A natural dentin extraction material and its preparation method and application

Abstract

The present invention relates to the technical field of oral restoration materials, and specifically to a natural dentin extraction material and a preparation method and application thereof. The method comprises the following steps performed in sequence: grinding dentin into dentin powder, and then freeze-drying to obtain the dentin powder to be digested; dispersing the dentin powder to be digested in an acidic pepsin solution to obtain a digestion system; then digesting, separating the solid and liquid to obtain the supernatant and adjusting the pH value to obtain a dentin extract; freezing and freeze-drying the dentin extract in sequence to obtain a dentin extract. This technical solution solves the technical problem of limited therapeutic effect of natural dentin extraction materials in the prior art, reduces the particle size of the material and enhances the biological activity. It can be used as a bone replacement material, root perforation repair material, pulp capping material, desensitization material for dentin hypersensitivity and material for promoting tooth remineralization, and is applied to medical practice operations, with an ideal prospect for promotion and application.

Description

Technical Field

[0001] The present invention relates to the technical field of oral restoration materials, and in particular to a natural dentin extraction material and a preparation method and application thereof. Background Art

[0002] Mammalian dentin, as a natural cell-free material, is extracted from healthy teeth of freshly extracted mammals and is considered to have good application prospects in the repair or regeneration of bone tissue and dentin. Several different types of dentin materials have been reported. The main ones are DDM (demineralized dentin matrix), TDM (treated dentin matrix), and CDM (cryopreserved dentin matrix). These materials are considered to have osteogenic / odontogenic effects and can be used for the repair or regeneration of bone tissue or dental tissue. TDM extracts contain a variety of extracellular matrix molecules (DSP, DMP-1, etc.), which can regulate cell proliferation and odontogenic differentiation. TDM has been shown to promote the differentiation of dental pulp cells into odontoblast-like cells. In addition, Chen et al. evaluated the effectiveness of TDM paste as a direct pulp capping agent in an animal model and found that TDM has good biocompatibility with pulp tissue and achieves the formation of reparative dentin. Demineralized dentin matrix DDM can improve the efficiency of bone induction. It has been verified that DDM can induce bone formation in an in vitro model of rodents. In a study on human teeth, the regenerative potential of DDM hydrogel as a direct pulp capping material was clinically evaluated with calcium hydroxide mineral trioxide polymer (MTA). The study found that DDM hydrogel preserved pulp vitality and achieved dentin regeneration, and it is considered to be a natural alternative to commercial pulp capping materials. These natural dentin extract materials can be used as alternatives to current commercial bone repair materials (such as artificial bone powder) or direct pulp capping materials (MTA or iRoot BP). In addition, they can also be used in bone tissue engineering or tooth tissue engineering to promote bone or dentin regeneration.

[0003] Currently, the dentin matrix materials produced are generally 100-300μm in size, which basically retains the basic structure of natural dentin, including dentinal tubules, peritubular dentin, and intertubular dentin. Although it has been proven to have the effect of bone repair and dentin repair, the large particles also bring many disadvantages, such as: it cannot be mixed into a uniform paste for direct pulp capping; if allogeneic transplantation occurs, there is a risk of causing immune response; because of the grinding process, the obtained particle size is uneven, and the particle size of the dentin extraction materials reported so far varies; the risk of infection; because the particles are too large, they cannot enter some narrow areas (such as dentinal tubules, which play a role in sealing dentinal tubules). In addition, because of the large size and the retention of the structure of dentin, it means that it is difficult to be absorbed by the body and replaced by the body's new tissue, and it is difficult to form real bone tissue or the recipient's own tooth tissue. Existing natural dentin extraction materials are difficult to achieve ideal therapeutic effects in some cases. There is an urgent need to develop a new type of material with osteogenic / dentinogenic effects. This material can be used as a bone replacement material, root perforation repair material and pulp capping material. It can also be used for desensitization treatment of dentin hypersensitivity and promote tooth remineralization. Summary of the invention

[0004] The present invention is intended to provide a method for preparing a natural dentin extraction material to solve the technical problem that the treatment effect of the natural dentin extraction material in the prior art is limited.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] A method for preparing a natural dentin extraction material comprises the following steps performed in sequence:

[0007] S1: grinding the dentin into dentin powder, and then freeze-drying it to obtain the dentin powder to be digested;

[0008] S2: dispersing the dentin powder to be digested in an acidic pepsin solution to obtain a digestion system; then performing digestion treatment, solid-liquid separation, taking the supernatant and adjusting the pH value to obtain a dentin extract;

[0009] S3: The dentin extract is subjected to freezing treatment and freeze-drying treatment in sequence to obtain a dentin extract.

[0010] The technical solution also provides a dentin extract prepared by a method for preparing a natural dentin extraction material.

[0011] The technical solution also provides the use of dentin extract in preparing bone replacement materials, root perforation repair materials, pulp capping materials, desensitizing materials for dentin hypersensitivity and materials for promoting tooth remineralization.

[0012] The principle and beneficial effects of this technical solution are:

[0013] By treating the dentin powder with pepsin in an acidic environment, a DDME (Digested dentin matrix extract) with a smaller particle size was obtained, which destroyed the dentinal tubule structure and the peritubular and intertubular dentin structure, overcoming the problems of the existing dentin extraction materials, such as the uneven particle size, the inability to enter some narrow areas, the difficulty in being absorbed by the body and being replaced by the body's new tissue, and the limited osteogenesis-promoting effect. The DDME prepared in this scheme has an uneven surface, which ensures that more active functional groups and polypeptide structures are exposed. Moderate pepsin degradation, which then divides the protein into stable domains, enhances the material's activity in promoting osteogenesis.

[0014] When the dentin extract of this scheme is used as a replacement material, root perforation repair material, pulp capping material, desensitization material for dentin hypersensitivity, and material to promote tooth remineralization, the DDME layer will be gradually degraded and occupied by new restorative dentin. DDME can be degraded and replaced by its own tissue, and it is an ideal repair material. Under the treatment of DDME, demineralized dentin is remineralized, and the dentinal tubules are sealed, and the mineralized sealant cannot be washed away by an ultrasonic cleaner. This shows that the DDME prepared by this scheme is very suitable for dentinal tubule sealing, promoting the remineralization of demineralized dentin, and is used to treat dentin hypersensitivity and the remineralization of demineralized dentin. In addition, a preliminary analysis of the mechanism of action of DDME was conducted, and it was found that the DDME prepared by this technical scheme promotes odontogenesis by promoting the Yap signaling pathway. The DDME of this scheme can act as a YAP1 protein nuclear entry promoter.

[0015] Furthermore, in S2, the pH value of the acidic pepsin solution is 1-2.

[0016] Furthermore, the concentration of pepsin in the acidic pepsin solution is 0.001-0.01 g / mL.

[0017] Furthermore, the concentration of the dentin powder to be digested in the acidic pepsin solution is 0.001-0.05 g / mL.

[0018] Furthermore, in S2, the digestion treatment method is: stirring the digestion system at a temperature of 37° C. for 24-48 hours.

[0019] Furthermore, in S2, the method for separating the solid from the liquid and obtaining the supernatant is: centrifuging at 1500-3000 rpm for 5-10 min at 4°C to obtain the supernatant.

[0020] Furthermore, in S2, the method for adjusting the pH value is: first adjusting the pH value of the supernatant to 10-12, and then adjusting it to 7.2.

[0021] Furthermore, in S1, the particle size of the dentin powder was 80-200 μm.

[0022] In summary, the beneficial effects of this technical solution are as follows:

[0023] (1) The dentin extract particles prepared by this technical solution are smaller and more uniform, with a particle diameter of about 7-8 μm. The preparation method has good reproducibility and can stably obtain small and uniform dentin extract particles in batches. Due to the reduction in particle size, DDME and liquid are mixed into a paste, which is more uniform and has better operability than traditional dentin extraction materials (DDM). It can be used as a direct pulp capping material in vital pulp preservation treatment.

[0024] (2) This technical solution destroys the dentinal tubule structure and the peri-tubular and inter-tubular dentin structure, enzymatically hydrolyzes the protein components of the dentin into polypeptides, reduces the antigenicity of the dentin extraction material, and reduces the immune response in allogeneic transplantation.

[0025] (3) The test results of the materials showed that the DDME prepared by this scheme had an uneven surface, with more active functional groups exposed and peptide structures exposed. In addition, Fourier infrared spectroscopy and X-ray photoelectron spectroscopy showed that DDME had a spectrum similar to that of traditional dentin extraction materials. It has retained the components of the dentin matrix while reducing its size. Protein analysis of DDME showed that it contained the main matrix proteins of dentin.

[0026] (4) Due to the reduction in particle size and other reasons, the dentin extraction material DDME prepared by this method can be partially degraded and replaced by new tissue (bone or dentin), which is different from the traditional large-particle dentin extraction material. In addition, the dentin extraction material prepared by this method has good biocompatibility and has potential application value in bone tissue / dentin repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the overall preparation flow chart of dentin extract (DDME) in Example 1.

[0028] Figure 2 This is a general picture of the dentin extract (DDME) of Example 1.

[0029] Figure 3 This is a comparison diagram of the appearance of the dentin extract (DDME) of Example 1 and the dentin extract material of the prior art.

[0030] Figure 4 The electron microscope images of the dentin extract (DDME) of Example 1 and the dentin extract material of the prior art are shown.

[0031] Figure 5 The particle size distribution of the dentin extract (DDME) of Example 1

[0032] Figure 6 The XPS and FITR images of the dentin extract (DDME) of Example 1 and the prior art dentin extract material are shown.

[0033] Figure 7 The experimental results of detecting cell proliferation by CCK-8 method in Experimental Example 1 (* indicates significant difference compared with blank control).

[0034] Figure 8 The WB detection results and statistical charts of Experimental Example 1.

[0035] Fig. 9 This is the flow cytometry test result of human dental pulp cells under normal conditions in Experimental Example 1.

[0036] Fig.10 This is a microscopic picture of human dental pulp cells under normal conditions in Experimental Example 1.

[0037] Fig.11 These are the microscopic images and fluorescent photos of human dental pulp cells treated with different concentrations of DDME in Experimental Example 1 and cell survival.

[0038] Fig.12 The experimental results of Experimental Example 2 show the effect of DDME on promoting osteogenesis and odontogenesis in a rat model (scale bar: 100 μm).

[0039] Fig.13 The experimental results of Experimental Example 2 show the effect of DDME on promoting osteogenesis and odontogenesis in the nude mouse model (scale bar: 100 μm).

[0040] Fig.14 These are the experimental study results of Experimental Example 3 on the effects of DDME on promoting dentin mineralization and sealing dentinal tubules.

[0041] Fig.15 This is a heat map of the gene expression analysis of human dental pulp cells by DDME in Experimental Example 4.

[0042] Fig.16 This is a statistical chart of the GO analysis results of Experimental Example 4 showing the effect of DDME on gene expression in human dental pulp cells.

[0043] Fig.17 This is a statistical chart of the KEGG analysis results of the effect of DDME on the signaling pathway of human dental pulp cells in Experimental Example 4.

[0044] Fig.18This is an immunofluorescence analysis image of the effect of DDME on the expression of YAP1 protein in human dental pulp cells in Experimental Example 4.

[0045] Fig.19 These are WB images and statistical graphs of the effect of DDME on the expression of YAP1 and DMP-1 proteins in human dental pulp cells in Experimental Example 4.

[0046] Fig. 20 These are the macroscopic photographs and microscopic images of the dentin extraction material obtained by treating the dentin powder with hydrochloric acid in Comparative Example 2.

[0047] Fig.21 These are the macroscopic photographs and microscopic images of DDME obtained by treating dentin powder with hydrochloric acid, pepsin and pancreatin in Comparative Example 3.

[0048] Fig. 22 The WB test results of Comparative Example 3 showing the effect of DDME on the expression of osteogenesis-related proteins obtained by treating dentin powder with hydrochloric acid, pepsin and pancreatin. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0050] Embodiment 1:

[0051] The general process flow of this technical solution is as follows:

[0052] (1) Collect fresh healthy teeth (human, cow, pig teeth, etc.), scrape off the soft tissue, use a high-speed mobile phone and diamond grinding needle to grind (ensure cooling water during the process) to grind off the periodontal membrane, cementum and crown, leaving only the dentin at the root. Grind the dentin into small pieces. The above process of obtaining dentin is a conventional method in the prior art and will not be described in detail here.

[0053] (2) Use a cryogenic grinding machine (-50 to -40°C, 60-70 Hz) to grind the dentin block (appropriate protease inhibitors can be added to prevent degradation of dentin proteins in the sample, but not pepsin inhibitors) into 80-200 μm dentin powder, and use a freeze dryer for conventional freeze drying for 24 hours (constant temperature freeze drying, cold trap temperature -50°C) for subsequent processing.

[0054] (3) Prepare an acidic pepsin solution (pH = 1-2, pepsin is a conventional biochemical reagent that can be obtained through commercial channels, such as: Sigma P6887; Solarbio P8390; the concentration of pepsin ranges from 0.001 to 0.02 g / mL), put an appropriate amount of dentin powder into the acidic pepsin solution, the concentration of the dentin powder is 0.001-0.05 g / mL, and stir it thoroughly (400 rpm, using a magnetic stirrer) at 37°C for 24-48 hours (when most of the dentin powder is dissolved, stop the treatment) to obtain a dentin digestion solution, centrifuge it at 1500-3000 rpm for 5-10 minutes at 4°C, and take the supernatant. This step can basically dissolve the dentin powder completely, and the undissolved part will be removed as a precipitate after centrifugation. At 4°C, adjust the pH to 10 with NaOH solution to inactivate pepsin, and then add dilute hydrochloric acid to adjust the pH to 7.2 to obtain a dentin extract.

[0055] (4) After the extract was stored at -80°C for 24 hours, it was freeze-dried for 48-72 hours (constant temperature freeze drying, cold trap temperature -50°C, freeze drying using a conventional laboratory freeze dryer, freeze vacuum drying was turned on to absorb the moisture of the sample) to obtain dentin extract (DDME). The obtained dentin extract particles have a smaller diameter and are more uniform. For the overall preparation process, see Figure 1 .

[0056] More specifically, the operation mode of this embodiment is as follows:

[0057] Single-rooted premolars that were healthy, aged 12-25 years old, and extracted for orthodontic reasons were selected in the clinic. They were washed with PBS buffer and set aside for later use. They could also be stored at -80°C. Using a high-speed handpiece and diamond bur, the root periodontal membrane and cementum of the ex vivo tooth were removed, the crown was removed, and only the root dentin was retained to obtain a dentin block. Then, a cryo-grinder was used to grind the dentin block into powdered dentin, freeze-dried for 24 hours, and left for subsequent processing. 1g of dentin powder and 0.1g of pepsin were weighed and added to 100ml of 0.1M dilute hydrochloric acid. The solution was stirred at 400rpm at a constant temperature of 37°C and digested for 24h. When most of the dentin powder was dissolved, the dentin digestion solution was collected. Using a low-temperature high-speed centrifuge, centrifuge at 3000rpm for 5min at 4°C and take the supernatant. At 4°C, 1M sodium hydroxide solution was added to the supernatant and adjusted to pH10 to inactivate pepsin. At this time, a large amount of white precipitate was seen. Then add dilute hydrochloric acid to adjust the pH to 7.2, the precipitation is greatly reduced, and the dentin extract is obtained. Then transfer to -80℃ for 24 hours, and freeze-dry for 48 hours to obtain white powdery human dentin extract DDME (Digesteddentin matrix extract).

[0058] See the DDME general picture Figure 2 , see the comparison pictures of DDME and DDM Figure 3 (DDME on top, DDM on bottom). For a comparison of the microstructures of DDME and DDM, see Figure 4 . DDM is an existing technical material (demineralized dentin matrix), which was prepared according to the literature (MehrvarzfarP, AbbottPV, MashhadiabbasF, VatanpourM, TourSavadkouhiS. Clinical and histological responses of human dental pulp to MTAand combined MTA / treateddentin matrix in partial pulpotomy. Aust Endod J.2018;44:46–53.). The preparation method of DDM is as follows: human dentin is ground into dentin powder, and then decalcified with EDTA solution in sequence: 17%EDTA 5min, 10%EDTA 5min, and 5%EDTA 10min. It can be seen that the traditional dentin material completely retains the structure of dentinal tubules, peritubular dentin and intertubular dentin, while DDME completely destroys the basic structure of dentin, presenting a structure with an uneven surface and many gaps. Dentin matrix materials (DDM) in the prior art are prepared by mechanical grinding and demineralization, and have large particles and retain the structure of dentin (e.g., dentinal tubules, peritubular dentin, and intertubular dentin, etc.). The DDME prepared by this technical solution has small particles, destroys the dentinal tubule structure and peritubular and intertubular dentin structure, has an uneven surface, and ensures that more active functional groups and polypeptide structures are exposed.

[0059] After testing, the average particle size of DDME prepared in this example is about 8 μm. Figure 5 (The abscissa is particle size in nm, and the ordinate is intensity). The Pop spectrum comparison of the DDME (DDME) of this scheme and the dentin matrix material (demineralized dentin matrix DDM) of the prior art is shown in Figure 6 (XRD, XPS, FITR; upper curve DDME, lower curve DDM), both have similar spectra, indicating that DDME prepared according to this protocol retains the characteristic components of the dentin matrix while reducing its size.

[0060] Experimental Example 1: In vitro experiment

[0061] This experimental example studied the effect of DDME on human dental pulp cells in vitro and found that DDME has the effect of promoting osteogenesis / odontogenesis in vitro. Human dental pulp cells were isolated from teeth extracted from healthy people due to orthodontic reasons by conventional means of existing technology. The general process of obtaining dental pulp stem cells is as follows: remove the pulp tissue from the freshly extracted tooth and put it into a 1.5mL centrifuge tube containing PBS, rinse it 3 times with PBS containing 1% penicillin / streptomycin / amphotericin B; cut the tissue into pieces with scissors, and try to cut it into pieces less than 1mm. 3 The small pieces were centrifuged at 1000 rpm for 5 min, the supernatant was removed, and the excess PBS rinse solution was aspirated; 3 mg / mL type I collagenase solution was added, and the volume of the collagenase solution was required to be at least 10 times the volume of the tissue block. After sufficient shaking with an oscillator, it was placed in a 37°C water bath for 40 min; a-MEM culture medium containing 10% FBS was added to terminate the digestion; centrifuged at 1000 rpm for 5 min, the supernatant was removed; a small amount of FBS was added, the tissue block was transferred to a T25 culture flask, 1 mL of complete culture medium was added, and human dental pulp cells were obtained after the cells crawled out. Human dental pulp cells were inoculated in a 96-well plate at a density of 5000 cells / well. After culturing in α-MEM medium containing 10% fetal bovine serum for 4-6 hours, DDME was added to the medium to make the final concentration of DDME 0.2, 0.4, 0.6, 0.8, 1 mg / mL (1×, 2×, 3×, 4× and 5×). No DDME was added to the blank control (con), and the culture medium was replaced with fresh culture medium in the routine process. After culturing cells with DDME for 24-72 hours, the cell proliferation of human dental pulp cells under different DDME concentrations and different culture times was detected by CCK-8 method. The experimental results are shown in Figure 7 After culturing cells with DDME for 7 days, cells from each group were taken to detect the expression of proteins related to odontogenesis and osteogenic differentiation (DMP-1, RUNX2, COLI, YAP1) in the cells using WB method, and the protein band signal intensity was identified and calculated by software to form a bar chart ( Figure 8 ). Fig. 9 The results of flow cytometric analysis of the phenotype of normal human dental pulp cells. Fig.10 Micrograph of primary culture of normal human dental pulp cells. Fig.11 The following are the microscopic cell photos (top) and fluorescence images (bottom, fluorescent staining shows live / dead) of blank control, 1×, 2×, 3×, 4× and 5× treated cells after 1 day of culture. Live / dead cell survival staining shows that 0.2-1 mg / mL DDME does not affect cell survival and has good biocompatibility for dental pulp cells (green represents live cells and red represents dead cells).

[0062] The results of CCK-8 and WB experiments showed that treating cells with low concentrations of DDME would not affect cell proliferation. However, as the concentration of DDME increased, it would inhibit the proliferation of dental pulp cells to a certain extent, but would significantly promote the expression of proteins related to osteogenic differentiation in the cells. Fig.11 The cell microscopic images show that DDME has no obvious toxicity to dental pulp cells and does not affect cell survival. However, more opaque mineralized nodule-like structures can be seen after treatment, that is, after DDME treatment, the cell morphology changes and mineralized nodules are formed.

[0063] Experimental Example 2: In vivo experiment

[0064] (1) Nude mouse back subcutaneous transplantation model

[0065] The model making process is as follows:

[0066] 1) 1×10 6 / mL human dental pulp stem cells and 0.2mL of DDME or HA-TCP (a classic osteogenic material) were mixed in a 15mL centrifuge tube, mixed thoroughly, and complete culture medium was added to 10mL. The mixture was incubated in a cell culture incubator for 6 hours, centrifuged at 1500rpm for 10 minutes, and the supernatant was removed to precipitate a mixture of human dental pulp cells and materials.

[0067] 2) Under isoflurane anesthesia, incisions were made on both sides of the midline of the nude mice's back, and DMME+human dental pulp cells or HA-TCP+human dental pulp cells mixture (about 0.2 mL at each implantation point) were implanted. The incisions were sutured, and then the nude mice were killed 8 weeks later. The samples were collected, fixed, decalcified, dehydrated, embedded in paraffin, sectioned, and stained with HE, Masson Trichrome, and immunohistochemistry (AQP4 and CD31).

[0068] The experimental results can be found in Fig.13 (The arrow points to the new dentin). HE staining shows that both implants can form obvious mineralization (hard tissue), and the mineralization formed in the DDME group is more similar to dentin. Masson staining results show that the mineralization formed in both groups contains more collagen components (dyed blue), and the blue is more obvious in the DDME group. There is a layer of AQP4-positive cells around the mineralization formed by DDME. The above results prove that DDME can induce the formation of mineralized tissue in animals, and compared with the classic material HA-TCP, the mineralization it forms is closer to dentin.

[0069] (2) Rat in situ dental pulp regeneration model:

[0070] The model making process is as follows:

[0071] 1) Under isoflurane inhalation anesthesia and local infiltration anesthesia with blue hemp, the upper and lower jaws of the rats were opened with a homemade opener to expose the maxillary first molar. The pulp was opened from the occlusal surface with an FG1 / 2 ball drill, and the coronal pulp in the pulp chamber was gradually removed. The residual pulp in the pulp chamber was cleaned with a probe and a micro-curette. A small cotton ball soaked in 1% sodium hypochlorite was placed in the pulp chamber for 3 minutes, and then rinsed with saline, and the small cotton ball absorbed excess water.

[0072] Mix DDME powder with 10% PVP to make it into a paste, and use it before it solidifies. Place DDME paste in the pulp cavity to ensure that DDME completely covers the exposed pulp and surrounding dentin. Use a cotton ball to gently press and absorb excess liquid. Place iRootBPplus on the upper layer of DDME paste. After it is initially solidified, use Songfeng flowing resin to seal the cavity. Avoid filling too high when filling. Use a small grinding head to slightly flatten the cusps of the opposing teeth to avoid excessive occlusal contact.

[0073] Then, the rats were killed after 4 and 8 weeks, and their maxillae were removed and fixed. One part was subjected to microCT, and the rest was decalcified, dehydrated, paraffin-embedded, sectioned, and stained with HE, Masson Trichrome, and immunohistochemistry (AQP4, CD31, and CD14).

[0074] The experimental results can be found in Fig.12 New dentin was formed in both groups. In the experimental group, the DDME layer was degraded and occupied by new reparative dentin, proving that DDME can be degraded and replaced by its own tissue. The new dentin contains a large amount of collagen (blue). There was no obvious inflammation in the pulp tissue, no inflammatory cell infiltration, and AQP4-positive odontoblasts.

[0075] Experimental Example 3: Experiment on the effect of DDME on dentin hypersensitivity

[0076] Freshly extracted healthy teeth (wisdom teeth) were obtained, and dentin slices with a thickness of about 1 mm were prepared using a hard tissue slicer. The slices were demineralized to obtain demineralized dentin. The demineralization method was to use 17% EDTA for decalcification for two weeks. The demineralized dentin was treated with DDME solution prepared with PBS containing 10% PVP as a solvent for 14 days, and then rinsed with an ultrasonic cleaner for 30 minutes. Then, the remineralization of the demineralized dentin after treatment and the closure of the dentinal tubules were observed under a scanning electron microscope.

[0077] The experimental results can be found in Fig.14Under the treatment of DDME, demineralized dentin remineralized and the dentinal tubules were sealed. The effect increased with the increase of DDME concentration, and the mineralized sealant could not be washed away by an ultrasonic cleaner. This shows that the DDME prepared in this scheme is very suitable for sealing dentinal tubules, promoting the remineralization of demineralized dentin, and being used to treat dentin hypersensitivity and remineralize demineralized dentin.

[0078] Experimental Example 4: Preliminary Analysis of Mechanism of Action

[0079] To conduct a preliminary analysis of the mechanism of action of DDME, human dental pulp cells were treated with DDME at a final concentration of 0.2 mg / mL for 7 days, and a blank control (con) without DDME was set up at the same time, and then gene expression was compared and signal pathway analysis was performed. The experimental results can be found in Fig.15 (heat map), Fig.16 (GO analysis) and Fig.17 (KEGG analysis). Fig.18 Immunofluorescence images (YAP1 in red, cell nuclei stained with DAPI in blue). Fig.19 The WB images and quantitative analysis results. DMP-1 is dentin matrix protein 1, and YAP1 is Yes-associated protein 1 (YAP1), which is a molecule in the Hippo pathway. YAP1 (activated state) can promote cell proliferation, metastasis, survival, and maintain stem cell activity. Verteporfin is a YAP1 inhibitor. The use of DDME can promote the expression of YAP1 and DMP-1, while the YAP1 inhibitor Verteporfin can offset the expression-promoting effect of DDME to a certain extent. It can be seen from the experimental results that the DDME prepared by this technical solution promotes tooth formation by promoting the Yap signaling pathway, which preliminarily clarifies the mechanism of DDME action.

[0080] Comparative Example 1

[0081] Existing dentin matrix materials are not easily absorbed by the body. The mineralized tissue formed by implantation is the material itself, not the mineralized tissue formed by the body itself, which limits the application of dentin matrix materials. The inventor analyzed the possible reasons for the above phenomenon: the current dentin matrix materials are mainly prepared by mechanical grinding, the size is too large, and the structure of dentin is retained, which is difficult to be absorbed by the body and replaced by its own new bone tissue. After analysis, the inventors think about achieving the performance of dentin matrix materials that can be absorbed by the body from the following aspects: removing the basic structure of dentin powder, destroying the quaternary structure of protein, reducing the immunogenicity of dentin materials, and reducing the size of materials, so that they can be absorbed and degraded by the body and replaced by its own new tissue.

[0082] The above is only the theoretical analysis of the inventor. How can we prepare a material that can be degraded by the body, replaced by its own new tissue, and has the function of tooth formation / osteogenesis? We need to find specific and feasible means of operation. The inventor has conducted a large number of experimental analyses to explore methods that can achieve the effects of removing the basic structure of dentin powder, destroying the quaternary structure of protein, reducing the immunogenicity of dentin materials, and reducing the size of materials.

[0083] This comparative example shows the inventor's attempt to strengthen the grinding method to reduce the material size and destroy the basic structure of the dentin powder. The inventor used a higher-performance freezing grinder to try to grind the dentin block into finer particles. However, after particle size analysis, it was found that the minimum particle size of the dentin powder after grinding alone can only reach the range of 70-80μm, and the particle size distribution of the particles is relatively wide, and the size of the particles is not completely uniform. At the same time, through microscopic observation, the tubules and inter-tubular structures of the dentin are still retained. The dentin matrix material obtained in this way cannot be dissolved by PBS or culture medium, resulting in its low bioavailability and limited administration method. Such dentin matrix materials cannot be absorbed and degraded by body tissues for a long time after implantation, and it is difficult to be replaced by the body's own tissues to form real self-tissues. Although the particle size of the dentin matrix material can be reduced from 100-300μm to 70-80μm by strengthening the grinding method, it still cannot meet the application requirements.

[0084] Comparative Example 2

[0085] There are many protein components in the dentin matrix. From the perspective of retaining the functionality of dentin protein, the first thing that comes to mind is demineralization / decalcification to dissolve hydroxyapatite (remove inorganic components) to expose its active protein components. This comparative example tried the following methods to remove inorganic components, reduce product size and immunogenicity:

[0086] In this comparative example, the inventor used 17% EDTA to decalcify the dentin powder (see Example 1 for the preparation method). The specific method is: soak the dentin powder in a 17% EDTA solution at a ratio of 1g:100mL for one week (the EDTA solution is replaced every two days). The purpose of the above treatment is that chelating agents such as EDTA can decalcify the dentin powder. After the above treatment, although the structure of the dentin surface can be exposed after decalcification, the internal structure of the dentin is not affected, and the original structure of the dentin is not destroyed, that is, the tubules and intertubular structures of the dentin are still retained, and EDTA has a slow effect on the dentin. Even if it is treated for one year, the size of the dentin powder cannot be reduced, and the application requirements cannot be met.

[0087] In this comparative example, the inventors also tried strong acid (hydrochloric acid) and weak acid (formic acid) to achieve demineralization of dentin powder and adjust the product particle size. Dentin powder was soaked in 0.1M hydrochloric acid at a ratio of 1g:100mL for 24h (refer to the operation method in Example 1, the only difference is that the enzyme treatment in step (3) was changed to acid treatment). The experimental results show that short-term hydrochloric acid treatment cannot completely dissolve the dentin powder, and the size is not significantly reduced. Hydrochloric acid obviously makes the dentin powder completely demineralized, and the original structure of dentin is destroyed (tubules and intertubular structures of dentin, etc.). However, by increasing the volume of hydrochloric acid and extending the treatment time, hydrochloric acid treatment can dissolve hydroxyapatite in dentin and expose demineralized collagen fibers, but cannot further hydrolyze dentin matrix proteins into polypeptides. Therefore, although hydrochloric acid treatment can destroy the original structure of dentin, the biological activity of the dentin matrix material prepared by hydrochloric acid treatment alone is not ideal and cannot meet the application requirements. However, weak acids such as formic acid take a long time to process, are not effective in dissolving dentin powder, and cannot meet application requirements.

[0088] The dentin powder was soaked in 0.1M hydrochloric acid at a ratio of 1g:100mL for 24h. The other preparation processes were as described in Example 1 to obtain the dentin extraction material. The general photo and microscopic image of the dentin extraction material are shown in Fig. 20 .Will Fig. 20 The general picture on the left and Figure 2 , 3 Compared with the general picture of (DDME obtained in Example 1), the dispersion of the dentin extraction material obtained in this comparative example is obviously worse than that in Example 1, aggregated into lumps, and the particle size distribution is very uneven, with a large difference between particles with larger particle sizes and particles with smaller particle sizes. The average particle size of the dentin extraction material in this comparative example is larger than that in Example 1. The dentin extraction material obtained in this comparative example was observed by electron microscopy. For the convenience of observation, the relatively loose and dispersed small particles in DDME were specially selected, see Fig. 20 The middle and right figures. This comparative example is different from Example 1 and does not show Figure 4 The more uneven surfaces shown cannot ensure more exposure of active functional groups and polypeptide structures, thereby reducing its biological activity.

[0089] Comparative Example 3

[0090] This comparative example attempts to use different enzymes to treat dentin powder in order to digest the dentin powder and destroy the original structure of dentin, and reorganize the components of dentin. The preparation methods of dentin matrix materials in the prior art are mainly grinding and demineralization (partial or complete), and there is no prior art report on the enzymatic hydrolysis of dentin materials. The inventor introduced enzymatic hydrolysis into the preparation process of dentin matrix materials for the first time and obtained DDME with ideal performance. According to the common knowledge in the field, there are many protein components in the dentin matrix. From the perspective of retaining the functionality of dentin protein, it is not expected to use proteases to destroy the structure of dentin protein. In this technical solution, the technical solution of enzymatic hydrolysis of dentin powder is used to improve the effect of DDME materials and overcome the prejudice of the prior art.

[0091] Considering the characteristics of dentin, the dentin matrix proteins are mainly collagen, non-collagen, and proteoglycans (such as chondroitin sulfate and a small amount of keratan sulfate). Therefore, the inventors tried to treat dentin powder with a variety of proteases. Proteases are enzymes that catalyze the hydrolysis of proteins, and there are many types, such as pepsin, trypsin, collagenase, cathepsin, papain, and subtilisin. Proteases can hydrolyze proteins into polypeptide chains and some amino acids and destroy their spatial structure, but they will not completely hydrolyze the proteins and lose their functions. Proteases have strict selectivity for the reaction substrates they act on. A protease can only act on certain peptide bonds in protein molecules, such as the peptide bonds formed by trypsin catalyzing the hydrolysis of basic amino acids.

[0092] In view of the characteristics of dentin, the inventors included and compared several digestion schemes: 1) type I collagenase (3 mg / mL, pH7, 37°C, treated for 48 hours), 2) pepsin (see Example 1), 3) pancreatin (0.25%, pH8, 37°C, treated for 48 hours), 4) chymotrypsin (2 mg / mL, pH7, 37°C, treated for 48 hours), 5) enteropeptidase (5000 U / mL, pH8, 37°C, treated for 48 hours), 6) pepsin first (2 mg / mL, pH1-2, 37°C, treated for 48 hours), then pancreatin (after adjusting the pH value, 0.25%, pH8, 37°C, treated for 48 hours), 7) collagenase (3 mg / mL, pH7, 37°C, treated for 48 hours) + pancreatin (after adjusting the pH value, 0.25%, pH8, 37°C, treated for 48 hours). The specific operation process is shown in Example 1. The difference between different experimental groups and group 2) lies in the type of enzyme used in step (3). It was found that schemes 1), 3), 4), 5), and 7) act slowly and cannot effectively dissolve most of the dentin powder within the above-mentioned treatment time. The inventors also tried to extend the action time, but still could not dissolve most of the dentin powder. Therefore, 1), 3), 4), 5), and 7) cannot be used for the preparation of DDME. For the remaining schemes 2) and 6), the inventors further studied and analyzed the dentin matrix materials prepared by digestion of these two schemes. The effects of the prepared materials were studied by SEM, XRD, XPS, FITR, particle size analysis, proteomics analysis, etc. Among them, group 2) destroyed the original structure of the dentin matrix while basically retaining the characteristics of the dentin matrix components. The particle size was basically controlled in the range of 7-8μm, the particles were uniform, and the particle size distribution range was narrow. XPS and FITR showed that the prepared material had a similar spectrum to the natural dentin matrix. The treatment method of group 2) destroyed the quaternary structure of dentin collagen, and more functional groups were exposed. Preliminary research results show that the material particles obtained after treatment in Scheme 2) are 7-8μm. Scheme 6) The general and microscopic images of DDME after treatment can be found in Fig.21 . Fig.21 The left schematic diagram shows that the DDME powder obtained by Scheme 6) is aggregated into lumps, and the dispersion of the powder is not ideal. Fig.21 The micrographs in the middle and right select the relatively loose and dispersed small particles in the DDME powder. Unlike Example 1, it does not show Figure 4 The more uneven surfaces shown cannot ensure more exposure of active functional groups and polypeptide structures, thus reducing the activity of DDME.

[0093] Subsequently, the inventors conducted further research and evaluation on the prepared dentin materials, and evaluated the effects of the materials on cell survival and proliferation, as well as the effects on odontogenesis and osteogenesis of human dental pulp cells (see Experimental Example 1 for experimental methods). The effect of the dentin matrix material extracted by pepsin alone in Scheme 2) is better than that of the dentin matrix material extracted by the combined pepsin + trypsin in Scheme 6). At the same time, it has been confirmed in animal experiments that the dentin matrix material extracted by Scheme 2) has better ability to form new dentin and stronger ability to form mineralized tissue, whether it is a rat in situ direct pulp capping model or a mouse back subcutaneous transplantation model. For example, referring to the method of Example 1, the effect of DDME on human dental pulp cells in vitro was detected, and the WB method was used to detect the expression of proteins related to odontogenesis and osteogenesis in the cells. The experimental results are shown in Table 1. Fig. 22 The experimental results showed that the dentin matrix material prepared by hydrolysis with two proteases in Scheme 6) did not have the effect of osteogenesis and odontogenesis. Fig. 22 It shows that the DDME prepared by Scheme 6) does not have the ability to enhance the expression of odontogenic related proteins (dentine sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP-1)). Comparative Example 1 shows that the selection of enzyme type is very critical to the maintenance of DDME activity. The pancreatic enzyme treatment on the basis of pepsin will lead to excessive enzymatic hydrolysis of proteins into polypeptides without odontogenic activity. However, the pepsin treatment of this technical solution hydrolyzes some peptide bonds, moderate enzymatic hydrolysis, and moderately enhances the activity of DDME, thereby enhancing the tooth mineralization and osteogenesis effects of DDME, and obtaining unexpected technical effects.

[0094] The inventors further analyzed the principle of increasing the effect of DDME by enzymatic treatment. The use of appropriate proteases destroys the basic structure of the dentin matrix, attacks the peptide chains exposed to the outside, and then cuts the protein into stable domains, and degrades the loops and non-structural domains of the protein exposed to the outside, thereby improving the effect. Although the use of pepsin acidic solution to enzymatically hydrolyze dentin powder will reduce the particle size and immunogenicity, it will also destroy the basic structure of dentin and degrade or inactivate some proteins. Some proteins or their peptide chains (such as DSP, DPP, TGF-beta, etc.) in dentin cannot be detected. However, the use of this technical solution can minimize the damage to the protein components in dentin while obtaining a dentin matrix material of suitable size and biological function. This solution will destroy the collagen fibers and the quaternary structure of the protein in the dentin, but will not destroy the triple helix structure of collagen, which plays an important role in biological remineralization. Therefore, the DDME prepared by this solution is very suitable for dentinal tubule sealing and for the treatment of dentin hypersensitivity; it also helps the remineralization of demineralized dentin. To produce the above-mentioned effects, the selection of pepsin plays a crucial role in the present technical solution, and an unexpected technical effect is obtained.

[0095] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing a natural dentin extraction material, characterized in that: The method includes the following steps: S1: grinding the dentin into dentin powder, and then freeze-drying it to obtain the dentin powder to be digested; S2: dispersing the dentin powder to be digested in an acidic pepsin solution to obtain a digestion system; then performing digestion treatment, solid-liquid separation, taking the supernatant and adjusting the pH value to obtain a dentin extract; the pH value of the acidic pepsin solution is 1-2; the concentration of pepsin in the acidic pepsin solution is 0.001-0.02 g / mL; the concentration of the dentin powder to be digested in the acidic pepsin solution is 0.001-0.05 g / mL; the digestion treatment method is: stirring the digestion system at a temperature of 37°C for 24-48 hours; The method for separating the solid from the liquid and taking the supernatant is: centrifuging at 1500-3000 rpm for 5-10 min at 4°C to take the supernatant; S3: The dentin extract is subjected to freezing treatment and freeze-drying treatment in sequence to obtain a dentin extract.

2. The method for preparing a natural dentin extraction material according to claim 1, characterized in that: In S2, the method for adjusting the pH value is: first adjusting the pH value of the supernatant to 10-12, and then adjusting it to 7.

2.

3. The method for preparing a natural dentin extraction material according to claim 1, characterized in that: In S1, the particle size of the dentin powder was 80-200 μm.

4. The dentin extract prepared by the method for preparing a natural dentin extraction material according to any one of claims 1 to 3.

5. Use of the dentin extract according to claim 4 in the preparation of bone replacement materials, root perforation repair materials, pulp capping materials, desensitizing materials for dentin hypersensitivity and materials for promoting tooth remineralization.

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