Summary of the invention:
The objective of the invention is to overcome existing above-mentioned deficiency in the prior art, pulp-cap of a kind of new biologically active and preparation method thereof is provided.
The pulp-cap of biologically active provided by the invention, comprise dentin lixiviating solution and dentin powder, described dentin lixiviating solution is that dentin is organized in cultivation gained supernatant in the culture medium, and described dentin powder is that the dentin behind culture medium culturing grinds the gained powder.
In the pulp-cap of above-mentioned biologically active, described culture medium can be preferably α-MEM culture medium for suitable culture medium commonly used of cultivating the dentin tissue.
A kind of preparation method of the pulp-cap of biologically active provided by the invention may further comprise the steps:
(1) gets healthy tissue of tooth, clean, remove periodontal membrane, cementum and dental pulp, sterilization with PBS liquid;
(2) step (1) gained tissue of tooth is ground to form graininess after freezing;
(3) place culture medium to cultivate step (2) gained graininess tissue of tooth;
(4) get step (3) and cultivate the gained supernatant liquid filtering, filtering the filtrate that obtains is dentin lixiviating solution (hereinafter referred lixiviating solution);
Get dentin granule after the cultivation and dry after with the PBS centrifugal elutriation, grind into powder is the dentin powder.
In the preparation method of above-mentioned biologically active pulp-cap, remove described in the step (1) periodontal membrane, cementum and dental pulp method can for: with 20000 rev/mins dentistry low speed mobile phones with corona and the amputation of the part tip of a root, with 300,000 rev/mins dental high-speed mobile phone periodontal membrane, cementum are removed then, pull out the in-house dental pulp of residual tooth, filing expanding needle expands file root inside pipe wall and removes clean dental pulp.
In the preparation method of above-mentioned biologically active pulp-cap, described in the step (1) sterilization method can for: the tissue of tooth that will remove periodontal membrane, cementum and dental pulp places deionized water, soaks sonic oscillation 30 minutes 30 minutes-1 hour; Repeat 3-6 time; Take out tissue of tooth, in the two anti-solution that contains 50 U/ml penicillins, 50 mg/ml streptomycins, soaked 72 hours.
In the preparation method of above-mentioned biologically active pulp-cap, the freezing method of tissue of tooth described in the step (2) can for: tissue of tooth was put into 4 ℃ of freezing 2-3 of refrigerator hours, change-20 ℃ of freezing 2-3 of refrigerator hours over to, put into-80 ℃ of freezing 2-3 of refrigerator hours, and put at last after liquid nitrogen 20-30 minute and take out.
In the preparation method of above-mentioned biologically active pulp-cap, graininess tissue of tooth described in the step (3) be 0.20-0.30mm.
In the preparation method of above-mentioned biologically active pulp-cap, culture medium described in the step (3) is α-MEM culture medium.
In the preparation method of above-mentioned biologically active pulp-cap, the consumption of culture medium described in the step (3) is that every 20g dentin granule uses 100ml α-MEM culture medium, and condition of culture is 37 ℃, 5%CO
2
In the preparation method of above-mentioned biologically active pulp-cap, the employed filter of supernatant liquid filtering described in the step (4) is 0.22 μ m filter.
In the preparation method of above-mentioned biologically active pulp-cap, the particle diameter of powder is 0.20-0.30mm described in the step (4).
Treat dark dental caries and should preserve vital pulp as much as possible,, keep the performance of masticatory function so that tissue of tooth can obtain nutrition supply.Because clinical Ca commonly used (OH)
2May there be unrestricted deficiency of inducing mineralising in pulp-cap, makes root canal subsequently be difficult to finish smoothly, and therefore, those skilled in the art expect to find the pulp-cap that can form reparative dentin or biologically active of alternative calcium hydroxide.
Contain in the dentin bone sialoprotein (Bone Sialoprotein, BSP), bone morphogenetic protein(BMP) (Bone Morphogenetic Protein, BMP), transforming growth factor-beta (Transforming Growth Factor-β, TGF-β).Goldberg is used for rat molar with BSP as pulp-cap, discovery is in the time of 28 days, osteodentin and reparative dentin (M. Goldberg1 appear, N. Six, et al, Application of Bioactiye Molecules in Pulp-capping Situations, Adv Dent Res, August, 2001,15:91-95).Hunter and Goldberg point out that BSP can not only promote symphysis, also can promote the growth of hydroxyapatite crystal.(Hunter GK, Goldberg HA. Nucleation of hydroxyapatite by bone sialoprotein. Proc Natl Acad Sci USA1993 play a crucial role in Dentinal mineralization process; 90:8562 – 8565.).(Bone Morphogenetic Protein BMP) is a kind of bone Induced substance to bone morphogenetic protein(BMP), can induce new bone formation.Use BMP to cover the formation of observing reparative dentin (the Saito T of marrow test energy success in animal body in recent years, Ogawa M, et al, Acceleration effect of human recombinant bone morphogenetic protein-2 on differentiation of human pulp cells into odontoblasts, J Endodontics, 2004,30 (4) 205-8).Transforming growth factor-beta (Transforming Growth Factor-β, TGF-β) expresses in the odontoblast in Dentinal growth course, when dentin sustains damage, can control and newly be divided into dentin cell's hypertrophy and differentiation, stimulate the formation of reparative dentin.Usefulness TGF-β such as Wen Lingying and hydroxyapatite are mixed for the lid marrow of healthy domesticated dog, promptly have dentinal matrix to form in 4 weeks of postoperative, and the tubulose dentin appears in 8 weeks of postoperative.Postoperative 12 week formation dentine bridge (effect that Wen Lingying, Fang Jun, Jia Dong etc., exogenous TGF-β induce reparative dentin to form in vivo, practical stomatology magazine, 2000,16(6): 426-429).
Though existing usefulness BSP, BMP or TGF-β are as the bibliographical information of pulp-cap in the prior art, but existing single somatomedin is induced (comprising BSP, BMP, TGF-β) formation of reparative dentin, its structure and intensity is not further research as yet, at present, it is not clear that these somatomedin influence the mechanism that dentin forms, so, abandon all the other somatomedin and adopt single somatomedin may reduce its mutual synergism, natural form and intensity after influencing dentin and forming.
The present invention utilizes dentin to be rich in the abundant somatomedin that can promote into the dentin growth and albumen as induced liquid, mixing with self dentin powder as a kind of novel pulp-cap, the present invention has extracted nearly all active substance in the dentin, utilize these somatomedin to make up Dentinal generation, can keep its mutual synergism as far as possible.Biological activity pulp-cap of the present invention has the favorable tissue compatibility, and is nontoxic, and can induce pulp cells to break up to dentin cell, has good application prospects.
The specific embodiment
The invention will be further described below in conjunction with example, but the present invention is not limited to following embodiment.Operation is all carried out in strict gnotobasis in the example.
Embodiment
In the present embodiment, the no pathological changes premolars of employed tooth for pulling out, healthy impacted tooth, accessory tooth and the deciduous teeth perhaps pulled out because of the orthodontic treatment conceptual design.
The pulp-cap of the biologically active of enumerating in the present embodiment, comprise dentin lixiviating solution and dentin powder, described dentin lixiviating solution is that dentin is organized in cultivation gained supernatant in α-MEM culture medium, and described dentin powder is that the dentin behind α-MEM culture medium culturing grinds gained.
The preparation method of the pulp-cap of the biologically active of enumerating in the embodiment of the invention is as follows:
(1), the tooth of pulling up is soaked in the PBS buffer solution and cleans 3 times, with 20000 rev/mins NSK dentistry low speed mobile phones with corona and the amputation of the part tip of a root, with 300,000 rev/mins NSK high speed handpiece periodontal membrane, cementum are removed subsequently, pull out the in-house dental pulp of residual tooth, filing expanding needle expands file root inside pipe wall and removes clean dental pulp;
Tissue of tooth is placed deionized water, soaked 30 minutes-1 hour, sonic oscillation is about 30 minutes subsequently, 3-6 time so repeatedly;
Take out tissue of tooth, in the two anti-solution that contains 50 U/ml penicillins, 50 mg/ml streptomycins, soaked 72 hours.
(2), step (1) gained tissue of tooth being put into 4 ℃ of refrigerators placed 2-3 hour, changing-20 ℃ of refrigerators then over to placed 2-3 hour, changing-80 ℃ of refrigerators again over to placed 2-3 hour, put into-70 ℃ of liquid nitrogen at last and place taking-up after about 30 minutes, put into dismembyator immediately and be ground into graininess.
(3), will step (2) add in α-MEM culture medium and cultivated 3-5 days, condition of culture is 37 ℃, 5%CO
2, the culture medium consumption is that every 20g dentin granule uses the 100ml culture medium.
(4), get supernatant, 0.22 μ m filter filters, the supernatant that obtains after the filtration is the dentin lixiviating solution;
Dentin granule after cultivating with oven dry behind the PBS centrifugal elutriation 3 times, is ground to form the powder of mean diameter 0.25mm size, and oxirane disinfection is standby.
Get the made dentin lixiviating solution of getting ready of step (4) (ISO 10993 standards), use the EILSA test kit, the bioactie agent in the dentin lixiviating solution is carried out assay, triplicate is averaged, and the result is as follows:
COL-1:Collogen-1, a collagen type (18.3725 ± 1.0585 ug/L)
TGF-β: Transforming Growth Factor-β, transforming growth factor-beta (1.16033 ± 0.08315 ug/L)
DSP:Dentin Sialoprotein, dentin sialoprotein (52.754 ± 3.383 pg/L)
DMP1:Dentin Morphogenetic Protein, dentin bone morphogenetic protein (571.859 ± 38.183 pg/L)
BGN:Biglycan, biglycan (353.72 ± 2.040 pg/L)
DCN:Decorin, decorin (405.129 ± 1.0416 pg/L)
The block diagram that three measurement result meansigma methodss are drawn out is seen accompanying drawing 1, accompanying drawing 2.
Present embodiment powder and lixiviating solution are pressed furnishing pasty state after the 1:1 mixed, be covered in nearly marrow or reveal the marrow place, the glass-ion cement spit of fland is sealed temporarily, 6-8 further consultation after week, according to X-ray film, visible reparative dentin forms, at this moment, remove the segment glass ion and seal thing temporarily, stay 2mm to do rebasing thing, feasible afterwards resin fill or inlay prothesis.
The biological activity pulp-cap of present embodiment and the contrast of traditional calcium hydroxide for pulp-capping: get three dental caries, respectively as experimental group 1, experimental group 2 and contrast groups, experimental group 1, experimental group 2 use present embodiment dentin active material as pulp-cap, and matched group uses calcium hydroxide as pulp-cap.Imaging data is seen accompanying drawing 3, accompanying drawing 4 after 8 weeks, and the result shows that the existing neonatal tooth essence of experimental group forms, and contrasting also has newborn Dentinal formation, illustrates that this experimental group and matched group all have the newborn Dentinal ability of formation.The pulp-cap that present embodiment is described have can the instead of hydrogen calcium oxide as the potential of novel pulp-cap, and the advantage that do not possess of calcium hydroxide such as biologically active.
Application examples 1,The structure of people's dentin repair materials:
(1) after extraction people venous blood is determined safety through strict trace routine (detection of ABO/Rh blood group, the detection of HLA typing, syphilis antibody detection, special-shaped hepatitis antigen antibody test, CMV antibody test and HIV immune detection etc.), gathers the donor essential information.Comprise: retrieval code, name, age, identification card number, date of birth, race, address and contact method.
(2) select the no pathological changes premolars that to pull out because of the orthodontic treatment conceptual design, healthy impacted tooth, accessory tooth and the deciduous teeth perhaps pulled out.The tooth of pulling up is soaked in the PBS buffer solution cleans 3 times, the low speed mobile phone is removed with high speed handpiece corona and the amputation of the part tip of a root subsequently with periodontal membrane, cementum, pull out the in-house dental pulp of residual tooth, and filing expanding needle expands file root inside pipe wall and removes clean dental pulp.Material is placed deionized water, soaked 30 minutes-1 hour, sonic oscillation is about 30 minutes subsequently, 3-6 time so repeatedly.Take out material, be soaked in two anti-(50 U/ml penicillins, 50 mg/ml streptomycins) solution 72 hours.Material was put into 4 ℃ of refrigerator 2-3 hours, is changed over to-20 ℃ then and placed 2-3 hour, again material is put into subsequently-80 ℃ refrigerator 2-3 hour, in-70 ℃ of liquid nitrogen, place after about 30 minutes at last and take out, put into dismembyator immediately and be ground into Powdered; Add in the material that will process in α-MEM culture medium and cultivated 3 days, get supernatant, 0.22 μ m filter filters standby; To cultivate back material PBS centrifugal elutriation 3 times, oven dry dentin powder, oxirane disinfection is standby.
Only dentin powder and extracting solution need be mixed in proportion when (3) using, place nearly marrow or perforation of pulp chamber hole place.The glass-ion cement spit of fland is sealed temporarily, if observation 4-6 week is asymptomatic, and after X-ray film confirms that reparative dentin forms, feasible permanent filling.
Application examples 2,The structure of pig dentin repair materials:
(1) gathers the donor essential information: the title of pig, strain, pig age, body weight etc., and the laboratory animal laboratory animal medical certificate that provides the center to provide.
(2) under the local anaesthesia, pig is gone up the lower jaw labial teeth pull out, bloodstain and dirt are removed in PBS flushing 3 times.Low speed turbine is with corona and the amputation of part organization of root tips, and barbed broach takes out dental pulp, EDTA flushing root tube chamber, and the H file remains dental pulp with wall of the lumen and goes only and remove the part predentin.Remove periodontal membrane, cementum, the back tissue of tooth be will handle and sonic oscillation 30 minutes, 17%EDTA demineralization 10-40 minute will be soaked in the deionized water about 1 hour, deionized water rinsing one hour, then material is soaked in two anti-(50 U/ml penicillins, 50 mg/ml streptomycins) solution after 72 hours, put into liquid nitrogen about 30 minutes, putting into sterile grinder after the taking-up is ground to Powdered, put into α-MEM culture medium subsequently and cultivated 3 days, get supernatant, 0.22 μ m filter filters standby.Residue dentin PBS centrifugal elutriation 3 times takes out and is placed on drying machine drying, and oxirane is standby then.
When (3) using, need set up bad model of pig dental caries, then the present invention be mixed by a certain percentage back furnishing pasty state and be covered in the dental caries harm, rebasing filling, complete operation.
Application examples 3,The structure of rat dentin repair materials:
(1) gathers the donor essential information: the title of Mus, strain, Mus age, body weight etc., and the laboratory animal laboratory animal medical certificate that provides the center to provide.
(2) the disconnected neck of adult rat is put to death, get Upper Anterior Teeth and grind one's teeth in sleep, under the Stereo microscope with enamel, periodontal membrane, cementum is removed, and intercepting part root of the tooth and corona use Syringe needle Irrigator that dental pulp is gone out, the H file is handled the root inside pipe wall, EDTA flushing root pipe, PBS flushing 3 times is soaked in two anti-(50 U/ml penicillins with material, 50 mg/ml streptomycins) in the solution 3 days, with the material oven dry, put into liquid nitrogen about 30 minutes, the dismembyator grinding-material is to Powdered, add α-MEM culture medium then, cultivated 3 days, and extracted lixiviating solution, 0.22 μ m filter filters standby, residue dentin powder PBS centrifugal elutriation 3 times, dry for standby.
(3) set up bad model of rat dental caries, use the present invention, dentin powder and lixiviating solution are reconciled into pasty state according to certain ratio, place nearly marrow or perforation of pulp chamber place, row dental defect plombage, end operation.
Application examples 4,The structure of primate (monkey) dentin repair materials:
(1) gathers the donor essential information: the title of monkey, strain, monkey age, body weight etc., and the laboratory animal laboratory animal medical certificate that provides the center to provide.
(2) with the sheet that pans after the permanent monkey anesthesia, choosing has the permanent monkey of wisdom teeth as experimental subject.The general anesthesia intubate is pulled out the lower jaw block teeth of monkey, is soaked in two anti-solution standby.After drawing materials, be for further processing immediately: PBS washes tooth, blood and dirt are rinsed well, mobile phone is with the corona amputation at a slow speed, high speed handpiece is removed periodontal membrane and cementum, and barbed broach takes out dental pulp, EDTA flushing root pipe, the H file is handled the root inside pipe wall, and its dental pulp and predentin are removed fully.Subsequently material is soaked in the deionized water,, removes spot with ultrasonator vibration 30 minutes.Use EDTA to carry out the gradient demineralization, PBS flushing 3 times is steeped in two anti-(50 U/ml penicillins, 50 mg/ml streptomycins) in the solution, take out material after 72 hours, PBS washes once more, after the oven dry material is put into liquid nitrogen, and use the grinding apparatus that material is ground into powder, and add α-MEM culture medium, cultivated 3 days, extract lixiviating solution, 0.22 it is standby that μ m filter filters, residue dentin powder PBS centrifugal elutriation 3 times, dry for standby.
When (3) using, permanent monkey anesthesia be detected oral hygiene, if any suffering from tooth, can repair, if there are dark dental caries detritus can be removed, and remove inorganic glaze, this material reconciles into paste-like and is covered in dark dental caries place after my mixed of extract then, the dental body filled art of rebasing back row.Then can not make up bad model of dental caries if there is the tooth of suffering from the oral cavity, on bad model of dental caries, carry out the experimental research of this material.