology Energy.

Embodiment 2: by the regeneration of chemotaxis；Supplementing of the PDGF induction of alveolus stem cell/progenitor cell

Stem cell/progenitor cell separates from numerous tissues.Known bone marrow is the rich sources of stem cell/progenitor cell One of, including both hematopoietic stem cell (HSC) and mescenchymal stem cell/stromal cell (MSC).Although fibroblast sample MSC First find in the bone marrow of long bone in nineteen seventies, but found that the bone marrow of face alveolar bone contained with long later The cell that bone MSC is similar, but it is likely to be of the bigger effect towards the differentiation of at least osteogenic.Due to alveolus MSC derived from Neural crest/mesenchymal cell, in embryo's origin, the attached MSC from mesoderm-derivative is different, and MSC is passed through in the present embodiment research Chemotaxis make the new ant algorithms of tissue regeneration.Dental pulp is the unique soft tissue of tooth and keeps tooth as organ Dynamic equilibrium.Root canal is one of modal dental procedure, wherein eradicates pulp tissue alive, and by the heat of biologically inert Plastic material replaces.Tooth after root pipe is deprived of viability biology, thus, it is easy to infect, fracture and damage.Due to tooth Marrow is connected with alveolus bone marrow, and inventor thinks can supplement alveolus MSC so that pulp tissue regenerates.

The multiple healthy patients extracted by the tooth for medical science needs obtain little alveolar bone sample.Monokaryon and attachment Slight culture-the expansion of cell.First sieved the MSC (p3) passed through in early days by immunocytochemistry and flow cytometry, send out Now express Stro-1, CD 105, CD73, CD44 and CD90, but do not express CD34.Alveolus MSC is the most chemically defined Jie In matter differentiation become osteogenic, become fatty, become cartilage with become flesh sexual cell.At multiple cytokines and the shadow of somatomedin Under sound, transwell insertion system is studied the migration of alveolus MSC.

When multiple time points describe cell migration in detail, the PDGF β β of 50ng/ml is the most significant.Confirm expression of receptor.

These find the stem cell/progenitor cell benefit towards tissue regeneration of explanation inducing endogenous and/or transplanting together Fill.

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In view of above description, it can be seen that achieve some advantages of the present invention and achieve further advantage.

Claims (20)

1. an acellular mammalian tooth shape support, described support comprises

Host material；With

Compositions, it comprises CXCL12 (SDF1) and BMP-7 (BMP-7)；

Wherein, described compositions is chemotaxis and osteogenic, one-tenth dentine, one-tenth enamel or one-tenth cementum,

In described compositions is distributed to the host material of tooth tooth shape support or on；

When contacting with the dental tissue comprising CFU-GM, described compositions lures that CFU-GM moves in tooth tooth shape support into；And

Tooth tooth shape support does not comprise living cells before implantation.

Support the most according to claim 1, wherein said support has people's front tooth, people's canine tooth, people's bicuspid or the shape of people's molar Shape.

Support the most according to claim 1, wherein said compositions is encapsulated in slow release encapsulation vehicle and is distributed to tooth shape and props up In the host material of frame or on.

Support the most according to claim 1, wherein said compositions also includes the somatomedin (PDGF) of platelet-derivative, interior Skin cell growth factor (ECGF), conversion growth factor-β l (TGF-β l), epidermal growth factor (EGF), hepatocyte growth factor (HGF), the bone morphogenetic protein in addition to BMP-7 (BMP), TGF-β, GDF (GDF), insulin-like growth factor Son-1 (IGF1), VEGF (VEGF), fibroblast growth factor (FGF), dentine matrix albumen, dentine saliva Gland albumen, bone salivary gland albumen, amelogenin or integrin.

Support the most according to claim 1, wherein chemotaxis somatomedin is SDF1, and osteogenic, one-tenth dentine, one-tenth enamel Property or become cementum somatomedin be BMP-7.

Support the most according to claim 5, wherein:

I () BMP-7 is 10ng/g support-1000 μ g/g support in the bracket, and SDF1 be in the bracket 10ng/g support- 1000 μ g/g supports；Or

(ii) BMP-7 is about 100 μ g/g supports in the bracket, and SDF1 is about 100 μ g/g supports in the bracket.

Support the most according to claim 6, wherein said host material comprises bone conduction material.

Support the most according to claim 7, wherein said host material comprises the mixture of ε-polycaprolactone and hydroxyapatite.

Support the most according to claim 1, described support comprises:

Diameter is between 50-500 μm, or the microchannel of 200 μm；With

Gel, in wherein gel is introduced into microchannel, and described chemotaxis and osteogenic, becomes dentine, becomes enamel or become tooth Sclerotin compound embeds in this gel.

Support the most according to claim 1, described support comprises atresia corona.

11. supports as claimed in one of claims 1-10, described support comprises selected from two or more following features:

I () described support has people's front tooth, people's canine tooth, people's bicuspid or the shape of people's molar；

(ii) in described compositions is distributed to the host material of tooth tooth shape support in being encapsulated in slow release encapsulation vehicle or on；

(iii) described compositions also include the somatomedin (PDGF) of platelet-derivative, endothelial cell growth factor (ECGF) (ECGF), Conversion growth factor-β l (TGF-β l), epidermal growth factor (EGF), hepatocyte growth factor (HGF), bone shape in addition to BMP-7 State generation albumen (BMP), TGF-β, GDF (GDF), insulin-like growth factor-i (IGF1), blood vessel endothelium are raw The long factor (VEGF), fibroblast growth factor (FGF), dentine matrix albumen, dentine salivary gland albumen, bone salivary gland albumen, tooth Glaze albumen or integrin；

(iv) chemotaxis somatomedin is SDF1, and osteogenic, one-tenth dentine, one-tenth enamel or one-tenth cementum somatomedin For BMP-7；

V () BMP-7 is 10ng/g support-1000 μ g/g support in the bracket, and SDF1 be in the bracket 10ng/g support- 1000 μ g/g supports；

(vi) BMP-7 is about 100 μ g/g supports in the bracket, and SDF1 is about 100 μ g/g supports in the bracket；

(vii) described host material comprises bone conduction material；

(viii) described host material comprises the mixture of ε-polycaprolactone and hydroxyapatite；

(ix) described host material comprises 80% weight polycaprolactone and the mixture of 20% weight hydroxyapatite；

X () described support comprises diameter microchannel between 50-500 μm；

(xi) described support comprises the microchannel of a diameter of 200 μm；

(xii) described support comprises microchannel and gel, and wherein said gel is introduced in microchannel, and described chemotaxis with become Bone, one-tenth dentine, one-tenth enamel or one-tenth cementum compound embed in this gel；

(xiii) described support comprises atresia corona；And

(xiv) described support comprises the microchannel of a diameter of 200 μm, and described microchannel comprises the glue being incorporated in described microchannel Former protein gel, the slow release encapsulation vehicle encapsulating described compositions is embedded into the collagen gel being introduced in microchannel In；Described compositions comprises the BMP-7 that concentration is 100ng/ml gel and the SDF1 that concentration is 100ng/ml gel, and described Frame comprises atresia corona.

12. support according to claim 11, wherein said bone conduction material is hydroxyapatite.

13. supports according to claim 1, wherein said host material comprises 80wt% ε-polycaprolactone and 20wt% hydroxyl phosphorus Lime stone.

14. supports according to claim 9, wherein said gel is collagen gel.

15. supports as claimed in one of claims 1-10 are for lacking the goods of tooth in the mouth for the treatment of mammal Purposes in manufacture, wherein absence of tooth and in mouth disappearance tooth position there is alveolus, wherein:

Described support has in shape the implanted alveolus of losing tooth；And

Modeled by the tooth that computer-aided design (CAD) is disappearance, and synthesize described support with bioplotter；Or

Described support has a shape of people's first molar, and to but second mortar of opposite side at mouth similar with first molar Tooth carries out CT scan, and CAD utilizes the CT scan data of second molar to design described support.

The method of the tooth scaffold any one of 16. manufacturing claims 1-10, described method comprises:

Manufacture the support of mammalian tooth shape；With

By at least one chemotaxis compound and at least one osteogenic, become dentine, become enamel or become cementum chemical combination Thing introduces described support.

17. methods according to claim 16, wherein said support is Acellular matrix.

18. methods according to claim 16, wherein said support is in being similar in mammal lack the shape of tooth.

19. methods according to claim 16, wherein said method also includes using computer-aided design (CAD) preparation disappearance The model of tooth also synthesizes described support with bioplotter.

20. methods according to claim 16, wherein said support has the shape of people's first molar and carries out being similar to first But the molar CT scan of second molar of the opposite side at face, wherein use the CAD quilt of the CT scan data of second molar For designing described support.