CN111407755A - Use of retinoic acid in tooth development - Google Patents

Abstract

The invention relates to the field of biomedicine, and discloses application of retinoic acid and a retinoic acid inhibitor in preparation of a tooth treatment drug, wherein the retinoic acid can promote pharyngeal tooth mineralization, and the retinoic acid inhibitor can inhibit proliferation and migration of cranial nerve spine cells, so that regulation and control of early development of teeth are realized. The retinoic acid and the inhibitor thereof can be used as a novel tooth regeneration drug to be applied to clinical practice.

Description

Application of retinoic acid in tooth development

technical field

The invention relates to the technical field of biomedicine, in particular to the application of retinoic acid in the preparation of a tooth regeneration medicine.

Background technique

Teeth are the hardest organs in the human body, and they are also the organs that cannot be regenerated after injury. Since the teeth are located at the forefront of the digestive tract, chewing and grinding food is conducive to further digestion and absorption of food. Therefore, once the teeth are damaged, it will greatly affect the intake of nutrients and thus affect the normal operation of the body's organs. Tooth loss due to dental caries, periodontitis and trauma will seriously reduce people's quality of life and even affect the health of the whole body. Therefore, the repair of tooth damage is a topic that researchers have been diligently studying. At present, the restoration of tooth damage mainly relies on the filling and covering of biological materials such as resin and implant restoration after tooth loss. These restoration methods can quickly restore tooth function, but there are some drawbacks, such as high requirements for oral environment and technical sensitivity of clinicians. Large differences and other factors will lead to uneven restoration effects, poor sealing of the restoration edges, loosening and falling off, which further aggravates the adverse consequences of tooth damage and chronic oral inflammation. Therefore, seeking a safer and more reliable method for the restoration of missing teeth—tooth regeneration is the direction explored by all dental workers.

SUMMARY OF THE INVENTION

The present invention is intended to provide the application of retinoic acid in the preparation of tooth regeneration medicine, so as to realize tooth regeneration, and solve the problems of poor sealing edge of the restoration body and looseness and shedding in the conventional restoration methods in the prior art, which further aggravate tooth damage and chronic oral inflammation. And other issues.

In order to achieve the above purpose, the present invention adopts the following technical scheme: the application of retinoic acid in the preparation of tooth regeneration medicine.

The principle and advantages of this scheme are as follows: retinoic acid is the main active metabolite of vitamin A, and its synthesis is mainly by oxidation of retinol, the alcohol mode of vitamin A, to retinal by retinol dehydrogenase, and then by retinol. Xanthaldehyde is oxidized to retinoic acid by retinal dehydrogenase; it is degraded by cytochrome P450 isoenzyme; retinoic acid acts by binding to two nuclear receptors RAR and RXR, and RAR and RXR form a heterodimer in It binds to DNA at the retinoic acid reaction element (RARE). When retinoic acid signaling is not required for organ development, RAR and RXR dimers recruit co-repressors to inhibit the expression of target genes. When retinoic acid signaling is sufficient again, the receptor dimer and repressor depolymerize and reconcile Retinoic acid binding promotes the transcription of downstream target genes to regulate the development of various organs.

In the process of exploring the effect of retinoic acid on tooth development, the inventors accidentally discovered that retinoic acid signaling can affect the expression of genes related to tooth regeneration, mainly affecting the mineralization of teeth and regulating the proliferation and migration of cranial nerve spine cells. realized. The inventors used in situ hybridization, alizarin red staining, confocal microscopy detection, construction of retinoic acid-degrading enzyme and synthetase overexpression fish, and used specific injury and embryonic heat shock to verify and evaluate the effect of retinoic acid on tooth mineralization. , the proliferation and migration of cranial nerve spine cells, discovered the new role of retinoic acid, broadened the application scope of retinoic acid, and provided a theoretical basis for the research and development of tooth regeneration and restoration drugs.

Preferably, as an improvement, the tooth regeneration drug further includes a retinoic acid inhibitor, and the retinoic acid inhibitor acts after retinoic acid.

Using the above technical solution, the applicant accidentally discovered in the research process that the detection results of the pharyngeal tooth-specific marker gene expression showed that the expression of genes in the retinoic acid treatment group was slightly stronger than that in the control group, and there was no related gene expression in the inhibitor treatment group, indicating that Compared with functional enhancement, functional weakening has a greater impact on pharyngeal tooth development, and retinoic acid inhibitors affect subsequent tooth development by attenuating the expression of pharyngeal tooth-specific marker genes after tooth mineralization. Through the successive effects of retinoic acid and retinoic acid inhibitors, the purpose of promoting tooth development and regeneration is achieved.

By adopting the above technical scheme, the use of exogenous all-trans retinoic acid to explore the regulation effect of retinoic acid on tooth development is convenient, quick and easy to operate. Compared with other endogenous exploration methods such as constructing transgenic animals, the time required is short, and exogenous retinoic acid is safe and non-toxic, and can be purchased directly from biological companies.

By adopting the above technical scheme, using diethylaminobenzaldehyde to inhibit retinoic acid signal so as to explore the regulation effect of retinoic acid on the tooth development process is convenient, quick and easy to operate. Compared with other endogenous exploration methods such as the construction of transgenic animals and gene knockout, the time required is short, and the appropriate concentration of treatment solution can be prepared according to actual needs to achieve different degrees of inhibition of retinoic acid signals, and can be directly obtained from biological Company purchase, convenient and easy to operate.

Preferably, as an improvement, the teeth are pharyngeal teeth, and retinoic acid and its inhibitor are prepared into pharmaceutically acceptable dosage forms according to conventional pharmaceutical preparation methods.

By adopting the above technical scheme, pharyngeal teeth are also called gill teeth, hypopharyngeal teeth and pharyngeal teeth. Teeth on the gill arches in bony fishes. For species with well-developed pharyngeal teeth, the jaw teeth are not developed; on the contrary, for species with well-developed jaw teeth, the pharyngeal teeth are poorly developed or not. The objects in this experiment are all pharyngeal teeth of zebrafish, which are more representative and can provide a reference for the subsequent research on the impact of holopoda. By preparing the retinoic acid and its inhibitor into different dosage forms, a suitable pharmaceutical dosage form can be reasonably selected according to different administration modes, the application scope is wide, and the selection space is large.

Preferably, as an improvement, the application of retinoic acid as a pharyngeal tooth mineralization accelerator.

Using the above technical solution, the effect of retinoic acid on tooth regeneration is to improve the ability of tooth regeneration by promoting tooth mineralization. The results of the inventor's alizarin red staining experiment showed that the zebrafish pharyngeal tooth mineralization after retinoic acid treatment enhanced.

Preferably, as an improvement, the retinoic acid inhibitor is the use of a cranial nerve spine cell migration inhibitor.

Using the above technical scheme, the migration of cranial nerve spinous cells has an important relationship with tooth development. After retinoic acid inhibitor treatment, the expression of the gene snail1a related to the epithelial-mesenchymal transition process of cranial nerve spinous cells has no significant change, while the cranial nerve spines The expression of cell migration-related gene cad is weakened, that is, retinoic acid inhibitor has the effect of inhibiting the migration of cranial nerve spine cells, and can be used as a cranial nerve spine cell migration inhibitor to realize the regulation of early tooth development.

Preferably, as an improvement, the retinoic acid inhibitor is the use of a cranial nerve spinous cell proliferation inhibitor.

Using the above technical scheme, the migration of cranial nerve spinous cells has an important relationship with tooth development. After retinoic acid inhibitor treatment, the expression of the gene snail1a related to the epithelial-mesenchymal transition process of cranial nerve spinous cells has no significant change, while the cranial nerve spines The expression of cell proliferation-related gene cad is weakened, that is, retinoic acid inhibitor has the effect of inhibiting the proliferation of cranial nerve spine cells, and can be used as a cranial nerve spine cell proliferation inhibitor to realize the regulation of early tooth development.

Preferably, as an improvement, the concentration of retinoic acid to promote the mineralization of pharyngeal teeth is: ≥3×10 ^-7 M.

Using the above technical scheme, after treatment with 3×10 ^-7 M retinoic acid, 5dpf embryos were collected, fixed and then stained with alizarin red, and the mineralization of pharyngeal teeth was significantly enhanced.

Preferably, as an improvement, the concentration of the retinoic acid inhibitor for inhibiting the migration of cranial nerve spine cells and the proliferation of cranial nerve spine cells is: 0.5×10 ^-7 M-1×10 ^-7 M.

Using the above technical scheme, when the concentration of retinoic acid inhibitor was 0.5×10 ^-7 M-1×10 ^-7 M, the results of in situ hybridization detection of zebrafish embryos treated with retinoic acid inhibitor showed that 48hpf and 56hpf dorsal There was no significant difference in the expression of the gene snail1a in lateral and lateral view, and only slightly decreased in lateral view at 72hpf; however, the expression of cad was significantly attenuated after 48hpf.

Description of drawings

Fig. 1 is the result of in situ hybridization of gene pitx2 after RA and DEAB treatment in the embodiment of the present invention 1;

Fig. 2 is the result of in situ hybridization of gene fth1b after RA and DEAB treatment in Example 1 of the present invention;

3 is the result of alizarin red staining at 5dpf after RA and DEAB treatment in Example 1 of the present invention;

Fig. 4 is the expression pattern diagram of snail1a after DEAB treatment in Example 2 of the present invention;

Fig. 5 is the cad expression pattern diagram after DEAB processing in the embodiment of the present invention 2;

Fig. 6 is the zebrafish Tg (dlx2b: Dendra2-NTR; hsp: aldh1a2-GFP) (a) and Tg (dlx2b: Dendra2-NTR; hsp: cyp26b1-GFP) (b) after pharyngeal tooth injury in Example 3 of the present invention Alizarin red staining results at 48h of regeneration;

Fig. 7 is the zebrafish Tg (dlx2b: Dendra2-NTR; hsp: aldh1a2-GFP) and Tg (dlx2b: Dendra2-NTR; hsp: cyp26b1-GFP) 24h and 48h confocal regeneration after pharyngeal tooth injury in Example 3 of the present invention Microscope observation results.

Detailed ways

Reagent Description:

Example 1: Pharyngeal tooth development in zebrafish treated with exogenous retinoic acid and its inhibitors

Experimental animals: Using zebrafish as a model animal, all zebrafish breeding and breeding strictly follow the standard experimental conditions stipulated by the regulations of the Experimental Animal Management Committee. The zebrafish embryos used in the experiment were soaked in 0.003% PTU to inhibit the production of melanin for easy observation.

Experimental reagents: The reagents used in the experiment include all-trans retinoic acid (RA), exogenous 4-diethylaminobenzaldehyde (DEAB), in situ hybridization related reagent (Roche), chromogenic solution (Roche), 0.5% Alizarin Red staining solution.

Experimental method: RA and DEAB powders were dissolved in 100% DMSO, respectively, and the final concentration of 1×10 ^-3 M was prepared as a stock solution and stored at -20°C. During embryo treatment, 3μl DEAB and 9ul RA stock solutions were added to each 30ml egg water to prepare treatment solutions with a final concentration of 1×10 ^-7 M and 3×10 ^-7 M, and the embryos were immersed in the treatment solution for a drug treatment time of 24hpf-36hpf. Control embryos were treated with 0.2% DMSO.

Whole embryo in situ hybridization:

(1) Collect 48hpf and 72hpf embryos after drug treatment and 48hpf and 72hpf embryos in DMSO control group, 20 embryos per tube, and fix overnight at 4% PFA at 4°C.

(2) Dehydrate the collected embryos, rinse 5 times with 100% anhydrous methanol for 30 min each time, and store the dehydrated embryos at -20°C overnight.

(3) The embryos were taken out and rehydrated. The rinsing solution was 75% anhydrous methanol/25% 1×PBT, 50% anhydrous methanol/50% 1×PBT, 25% anhydrous methanol/75% 1×PBT, 100% 1×PBT, each 1ml solution was rinsed at room temperature for 5min.

(4) Embryos were digested. After 1ml of 100% 1×PBT was rinsed for 5 minutes, proteinase K was prepared into a final concentration of 10ug/ml of digestion solution with 1×PBT solution, and 1ml of digestion solution was added to each tube. The corresponding periods were treated slowly (48hpf embryos were digested for 20min, 56hpf embryos were digested for 25min, and 72hpf embryos were digested for 30min).

(5) Rinse twice with 1 ml of 1×PBT solution for 5 min each time to remove residual proteinase K, fix the embryos with 1 ml of 4% PFA for 30 min, and then rinse 5 times with 1 ml of 1× PBT solution for 5 min each time.

(6) Hyb buffer was preheated in advance. After rinsing the embryos with 1ml of 50% 1×PBT/50% Hyb buffer, add 1ml of preheated Hyb buffer, and pre-hybridize in a 68.5°C water bath for 3-5h.

(7) Remove Hyb buffer, add preheated probe hybridization solution (200 μl Hyb buffer adds 3 μl probe), 200 μl per tube, hybridize overnight in 68.5°C water bath.

(8) The probes were recovered on the second day and stored at -20°C.

(9) Rinse the embryos in a 68.5°C water bath. The rinsing solution is preheated in advance, followed by 100% Hybbuffer, 25% 2×SSCT/75% Hyb buffer, 50% 2×SSCT/50% Hyb buffer, 75% Hyb buffer 2 × SSCT/25% Hyb buffer, 100% 2 × SSCT, each buffer was rinsed for 10 min, followed by 4 washes with pre-warmed 0.2 × SSCT, 15 min each time.

(10) Rinse the embryos at room temperature in the order of 75% 0.2×SSCT/25%1×MABT, 50%0.2×SSCT/50%1×MABT, 25%0.2×SSCT/75%1×MABT , 100% 1×MABT, each rinse once, each 1ml rinse for 5min.

(11) After rinsing, add 1 ml of 1× blocking solution to each tube of embryos and seal them for 2 hours at room temperature on a shaker. (12) Remove the 1× blocking solution. After the 1× blocking solution and the anti-Dig-AP antibody are prepared at a ratio of 1:1000, add 200 μl to each tube and incubate overnight at 4°C with a shaker.

(13) The secondary antibody is recovered on the third day, stored at 4°C, and can be reused twice.

(14) Rinse the embryos 8 times with 1ml 1×MABT solution for 15min each time. (15) The embryos were rinsed three times with freshly prepared NTMT solution for 5 minutes each time, and then the embryos were transferred to a 24-well plate, and each embryo was marked.

(16) According to the ratio of adding 20μl NBT/BCIP to each 1ml NTMT solution, prepare the color developing solution, add 500 μl color developing solution to each well, and develop color at 37°C in the dark. According to the working conditions of the probe, observe the color development under the microscope every 20 minutes. .

(17) When there is no significant difference in the color development status of the embryos after two consecutive observations, the color development can be stopped, the color development solution can be removed, and the 1ml stop solution can be rinsed 3 times for 5 minutes each time. After that, the embryos can be stored at 4°C and collected at selected times. image.

Alizarin red staining:

(1) Collect one tube of 5dpf stage embryos after drug treatment and control group, 20 embryos in each tube, and fix them with 1 ml of 4% PFA overnight at 4°C.

(2) Rinse twice with 1×PBST solution for 5 min each time, then peel off the embryonic heart and liver tissue under a microscope to expose the pharyngeal teeth completely.

(3) Dehydrate the embryos with 50% ethanol for 30 minutes at room temperature.

(4) 1 ml of staining solution was added to stain overnight in the dark at room temperature. The staining solution was 1 ml of 1×PBST/20ul of 0.05% alizarin red.

Zebrafish embryos were treated with RA and DEAB at 24hpf-36hpf. After the treatment was stopped, 48hpf, 56hpf and 72hpf embryos were collected, fixed and dehydrated, and used for the detection of whole embryo in situ hybridization. Here we select the probe for zebrafish pharynx Tooth-specific marker genes pitx2 and fth1b, the probes were provided by Zheng Xuedan, and it was verified that the probes work normally.

As shown in the in situ hybridization results shown in Figure 1 and Figure 2, in the 3×10 ^-7 M RA treatment group, the expression of the pharyngeal tooth marker gene pitx2 was not significantly different from the control group at 48hpf and 72hpf, while at 1 In ×10 ^-7 M DEAB treatment group, there was no pitx2 expression at 48hpf and 72hpf; in 3 × 10 ^-7 M RA treatment group, at 56hpf 72hpf, the expression of fth1b was not significantly different from the control group, 1×10 ^-7 In the M DEAB treatment group, no expression of fth1b was found at 56 hp and 72 hpf.

As shown in Figure 3, after drug treatment of embryos at 24hpf-36hpf, 5dpf embryos were collected and fixed for Alizarin Red staining. Alizarin red staining mainly stains mineralized tissues including teeth and bones. The results of alizarin red staining showed that in the control group, strong 4V ¹ mineralization and weak 5V ¹ cusp mineralization were seen, and in the 3×10 ^-7 M RA treatment group, the mineralization of the pharyngeal teeth of zebrafish Stronger than the control group, the mineralization of 4V ¹ was stronger in the RA group, and stronger cusp mineralization was seen in the 3V ¹ and 5V ¹ than the control group, while the 1×10 ^-7 MDEAB treatment group did not find the existence of mineralized pharyngeal teeth.

The above results indicate that retinoic acid can affect the development of pharyngeal teeth by affecting the expression of pitx2 and fth1b genes, and retinoic acid signaling can promote the mineralization of pharyngeal teeth.

Example 2: Changes in neural spine cell-related gene expression after retinoic acid signal inhibition

The PCR primer synthesis and DNA sequencing used in the experiment were completed by Beijing Liuhe Huada Biological Co., Ltd. TRIZOL (Life Technologies), chloroform (Sangon), isopropanol (Sangon), reverse transcription kit (Roche), rTaq DNA polymerase (TaKaRa), pGEM@T Easy Vector kit (Promega), DH5α competent strain (Invitrogen) ), agrose (Invitrogen), Gel extraction kit (Qiagen), DIG RNA labeling Kit (Roche), T7 RNA polymerase (Roche), DNase I enzyme (New England Biolabs), and other reagents were the same as in Example 1.

Total RNA was extracted from zebrafish embryos and cDNA and probes were synthesized. After purifying the probes, in situ hybridization experiments were performed. After 24hpf-36hpf zebrafish embryos were treated with retinoic acid signal inhibitors, 48hpf, 56hpf and 72hpf embryos were collected to detect snail1a and cad expression changes.

The results are shown in Figure 4 and Figure 5. After drug treatment, in situ hybridization was performed on 48hpf, 56hpf and 72hpf embryos. It was found that there was no significant difference in the expression of the gene snail1a in the dorsal and lateral views compared with the control group, and it was only visible in the lateral view of 72hpf. Slightly weakened expression; while the expression of gene cad was significantly different between the drug treatment group and the control group, at 48hpf, the expression of cad in the experimental group was significantly weaker than that in the control group, with the proliferation and migration of cranial nerve spine cells to the corresponding parts, the two groups were The difference of cad expression became smaller and smaller, but overall the cad expression of the experimental group was weaker than that of the control group.

The above results indicate that the expression of the gene snail1a related to the epithelial-mesenchymal transition in cranial nerve acanthocytes has no significant change, while the expression of the gene cad related to the proliferation and migration of cranial nerve acanthocytes is significantly attenuated in the drug-treated group, that is, retinoic acid signaling regulates zebrafish. The early development of pharyngeal teeth mainly plays a role by affecting the proliferation and migration of cranial nerve spinous cells.

Example 3: Effect of retinoic acid signal inhibition on regeneration of pharyngeal teeth in zebrafish

Construction of retinoic acid signal synthase and degradase overexpression fish: The CDS fragments of cyp26b1 and aldh1a2 were cloned from the zebrafish genome, and the plasmids without mutation and hsp70l-(-)-gfp were selected by TA cloning and plasmid screening and sequencing; cryaa -Cerulean subcloned, successfully constructed recombinant plasmids hsp:cyp26b1-GFP and hsp:aldh1a2-GFP. After microinjection into ABGO background zebrafish single-cell embryos and heat shock at 3dpf, the zebrafish embryos express green fluorescent protein throughout the body, that is, the overexpressed fish hsp:cyp26b1-GFP and hsp:aldh1a2-GFP were successfully constructed. After three consecutive generations of forward genetic screening to the F3 generation, the inheritance can be stably inherited.

MTZ-treated zebrafish embryos: Transgenic zebrafish Tg (dlx2b:Dendra2-NTR) were hybridized with hsp:cyp26b1-GFP and hsp:aldh1a2-GFP, respectively. After hybridization, the embryos were treated with 12mM MTZ solution, and the 3dpf zebrafish embryos were soaked. In the MTZ solution, the treatment time was 48hpf. After stopping the MTZ treatment at 5dpf, the embryos were heat-shocked at 38.5℃ for 30min, and then heat-shocked again after 24h of recovery. After 48h of recovery, the difference in pharyngeal tooth regeneration between the experimental group and the control group was observed.

The rest of the methods are the same as in Example 1.

48h after the regeneration of pharyngeal teeth injury, the embryos were collected and alizarin red staining was performed to observe the regeneration of pharyngeal teeth. After treatment, the mineralization of the regenerated pharyngeal teeth was significantly stronger than that of the control group. It can be observed in the results of alizarin red staining. In the control group, stronger 4V ¹ mineralization and weaker 3V ¹ and 5V ¹ cusp mineralization were observed. In the experimental group, the mineralization of 3V ¹ , 4V ¹ and 5V ¹ was stronger than that in the control group; in Tg(dlx2b:Dendra2-NTR; hsp:cyp26b1-GFP), the mineralization of the regenerated pharyngeal teeth was weaker than that in the control group, Alizarin red staining results can be observed that strong 4V ¹ mineralization and weak 3V ¹ and 5V ¹ mineralization are seen in the control group, while in the experimental group, 3V ¹ and 5V ¹ mineralization is not visible, and 4V ¹ The mineralization was also weaker than the control group.

Embryos were collected at 24h (R24h) and 48h (R48h) after the regeneration of pharyngeal tooth injury, and the pharyngeal teeth were peeled and completely exposed under the asana microscope to observe the regeneration of the pharyngeal teeth under a confocal microscope. The results are shown in Figure 7. Confocal microscope observation The results were consistent with the results of alizarin red staining. In Tg(dlx2b:Dendra2-NTR; hsp:aldh1a2-GFP), the strongly mineralized 4V ¹ , 3V ¹ and 5V ¹ were also stronger than the control group. In Tg (dlx2b:Dendra2-NTR; hsp:cyp26b1-GFP), the mineralization of 4V ¹ was weak, and the tooth germs of 3V ¹ and 5V ¹ were not yet mineralized. The results were consistent with the results of alizarin red staining.

The above results indicate that retinoic acid signaling affects the mineralization of regenerated pharyngeal teeth during the regeneration of zebrafish pharyngeal teeth, but does not affect the number and shape of regenerated pharyngeal teeth.

The above are only examples of the present invention, and common knowledge such as well-known specific technical solutions and/or characteristics in the solutions are not described too much here. It should be pointed out that for those skilled in the art, some modifications and improvements can be made without departing from the technical solution of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation of the present invention. effect and the applicability of the patent. The scope of protection claimed in this application shall be based on the content of the claims, and the descriptions of the specific implementation manners in the description can be used to interpret the content of the claims.

Claims (10)

1. The application of retinoic acid in the preparation of tooth regeneration medicine. 2. the application of retinoic acid according to claim 1 in the preparation of tooth regeneration medicine, it is characterized in that: described tooth regeneration medicine also comprises retinoic acid inhibitor, and after retinoic acid inhibitor acts on retinoic acid . 3. the application of retinoic acid according to claim 1 in the preparation of tooth regeneration medicine, it is characterized in that: described retinoic acid is exogenous all-trans retinoic acid, the molecular formula of all-trans retinoic acid It is: C ₂₀ H ₂₈ O ₂ , the structural formula is:

4. the application of retinoic acid according to claim 2 in the preparation of tooth regeneration medicine, it is characterized in that: described retinoic acid inhibitor is diethylaminobenzaldehyde, and the molecular formula of diethylaminobenzaldehyde is C ₁₁ H ₁₅ NO, the structural formula is:

5. the application of retinoic acid according to claim 2 in the preparation of tooth regeneration medicine, is characterized in that: described tooth is pharyngeal tooth, and described retinoic acid and its inhibitor are made according to the method for conventional pharmaceutical preparations A pharmaceutically acceptable dosage form. 6. The application of retinoic acid as pharyngeal tooth mineralization accelerator. 7. The application of a retinoic acid inhibitor as a cranial nerve spinous cell migration inhibitor. 8. The use of a retinoic acid inhibitor as a cranial nerve spinous cell proliferation inhibitor. 9 . The application of retinoic acid according to claim 6 in the preparation of a tooth regeneration medicine, wherein the concentration of the retinoic acid for promoting the mineralization of pharyngeal teeth is: ≥3×10 ⁻⁷ M. 10 . 10. the application of retinoic acid according to claim 7 or 8 in the preparation of tooth regeneration medicine, it is characterized in that: the concentration that described retinoic acid inhibitor suppresses cranial nerve spine cell migration and cranial nerve spine cell proliferation is: 0.5× ^{10-7M - 1} ×10-7M.

Images