CN102068318B - Manufacturing method of biological tooth root bracket material - Google Patents

Manufacturing method of biological tooth root bracket material Download PDF

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CN102068318B
CN102068318B CN 201010597640 CN201010597640A CN102068318B CN 102068318 B CN102068318 B CN 102068318B CN 201010597640 CN201010597640 CN 201010597640 CN 201010597640 A CN201010597640 A CN 201010597640A CN 102068318 B CN102068318 B CN 102068318B
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root
dental
tooth
biological
teeth
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CN 201010597640
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CN102068318A (en )
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李锐
杨波
田卫东
盛磊
郭维华
陈岗
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四川大学
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Abstract

The invention discloses a manufacturing method of a biological tooth root bracket material. The method comprises the following steps of: flushing teeth with phosphate buffered saline (PBS) in a sterile state for three times; removing dental crown parts by grinding with a carborundum disc, and keeping tooth root parts; pulling pulp tissues out of the tooth roots and repeatedly flushing the inside of root canals with physiological saline; gradually expanding the inside of teeth with a dental expander and repeatedly flushing the inside of the root canals with 5 to 17 percent of ethylene diamine tetraacetic acid (EDTA) solution; grinding outer side tissues of the tooth roots by grinding with a dental turbine and constructing a biological tooth root profile as required so as to obtain a bracket with a specific shape; preserving the teeth in the PBS and treating in an ultrasonic oscillator for 3 to 8 times; performing stepped demineralization on a dentine with EDTA with different concentrations in a magnetic stirrer with the rotating speed of between 100 and 500 revolutions per minute so as to obtain a dentine substrate; and preserving the dentine substrate in double-antibody-containingPBS. The biological tooth root bracket material has a tooth root profile, the thickness of 1mm to 3mm, the length of 1cm to 2.5cm and certain tooth inducing capability, comprises a large number of proteins and factors related to the manufacturing of teeth, can release the proteins and factors continuously, can better solve the problem of induction micro-environment, and contributes to applicationto tooth socket intra-alveolar orthotopic transplantation.

Description

生物牙根支架材料的构建方法 Construction root biological scaffold

技术领域 FIELD

[0001] 本发明公开了一种生物牙根支架材料的构建方法。 [0001] The present invention discloses a method of constructing a biological scaffold root.

背景技术 Background technique

[0002]目前,各种原因所致的牙齿缺失患病率随年龄增长而增加,到中老年患病率约为80%。 [0002] At present, due to various reasons missing teeth prevalence increases with age, the elderly prevalence rate of about 80%. 牙齿缺失与牙发育异常严重妨碍患者的咀嚼、语言等功能,并引起患者容貌畸形、认知缺陷、心理障碍,从而极大地危害了患者的身心健康,因此,寻求有效的治疗途径是迫切而艰巨的任务。 Tooth loss and tooth abnormalities seriously impede the function of chewing and language of the patient and cause the patient looks abnormal, cognitive defects, mental disorders, which greatly harm the patient's physical and mental health, and therefore, the search for effective therapeutic approach is urgent and arduous task.

[0003]目前的牙齿缺失均采用人工材料赝复体修复,存在着生理功能恢复有限、使用寿命短、损伤正常组织等缺陷,缺乏真正生理意义的修复。 [0003] The current lack of teeth are made of artificial materials prosthodontic rehabilitation, there is limited functional recovery, life is short, normal tissue damage and other defects, lack of real physiological significance of repair. 种植体修复缺失牙是目前治疗牙缺失的热点和重点,但种植体与牙周组织只能实现骨性结合,无法获得新生的牙周膜等缺点。 Implant missing teeth is the treatment of tooth loss of focus and emphasis, but the implant and periodontal tissue can only achieve osseointegration, you can not get periodontal film shortcomings newborn. 如何有效地在目前的修复方法基础上,获得新生的牙骨质、牙周膜和牙槽骨,是治疗牙缺失的关键。 How effectively in the current repair method on the basis of a new life cementum, periodontal ligament and alveolar bone, tooth loss is the key to treatment.

[0004] 支架材料和诱导微环境问题是进行牙根再生组织工程的重要问题。 [0004] scaffolds and induced micro-environment is an important issue root regeneration of tissue engineering. 近年来,随着对各种支架材料研究的深入,采用生物有机材料作为支架材料应用于牙根组织工程已成为研究热点,肠系膜、煅烧牛骨、牙本质基质等均有应用报道。 In recent years, along with a variety of in-depth study of the scaffold material, the use of bio-organic materials as scaffolds used in root tissue engineering has become a hot topic, mesenteric, calcined bone, dentin matrix and other applications have been reported. 对牙本质的组织学和蛋白组学的研究表明:在牙本质中存在多种对于牙齿发育和牙齿受到损伤后修复过程中重要的蛋白和因子,其中很多都有很强的表达或者较高的含量。 Histological study of dentin and proteomics show: There are a number of teeth and tooth development for the damage repair process important factor in protein and dentin, many of which have strong or high expression content. Guo等以大鼠的牙本质为研究对象,在牙本质周围成功的诱导了牙骨质、牙周膜以及神经血管的再生(Weihua Guo, Yong He,Xiaojun Zhang, et al, The use of dentin matrix scaffold and dental follicle cellsfor dentin regeneration, Biomaterials 2009 ;30: 6708-6723)。 Guo et dentin in rats as subjects, about the successful induction of a dentin cementum, periodontal ligament, nerve regeneration vessel (Weihua Guo, Yong He, Xiaojun Zhang, et al, The use of dentin matrix scaffold and dental follicle cellsfor dentin regeneration, Biomaterials 2009; 30: 6708-6723). Guo 等的研究显不:相对于其他生物支架材料,牙本质基质可以更好的诱导分化为成牙骨质细胞样细胞、成骨细胞样细胞以及成纤维细胞样细胞,形成类似天然的牙周膜样结缔组织和牙周膜/牙骨质复合体的结构,有利于生物牙根的重建,这是区别于其他生物支架材料的重要特征之一。 Guo et al study is not significant: with respect to other biological scaffold, dentin matrix can be better induced to differentiate into cementum-like cells, osteoblast-like cells and fibroblast-like cells, natural-like formation of periodontal connective tissue and periodontal membrane-like structure of the film / composite cementum, facilitate reconstruction of biological root, one of which is different from other important characteristics of biological material. 但该研究中所制备的牙本质基质为老鼠牙本质,与人类等其他物种的牙本质存在一定不同,构建出的组织为牙本质、无法设计成为牙根形的支架材料,并且,生物牙根材料在不能破坏牙本质基质中的重要生物活性物质,以保证这些生物活性物质可以持续的从材料中释放到周围环境中的同时,需尽量保持牙根的形状、保持该材料具有一定的力学强度并且充分脱矿,以利于牙髓和牙周组织的软组织和硬组织的共同构建。 However, dentin matrix prepared in this study was rat dentin, the dentin certain other different species including humans, dentin tissue construct, scaffold not designed to shape the root, and the root of biological material important not destroy the biologically active material in the dentin matrix, to ensure that they can continue the biologically active substance is released from the material to the surroundings at the same time, the need to keep the shape of the root, the holding material having a certain mechanical strength and sufficiently off work together to build the mine, in order to facilitate soft tissue dental pulp and periodontal tissue and hard tissue. 同时,Guo的研究中使用的异位的牙本质的再生,对于牙槽骨内的再生情况并没有进行相应的讨论。 Meanwhile, dentin regeneration study of Guo ectopic used for regeneration of alveolar bone and not in the corresponding discussion. 综上所述,该研究并未实现真正意义上的生物牙根支架材料的重建。 In summary, this study did not achieve reconstruction of biological scaffold root in the true sense.

发明内容 SUMMARY

[0005] 本发明的目的在于克服现有技术中所存在的上述不足,提供一种生物牙根支架材料的构建方法。 [0005] The object of the present invention is to overcome the above disadvantages present in the prior art, there is provided a method of constructing a biological scaffold root. 本发明的构建方法容易掌握、安全可行、成本低廉。 Construction method of the present invention is easy to grasp, is feasible and safe, and low cost.

[0006] 为了实现上述目的,本发明提供了以下技术方案:[0007] —种生物牙根支架材料的构建方法,包括以下步骤: [0006] To achieve the above object, the present invention provides the following technical solution: [0007] - Biological Species root scaffold construction method, comprising the steps of:

[0008] ( I)将供体新鲜的牙齿在无菌状态下,用PBS缓冲液冲洗; [0008] (I) of the donor fresh tooth under aseptic conditions, rinsed with PBS buffer;

[0009] (2)将步骤(I)所得牙齿在有喷水装置的牙科用慢速涡轮机上磨除牙冠部分,保留牙根部分,所述牙科用慢速涡轮机的转速为1000-10000转/分,所得牙根部分保存于含有PBS缓冲液的培养瓶或其他器皿中; [0009] (2) the step (I) obtained in the teeth with a dental apparatus with a water jet abrading slow turbine crown portion, retained root portion, said slow speed dental turbine 1,000 to 10,000 revolutions / points, the root portion of the resulting stored in culture flasks or other containers in PBS buffer containing;

[0010] (3)用牙科用拔髓针拔出步骤(2)所得牙齿牙根内的牙髓组织,用生理盐水反复冲洗牙齿根管内部3-8次,每次10-30晕升; [0010] (3) by pulling a dental broach pulled out in step (2) the resultant tooth pulp tissue in the root, a tooth root of the inner tube repeatedly washed with saline 3-8 times 10-30 liters halo;

[0011] (4)使用牙科用扩大机器将步骤(3)所得牙齿内部逐渐扩大,去除牙髓组织和前期牙本质等组织,此过程中使用5%-17%的EDTA溶液反复冲洗根管内部3-8次,每次10-30毫升; [0011] (4) a dental expansion machine in step (3) the resulting internal teeth gradually expanded, and removal of pulp tissue and other tissues predentin, this procedure using 5% to 17% EDTA solution repeatedly washed inside tubes 3-8 times 10-30 ml;

[0012] (5)使用牙科用涡轮机按照生物牙根外形磨除部分牙齿组织,以去除牙周组织和牙骨质等组织。 [0012] (5) a dental turbine mill according to biological tissue other part of the tooth root shape, to remove cementum and periodontal tissue and other tissues. 最终得到的材料厚度l_3mm,长度1-2. 5cm ; The finally obtained material thickness l_3mm, length 1-2 5cm.;

[0013] (6)将步骤(5)处理好的牙齿保存于PBS缓冲液中,于超声震荡器中处理3-8次,每次处理10_30min ; [0013] (6) The step (5) Handled teeth were stored in PBS buffer, treated in an ultrasonic shaker 3-8 times per treatment 10_30min;

[0014] (7)在转速为100-500转/分的磁力搅拌器中,使用不同浓度的EDTA对牙本质进行梯度脱矿,得到牙本质基质。 Magnetic stirrer [0014] (7) at a speed of 100-500 rev / min, different concentrations of EDTA on demineralized dentin gradient, to give dentin matrix.

[0015] 将步骤(7)处理好的牙本质基质保存于含有双抗的PBS缓冲液中备用,所述含双抗的PBS中青霉素浓度为50-500unit/mL,链霉素浓度为50_500unit/mL。 [0015] The step (7) for good dental matrix stored in PBS buffer containing bis standby antibody, PBS containing penicillin double antibody concentration 50-500unit / mL, streptomycin at a concentration of 50_500unit / mL.

[0016] 上述生物牙根支架材料的构建方法中,步骤(4)中所述使用牙科用扩大机器将步骤(3)所得牙齿内部逐渐扩大的方法为首先使用15号牙科手用普通镍钛锉扩大牙齿根管内部,20号普通镍钛锉扩大牙齿根管内部,然后从小号到大号使用Sx号牙科用扩大针扩大牙齿根管口部分,即依次用SI号、S2号、Fl号、F2号牙科用扩大针扩大牙齿根管口部分,必要时用F3号牙科用扩大针扩大牙齿根管口部分。 [0016] The method of constructing the above-described scaffold root biological material, in step (4) said expanded machine using dental step (3) the resulting internal teeth gradually expanded to first use a method 15 for expanding the common dental hand NiTi files internal tooth root canal, NiTi file 20, an ordinary expansion inside the tooth root canal, and then small to large in number Sx using expanded dental root canal reamer with the port portion, i.e., sequentially in SI No. No. S2, Fl number, F2 No. expand dental root canal reamer with the inlet portion, outlet portion expanded root canal reamer with, if necessary, with a number of dental F3. 步骤(5)所述使用牙科用涡轮机将步骤 Step (5) The use of a dental turbine of step

(4)所得牙根外侧磨除的方法为:使用金刚砂车针,按照生物牙根外形磨除部分牙齿组织,最终得到的材料厚度l_3mm,长度1-2. 5cm。 Method (4) resulting outer root abrading of: using diamond bur according biological profile abrading tooth root part of the tooth tissue, the resulting material thickness l_3mm, length 1-2 5cm..

[0017] 上述生物牙根支架材料的构建方法中,步骤(7)中所述梯度脱矿的方法为: [0017] The method of constructing the above-described scaffold root biological material, in step (7) in the demineralization of a gradient method:

[0018] 首先使用17%-25%的EDTA溶液脱矿处理5_15min,然后使用7%_15%的EDTA溶液脱矿处理5-15min,最后使用5%的EDTA溶液脱矿处理5_15min。 [0018] First, a 17% to 25% EDTA solution demineralization process 5_15min, then 7% _15% EDTA solution demineralization process 5-15min, and finally with 5% EDTA solution demineralization process 5_15min.

[0019] 牙本质作为一种生物材料,由于其本身具有一定的机械强度,可以作为一种新型的实现牙根再生的支架材料;而且牙本质本身来源于牙源性细胞,存在一些牙齿发育过程中重要的蛋白和因子,又可以做为一种细胞牙向分化的诱导微环境。 [0019] Dentin as a biological material, because of its own having certain mechanical strength can be used as a scaffold to achieve a new root regeneration; and odontogenic cells derived from dentine itself, there are some tooth development process important factors and protein, but also can be used as a cell-induced differentiation of dental microenvironment. 同时,由于各种临床需要而拔除的牙齿除了用于细胞培养外,牙齿的硬组织部分几乎都被当做医疗废物处理,而且牙本质又是牙齿的主要组成部分,这就很好的解决了来源问题。 At the same time, due to a variety of clinical needs in addition to the extracted tooth for cell culture, the hard part of the teeth of almost all organizations are treated as medical waste, and is a major component of dentin of the tooth, which is a good solution to source problem. 但是,利用牙本质基质作为生物牙根支架材料,需要尽量保持牙根的形状、并且充分脱矿,但又不能破坏牙本质基质中的重要生物活性物质,保证这些生物活性物质可以持续的从材料中释放到周围环境中,此外,还要保持该材料具有一定的力学强度。 However, with the root dentin matrix as a biological scaffold needs to keep the shape of the root, and will fully demineralized, but not destroy important biologically active substance dentin matrix, can guarantee that these biologically active substances in sustained release of material from the to the surroundings, in addition, also to maintain the material having a certain mechanical strength. 因而,本发明采用从小号到大号、逐渐使用牙科用扩大机将牙齿根管内部扩大,并采用梯度脱矿的方法对牙齿进行脱矿处理。 Accordingly, the present invention employs a small number to a large, gradual expansion machine using a dental tooth root of the inner tube to expand, and a gradient method for the demineralization of teeth demineralization process. 经上述方法,在没破坏牙本质基质中的重要生物活性物质的同时,尽量保持了牙根的形状、保持该材料具有一定的力学强度并且充分脱矿,有利于于牙髓和牙周组织的软组织和硬组织的共同构建,最终构建出具有生物活性的牙根支架材料。 By the method described above, while no destruction of vital dentin biologically active substance matrix, and try to keep the shape of the tooth root, the holding material having a certain mechanical strength and sufficient demineralized conducive pulp and periodontal tissue in soft tissue common Construction and hard tissues, root final construct a bioactive scaffold materials.

[0020] 本发明构建的牙本质基质支架材料的评价:①将处理好的牙本质基质至于培养基中、37°C孵育3天后,培养基清亮、无混浊,证明该材料可以使用,消毒过关,对该培养基进行分析,发现里面含有大量具备成牙相关的蛋白和因子,如TGF-β I、DMP-1、DSPP等。 [0020] Evaluation of dentine matrix scaffold material of the present invention is constructed: ① As dentin matrix sends the processed medium, 37 ° C 3 days of incubation, medium was clear, haze-free, prove that the material can be used, sterilized clearance , the culture medium was analyzed, and found to contain a large number of teeth provided factors and related proteins, such as TGF-β I, DMP-1, DSPP like. ②扫面电镜观察发现材料表面牙本质小管通畅,管间牙本质胶原充分暴露,细胞在材料表面生长良好。 Electron microscopy showed that the material ② scan plane surface smooth dentinal tubules, dentin between fully exposed collagen, the cells grew well on the surface of the material. ③细胞活性检测,发现该材料有利于细胞的增殖和生长。 ③ cell activity was found that the materials facilitate cell growth and proliferation.

[0021] 本发明的生物牙根支架材料的构建方法,可从废弃的人健康牙齿中获得大量的人牙本质基质,建立组织工程技术生物支架材料,充分利用有效资源。 [0021] Construction root biological scaffold material of the present invention can be obtained from discarded human healthy tooth dentin matrix in a large number of people, the establishment of the biological tissue engineering scaffolds, make full use of available resources. 根据本发明的方法,制备的生物牙根支架材料及诱导微环境,经复合细胞处理后可以诱导种子细胞分化为牙骨质、牙周膜、牙本质以及牙髓组织,实现真正意义的生物牙根的重建。 The method according to the present invention, the biological and root scaffolds prepared inductive microenvironment, cells were treated after the composite seed cells can be induced to differentiate into cementum, periodontal ligament, dentin and pulp tissue, root real significance of the biological reconstruction. 与已有其他一些支架材料相比,本材料的构建操作简单容易掌握,安全可行,成本低廉,并且可以提供种子细胞诱导分化的微环境。 Compared with other existing scaffold, construct a simple operation of this material is easy to grasp, is feasible and safe, inexpensive, and may provide seed cells differentiate microenvironment. 此外,该方法中所使用的牙齿均为因各种原因被拔除废弃的牙齿,这大大的节约了医疗资源。 In addition, the method used teeth were abandoned for various reasons, be plucked teeth, which greatly saves health care resources. 因此,该生物牙根支架材料及诱导微环境的制备有广泛的应用前景。 Thus, the biological scaffolds prepared root inductive microenvironment and have broad application prospects.

[0022] 本发明的生物牙根支架材料,首先具备牙根样的外形,更加接近天然牙齿的外形;其次,本发明制备的牙根支架材料中含有大量具备成牙相关的蛋白和因子,如TGF-βΙ、DMP-U DSPP,牙槽骨内部对于细胞、材料的成牙能力具有一定的诱导性,支架材料本身也具有一定的牙向诱导能力,两者相辅相成,可以更好的解决诱导微环境的问题,有利于将该生物牙根支架材料进行牙槽骨内移植。 [0022] Biological root scaffold of the present invention, first provided with root-like shape, closer to the outer shape of a natural tooth; secondly, root scaffold material prepared in the present invention contains a large amount is provided to the dental-related proteins and factors, such as TGF-βΙ , DMP-U DSPP, alveolar bone internal to the cell, the ability of the material to the tooth with a certain inducing scaffold material itself has some ability to induce the teeth, the two complement each other, the problem can be solved better microenvironment induced conducive to the root biological scaffold alveolar bone graft material. 总之,牙本质基质的制备为实现生物牙根的重建提供了新思路,对治疗牙齿缺失具有重要意义,而对牙本质基质的研究及临床应用则需要大量的牙本质基质这种支架材料。 In short, the preparation of dentin to achieve root reconstruction of biological provides a new idea, is important for the treatment of missing teeth, and the research and clinical application of dentin matrix of the dentin matrix that requires a lot of scaffold material.

[0023] 附图说明: [0023] BRIEF DESCRIPTION OF DRAWINGS:

[0024] 图I为本发明实施例I构建的人前牙牙本质基质支架材料; Human nature anterior teeth matrix scaffolds Example I [0024] I FIG constructed embodiment of the present invention;

[0025] 图2为本发明实施例I构建的人前牙牙本质基质支架材料的电镜扫描图; [0025] FIG 2 FIG SEM Example I Construction of human nature anterior teeth matrix scaffold material embodiment of the present invention;

[0026] 图3为本发明实施例I构建的人前牙牙本质基质支架材料的细胞生长图; [0026] FIG 3 Example I Construction of human cells of anterior teeth essentially matrix scaffold material embodiment of the invention FIG growth;

[0027] 图4为本发明实施例I构建的人前牙牙本质基质对细胞活性的影响; [0027] FIG. 4 Effect Example I Construction of human nature anterior teeth stromal cell viability embodiment of the invention;

[0028] 图5、6本发明实施例I构建的人前牙牙本质基质分泌的相关的蛋白和因子; Human nature anterior teeth matrix Example I [0028] Figures 5, 6 constructed embodiment of the present invention secreted proteins and related factors;

[0029] 图7为本发明实施例2构建的人前牙牙本质基质支架材料; [0029] FIG. 72 persons before the teeth of the nature of the matrix scaffold material embodiment of the present invention;

[0030] 图8为本发明实施例2构建的人前牙牙本质基质支架材料的电镜扫描图; [0030] FIG 82 FIG al SEM front teeth essentially constructed matrix scaffold material embodiment of the present invention;

[0031] 图9为本发明实施例2构建的人前牙牙本质基质支架材料的细胞生长图;图10为本发明实施例3构建的人前牙牙本质基质支架材料; Cells anterior teeth human nature matrix scaffold material [0031] Example 2 FIG. 9 constructed embodiment of the present invention FIG growth; 10 anterior teeth nature FIG matrix scaffold material of Example 3 Construction of the embodiment of the invention;

[0032] 图11为本发明实施例3构建的人前牙牙本质基质支架材料的电镜扫描图; [0032] Figure 11 SEM Example 3 Construction of human FIG anterior teeth essentially matrix scaffold material embodiment of the present invention;

[0033] 图12为本发明实施例3构建的人前牙牙本质基质支架材料的细胞生长图。 [0033] FIG 12 FIG Example 3 Construction of cell growth of human nature anterior teeth matrix scaffold material embodiment of the present invention.

具体实施方式 Detailed ways

[0034] 下面结合试验例及具体实施方式对本发明作进一步的详细描述。 [0034] The following examples and binding assay specific embodiments of the present invention will be further described in detail. 但不应将此理解为本发明上述主题的范围仅限于以下的实施例,凡基于本发明内容所实现的技术均属于本发明的范围。 However, this should not be understood that the present invention relating to the above-described range limited to the following examples, all based on the teachings of the present invention are achieved within the scope of the present invention. 实例中操作均在严格无菌环境中进行,为保证细胞不受污染,在所有使用的PBS, DMEM中均加有100U/ml青霉素和100U/ml链霉素。 Example operations are carried out in a strictly sterile environment, in order to ensure against contamination of cells, for use in all PBS, DMEM supplemented with both 100U / ml penicillin and 100U / ml streptomycin.

[0035] 实施例I、人前牙生物牙根支架材料及诱导微环境的制备:[0036] 本实施例列举的生物牙根支架材料的制备方法包括以下步骤: [0035] Example I, the preparation of human biological anterior teeth and root inducing scaffold microenvironment: [0036] The present method for preparing a biological scaffold root exemplified embodiment comprises the steps of:

[0037] (I)取健康前牙四颗,用PBS液冲洗三遍,保存于含有PBS的培养瓶或其他器皿中,封口膜密封瓶口并储存于4°C冰箱; [0037] (I) from healthy four front teeth, washed three times with PBS solution, stored in culture flasks or other containers containing PBS, sealed bottle parafilm and stored a refrigerator at 4 ° C;

[0038] (2)在有喷水装置的牙科用慢速涡轮机上安装金刚砂片,调整转速为3000转/分,使用金刚砂片磨除步骤(I)所得牙齿的牙冠部分,保留牙根部分并保存于含有PBS的培养瓶或其他器皿中,封口膜密封瓶口并储存于4°C冰箱; [0038] (2) mounted with a dental apparatus with a slow water turbine emery sheet, adjust the speed of 3000 rev / min, using emery sheet abrading step (I) The resulting tooth crown portion, and retained root portion stored in bottles or other containers containing culture in PBS, sealed bottle parafilm and stored a refrigerator at 4 ° C;

[0039] (3)使用牙科用拔髓针完整拔出步骤(2)所得牙齿牙根内的牙髓组织,并使用生理盐水反复冲洗根管内部3次,每次约20mL ; [0039] (3) a dental broach complete pull pulling step (2) the resultant tooth pulp tissue in the root, and repeatedly washed with physiological saline three times inside the root canal, each about 20 mL;

[0040] (4)使用牙科用扩大机器对步骤(3)所得牙齿进行牙齿内部处理,首先使用15号牙科手用普通镍钛锉扩大牙齿根管内部,20号普通镍钛锉扩大牙齿内部,然后从小号到大号使用Sx号牙科用扩大针扩大牙齿根管口部分,即依次用SI号、S2号、Fl号、F2号牙科用扩大针扩大牙齿根管口部分,并反复使用10%的EDTA溶液冲洗根管内部3次,每次IOmL ; [0040] (4) a dental expansion machine step (3) the resulting tooth for dental internal processing, first using 15 dental hand expand inside the tooth root canal by conventional NiTi files, number 20 an ordinary nickel-titanium file expanded inside the tooth, then small to large in number Sx use for expanding the dental root canal reamer port portion, i.e., sequentially in SI No. No. S2, Fl number, the F2 expanded dental root canal reamer with the port portion, and repeated using 10% EDTA solution rinse the inner tubes 3 times IOmL;

[0041] (5)使用牙科用涡轮机对步骤(4)所得的牙齿按照前牙牙根外形磨除部分牙齿组织,以去除牙周组织和牙骨质等组织。 [0041] (5) dental step (4) grinding the resulting tooth part of the tooth root tissues other anterior teeth in accordance with the outer shape of the turbine, to remove cementum and periodontal tissue and other tissues. 最终得到的材料厚度1mm,长度I. 5cm ; Finally obtained material thickness 1mm, length I. 5cm;

[0042] (6)将步骤(5)处理好的牙齿保存于PBS缓冲液中,并于超声震荡器中处理4次,每次处理IOmin ; [0042] (6) The step (5) Handled teeth were stored in PBS buffer and treated in an ultrasonic shaker four times, each treatment IOmin;

[0043] (7)在转速为100转/分的磁力搅拌器中,使用不同浓度的EDTA对牙本质进行梯度脱矿,首先使用15%的EDTA溶液脱矿处理5min,然后使用10%的EDTA溶液脱矿处理5min,最后使用5%的EDTA溶液脱矿处理5min ; Magnetic stirrer [0043] (7) at a speed of 100 rev / min, different concentrations of EDTA gradient demineralized dentin, first using 15% EDTA solution in demineralized treated 5min, then a 10% EDTA was treated demineralized 5min, and finally with 5% EDTA solution treated demineralized 5min;

[0044] 将步骤(7)处理好的牙本质基质保存于含有双抗(青霉素浓度为100unit/ml,链霉素浓度为100unit/ml)的PBS缓冲液中,使用时将上述所制作的牙本质基质按照临床要求和实际应用进行使用即可。 PBS buffer [0044] The step (7) stored in the Handled dentin matrix containing an anti-bis (concentration of penicillin 100unit / ml, streptomycin at a concentration of 100unit / ml) of, when using the above prepared tooth to be used according to the nature of the substrate requirements of the clinical and practical use.

[0045] 本实施例构建的牙本质基质支架材料的评价: Dentin matrix scaffold constructed in embodiment [0045] The present evaluation:

[0046] 处理好的牙本质基质至于DMEM培养基中、37°C孵育3天后,检验发现培养基清 [0046] As substrate dentin Handled DMEM medium, 37 ° C 3 days incubation, the medium clearance test showed

亮、无混浊,证明该材料可以使用,消毒过关。 Bright, no turbidity, to prove that the material can be used, sterilized clearance. 收集上述浸泡材料的培养基,并对该培养基进行分析,采用ELISA试剂盒检测相关蛋白和因子。 The immersing material collection medium, and the medium was analyzed using ELISA kit factors and related proteins. 结果发现培养基里面含有大量具备成牙相关的蛋白和因子,如TGF-β I、DMP-1、DSPP等,而未浸泡过材料的培养基中无上述相关蛋白和因子。 The results showed that the medium is provided which contains a lot of proteins related to the teeth and factors, such as TGF-β I, DMP-1, DSPP, etc., without the medium material soaked no such factors and related proteins.

[0047] ②将本实施例所构建的牙本质基质支架材料置于扫面电镜观察下观察,发现材料表面牙本质小管通畅,管间牙本质胶原充分暴露,细胞在材料表面生长良好。 [0047] ② The dentin matrix scaffolds constructed embodiment of the present embodiment is placed under the scan surface electron microscope observation, it was found the material surface smooth dentinal tubules, dentin collagen between fully exposed cells growing well on the material surface.

[0048] ③细胞活性检测,采用CCK-8实验方法,即收集使用了该材料进行培养的细胞和未使用该材料、只是用正常培养基培养的细胞,制备成细胞悬液,接种在96孔板中,每板IOOy 1,同时加入IOy 1CCK-8溶液,37°C孵育2小时后在450nm处使用酶标仪检测吸光度,检测细胞活性。 [0048] ③ cell activity, using experimental methods CCK-8, i.e., the material is collected using cultured cells and the material is not used, except that the normal cell culture medium to prepare a cell suspension were seeded in 96-well plate, each plate IOOy 1, while adding IOy 1CCK-8 solution, 37 ° C for 2 hours after the absorbance was measured using a microplate reader at 450nm, detection of cellular activity. 发现该材料有利于细胞的增殖和生长。 The material is found to facilitate cell growth and proliferation.

[0049] 实施例2、人前磨牙生物牙根支架材料及诱导微环境的制备: [0049] Example 2, preparation of biological material and scaffold root microenvironment induced human premolars:

[0050] 本实施例列举的生物牙根支架材料的制备方法包括以下步骤: [0050] The preparation method of biological scaffold root embodiments recited embodiment comprises the steps of:

[0051] ( I)取前磨牙四颗,用PBS液冲洗三遍,保存于含有PBS的培养瓶或其他器皿中,封口膜密封瓶口并储存于4°C冰箱。 [0051] (I) before taking four teeth, washed three times with PBS solution, stored in culture flasks or other containers containing PBS, sealed bottle parafilm and stored at 4 ° C refrigerator. [0052] (2)在有喷水装置的情况下,在牙科用慢速涡轮机上安装金刚砂片,调整转速6500转/分,使用金刚砂片磨除步骤(I)牙冠部分,保留牙根部分并保存于含有PBS的培养瓶或其他器皿中,封口膜密封瓶口并储存于4°C冰箱。 [0052] (2) In the case where there are water jet means, mounted on the dental turbine slow emery sheet, adjust the speed 6500 rev / min, using emery sheet abrading step (I) part of the crown, and retained root portion stored in bottles or other containers containing culture in PBS, sealed bottle parafilm and stored at 4 ° C refrigerator.

[0053] (3)使用牙科用拔髓针完整拔出步骤(2)所得牙齿牙根内牙髓组织,并使用生理盐水反复冲洗根管内部5次,每次约15ml。 [0053] (3) a dental broach complete pull pulling step (2) the resulting root tooth pulp tissue, physiological saline and repeatedly washed 5 times the internal tubes, each about 15ml.

[0054] (4)使用牙科用扩大机器进行牙齿内部处理,首先使用15号牙科手用普通镍钛锉扩大牙齿根管内部,20号普通镍钛锉扩大牙齿内部,然后从小号到大号使用Sx号牙科用扩大针扩大牙齿根管口部分,即依次用SI号、S2号、Fl号、F2号牙科用扩大针扩大牙齿根管口部分,并反复使用10%的EDTA溶液冲洗根管内部5次,每次20mL ; [0054] (4) internal teeth for dental treatment with expanding machine 15 is first used to expand the interior of a dental hand root canal file with an ordinary nickel-titanium, nickel-titanium file 20 common to expand inside the tooth, and then small to large in use Sx No. dental expanded by expanding the needle root canal opening portion, i.e., sequentially in SI No. No. S2, Fl number, F2 on dental enlarged tooth root canal orifice portion with a reamer, and repeatedly using 10% EDTA solution, rinse the inside of root canal 5 times, 20 mL each time;

[0055] (5)使用牙科用涡轮机对步骤(4)所得的牙齿按照前磨牙牙根外形磨除部分牙根组织,以去除牙周组织和牙骨质等组织。 [0055] (5) a dental turbine in step (4) obtained according premolar tooth root profile grinding root tissues other part, in order to remove cementum and periodontal tissue and other tissues. 最终得到的材料厚度I. 5mm,长度I. 5cm ; The final thickness of the material obtained I. 5mm, length I. 5cm;

[0056] (6)将步骤(5)处理好的牙齿保存于PBS缓冲液中,并于超声震荡器中处理4次, 每次处理20min。 [0056] (6) The step (5) Handled teeth were stored in PBS buffer and treated in an ultrasonic shaker four times, each treatment 20min.

[0057] (7)在转速为200转/分的磁力搅拌器中,使用不同浓度的EDTA进行梯度脱矿,20%的EDTA溶液脱矿处理5min,然后使用12%的EDTA溶液脱矿处理5min,最后使用5%的EDTA溶液脱矿处理5min。 [0057] (7) at a speed of 200 rev / min on a magnetic stirrer, using different concentrations of EDTA demineralized gradient, 20% EDTA solution and demineralized for 5min, then 12% EDTA solution for 5min demineralization Finally, the use of a 5% EDTA solution demineralization process 5min.

[0058] 将步骤(7)处理好的牙本质基质保存于含有双抗(青霉素浓度为100imit/ml,链霉素浓度为100unit/ml)的PBS中,使用时将上述所制作的牙本质基质按照临床要求和实际应用进行使用即可。 [0058] The step (7) stored in the Handled dentin matrix containing an anti-bis (penicillin concentration 100imit / ml, streptomycin at a concentration of 100unit / ml) in PBS, when produced using the above-described dental matrix It can be carried out in accordance with the clinical use requirements and practical application.

[0059] 本实施例构建的牙本质基质支架材料的评价: [0059] dentin matrix scaffold material embodiment of the present embodiment constructed Evaluation:

[0060] 处理好的牙本质基质至于DMEM培养基中、37°C孵育3天后,检验发现培养基清亮、无混浊,证明该材料可以使用,消毒过关。 [0060] As substrate dentin Handled DMEM medium, 37 ° C 3 days incubation, media test showed clear, haze-free, prove that the material can be used, sterilized clearance.

[0061] ②将本实施例所构建的牙本质基质支架材料置于扫面电镜观察下观察,发现材料表面牙本质小管通畅,管间牙本质胶原充分暴露,细胞在材料表面生长良好。 [0061] ② The dentin matrix scaffolds constructed embodiment of the present embodiment is placed under the scan surface electron microscope observation, it was found the material surface smooth dentinal tubules, dentin collagen between fully exposed cells growing well on the material surface.

[0062] 实施例3、人磨牙(多根牙)生物牙根支架材料及诱导微环境的制备: [0062] Example 3, Preparation of (multi-rooted teeth) Root biological scaffold and human molars inductive microenvironment:

[0063] 本实施例列举的生物牙根支架材料的制备方法包括以下步骤: [0063] The preparation method of biological scaffold root embodiments recited embodiment comprises the steps of:

[0064] (I)取健康磨牙四颗,用PBS液冲洗三遍,保存于含有PBS的培养瓶或其他器皿中,封口膜密封瓶口并储存于4°C冰箱; [0064] (I) from healthy four teeth, washed three times with PBS solution, stored in culture flasks or other containers containing PBS, sealed bottle parafilm and stored a refrigerator at 4 ° C;

[0065] (2)在有喷水装置的牙科用慢速涡轮机上安装金刚砂片,调整转速为4000转/分,使用金刚砂片磨除步骤(I)所得牙齿的牙冠部分,保留牙根部分并保存于含有PBS的培养瓶或其他器皿中,封口膜密封瓶口并储存于4°C冰箱; [0065] (2) mounted with a dental apparatus with a slow water turbine emery sheet, adjust the speed of 4000 rev / min, using emery sheet abrading step (I) The resulting tooth crown portion, and retained root portion stored in bottles or other containers containing culture in PBS, sealed bottle parafilm and stored a refrigerator at 4 ° C;

[0066] (3)使用牙科用拔髓针完整拔出步骤(2)所得牙齿牙根内的牙髓组织,并使用生理盐水反复冲洗根管内部7次,每次约20mL ; [0066] (3) a dental broach complete pull pulling step (2) the resultant tooth pulp tissue in the root, and repeatedly washed using saline inner tubes 7, each about 20 mL;

[0067] (4)使用牙科用扩大机器对步骤(3)所得牙齿进行牙齿内部处理,首先使用15号牙科手用普通镍钛锉扩大牙齿根管内部,20号普通镍钛锉扩大牙齿内部,然后从小号到大号使用Sx号牙科用扩大针扩大牙齿根管口部分,即依次用SI号、S2号、Fl号、F2号牙科用扩大针扩大牙齿根管口部分,并反复使用10%的EDTA溶液冲洗根管内部7次,每次20mL ; [0067] (4) a dental expansion machine step (3) the resulting tooth for dental internal processing, first using 15 dental hand expand inside the tooth root canal by conventional NiTi files, number 20 an ordinary nickel-titanium file expanded inside the tooth, then small to large in number Sx use for expanding the dental root canal reamer port portion, i.e., sequentially in SI No. No. S2, Fl number, the F2 expanded dental root canal reamer with the port portion, and repeated using 10% EDTA solution rinse the inside of root canal 7 times 20 mL;

[0068] (5)使用牙科用涡轮机对步骤(4)所得的牙根按照磨牙牙根外形磨除部分牙齿组织,以去除牙周组织和牙骨质等组织。 [0068] (5) a dental turbine in step (4) grinding the resulting root part of the tooth root tissues other teeth shape according to remove cementum and periodontal tissue and other tissues. 最终得到的材料厚度1_,长度Icrn ; 1_ resulting material thickness, length ICRn;

[0069] (6)将步骤(5)处理好的牙齿保存于PBS缓冲液中,并于超声震荡器中处理4次,每次处理20min ; [0069] (6) The step (5) Handled teeth were stored in PBS buffer and treated in an ultrasonic shaker four times, each treatment 20min;

[0070] (7)在转速为300转/分的磁力搅拌器中,使用不同浓度的EDTA对牙本质进行梯度脱矿,首先使用20%的EDTA溶液脱矿处理lOmin,然后使用10%的EDTA溶液脱矿处理lOmin,最后使用5%的EDTA溶液脱矿处理IOmin ; Magnetic stirrer [0070] (7) at a speed of 300 rev / min, different concentrations of EDTA gradient demineralized dentin, first using 20% ​​EDTA solution in demineralized treated lOmin, then a 10% EDTA was treated demineralized lOmin, and finally 5% EDTA solution in demineralized treated IOmin;

[0071] 将步骤(7)处理好的牙本质基质保存于含有双抗(青霉素浓度为100unit/ml,链霉素浓度为100unit/ml)的PBS缓冲液中,使用时将上述所制作的牙本质基质按照临床要求和实际应用进行使用即可将上述所制作的牙本质基质按照临床要求和实际应用进行使用即可。 PBS buffer [0071] The step (7) stored in the Handled dentin matrix containing an anti-bis (concentration of penicillin 100unit / ml, streptomycin at a concentration of 100unit / ml) of, when using the above prepared tooth the nature of the substrate used in accordance with clinical requirements and the practical application of the above can be produced using the dental matrix can be performed according to the clinical requirements and practical applications.

[0072] ①将处理好的亚本质基质至于培养基中、37°C孵育3天后,培养基清亮、无混浊,证明该材料可以使用,消毒过关。 [0072] ① sends the processed sub-matrix As for the nature of the medium, 37 ° C 3 days of incubation, medium was clear, haze-free, prove that the material can be used, sterilized clearance. ②扫面电镜观察发现材料表面牙本质小管通畅,管间牙本质胶原充分暴露,细胞在材料表面生长良好。 Electron microscopy showed that the material ② scan plane surface smooth dentinal tubules, dentin between fully exposed collagen, the cells grew well on the surface of the material.

Claims (3)

  1. 1. 一种生物牙根支架材料的构建方法,其特征在于包括以下步骤: (1)将供体新鲜的牙齿在无菌状态下,用PBS缓冲液冲洗; (2)将步骤(I)所得牙齿在有喷水装置的牙科用慢速涡轮机上磨除牙冠部分,保留牙根部分,所述牙科用慢速涡轮机的转速为1000-10000转/分,所得牙根部分保存于含有PBS缓冲液的培养瓶或其他器皿中; (3)用牙科用拔髓针拔出步骤(2)所得牙齿牙根内的牙髓组织,用生理盐水反复冲洗牙齿根管内部3-8次,每次10-30晕升; (4)使用牙科用扩大机器将步骤(3)所得牙齿内部逐渐扩大,去除牙髓组织和前期牙本质组织,此过程中使用5%-17%的EDTA溶液反复冲洗根管内部3-8次,每次10-30毫升; (5)使用牙科用涡轮机按照生物牙根外形磨除部分组织,以去除牙周组织和牙骨质组织,得到厚度为l_3mm、长度1-2. 5cm的支架材料; (6)将步骤(5)处理好的支架材料 1. A method of constructing a biological root scaffold material, comprising the steps of: (1) donor fresh tooth under sterile conditions, washed with PBS buffer; (2) the step (I) obtained teeth in dental apparatus there is a water abrade the crown portion slow turbine, retained root portion, said slow speed dental turbine 1,000 to 10,000 rev / min, the root portion of the resulting buffer stored in PBS containing culture bottle or other vessel; (3) with a dental broach pulled pulling step (2) the resultant tooth pulp tissue in the root, a tooth root of the inner tube repeatedly washed with saline 3-8 times 10-30 halo l; (4) a dental expansion machine step (3) the resulting internal teeth gradually expanded, and removal of pulp tissue predentin tissue, this procedure using 5% to 17% EDTA solution repeatedly washed inside the root canal 3- 8 times, each time 10-30 ml; (5) a dental turbine according to profile abrading tooth root portion biological tissue to remove cementum and periodontal tissue organization, a thickness of l_3mm, 1-2 5cm length of the stent. material; (6) the step (5) for a good scaffold 存于PBS缓冲液中,于超声震荡器中处理3-8次,每次处理10_30min ; (7)在转速为100-500转/分的磁力搅拌器中,使用不同浓度的EDTA对牙本质进行梯度脱矿,得到生物牙根支架材料。 In PBS buffer, treated in an ultrasonic shaker 3-8 times, each treatment 10_30min; (7) 100-500 rev / min on a magnetic stirrer, using different concentrations of EDTA in the speed of dentin gradient demineralization, to obtain root biological scaffold.
  2. 2.根据权利要求I所述的生物牙根支架材料的构建方法,其特征在于: 步骤(4)中所述使用牙科用扩大机器将步骤(3)所得牙齿内部逐渐扩大的方法为,首先使用15号牙科手用普通镍钛锉扩大牙齿根管内部,20号普通镍钛锉扩大牙齿根管内部,然后从小号到大号使用Sx号牙科用扩大针扩大牙齿根管口部分,即依次用SI号、S2号、Fl号、F2号牙科用扩大针扩大牙齿根管口部分。 The method of constructing a biological root I of the scaffold material as claimed in claim wherein: in step (4) said expanded machine using dental Step (3) The method of gradually increasing the internal teeth is obtained, using the first 15 No. dental root canal hand expanded internal ordinary NiTi files, NiTi file 20 common to expand inside the tooth root canal, and then small to large in number Sx using expanded dental root canal reamer with the port portion, i.e. sequentially with SI No. No. S2, Fl number, F2 on the dental root canal expanded with the expansion of needle port portion.
  3. 3.根据权利要求I或2所述的生物牙根支架材料的构建方法,其特征在于:步骤(7)中所述梯度脱矿的方法为,首先使用17%-25%的EDTA溶液脱矿处理5_15min,然后使用7%-15%的EDTA溶液脱矿处理5-15min,最后使用5%的EDTA溶液脱矿处理5_15min。 The root I or construct biological scaffold material according to claim 2, wherein: the step (7) in the demineralization of a gradient method, first using 17% -25% of the demineralization process EDTA solution 5_15min, then 7% -15% EDTA solution demineralization process 5-15min, and finally with 5% EDTA solution demineralization process 5_15min.
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