Improved hard tissue decalcification liquid and application thereof

Abstract

The invention provides an improved hard tissue decalcification solution and application thereof, and relates to the technical field of hard tissue decalcification, the improved hard tissue decalcification solution is obtained by mixing EDTA, ammonia water and water, and Ca < 2 + > ions are jointly chelated by the ammonia water and the EDTA in a water environment; the mass volume ratio of the EDTA to the ammonia water to the water is (140 to 280) g to (20 to 400) mL to (1000 to 2000) mL; the pH value of the improved hard tissue decalcification liquid is 6.5-7.5, the decalcification speed is accelerated by accurately controlling the pH value, the decalcification period is shortened by about 50%, it is guaranteed that antigen-antibody combination is not affected after decalcification, mild decalcification is achieved, and a high-quality sample is provided for subsequent histological research.

Description

Technical Field

[0001] The present invention relates to the technical field of hard tissue decalcification, and particularly relates to an improved hard tissue decalcifying solution and its application. Background Art

[0002] The research and application of hard tissue decalcifying solutions are an important field in histology, pathology, zoology, and forensic medicine, especially in the research of dental and bone tissues. Decalcification refers to the removal of calcium salts from hard tissues to facilitate sectioning and histological studies. This process is of great significance for understanding the development, diseases, and treatments of bones and teeth. Commonly used decalcifying solutions are mainly divided into acidic decalcifying solutions and non-acidic decalcifying solutions. Acidic decalcifying solutions, such as hydrochloric acid, formic acid, and nitric acid, achieve decalcification by dissolving calcium salts in hard tissues. Non-acidic decalcifying solutions, such as ethylenediaminetetraacetic acid (EDTA), achieve decalcification by chelating calcium ions.

[0003] Currently, the research on tooth decalcification mainly focuses on dentin and pulp pathology. Since teeth contain a large amount of calcium salts, the decalcification process needs to be carefully handled to maintain the integrity of tooth tissues. Research has shown that EDTA is widely used in tooth decalcification due to its chelating effect, especially in studies that require maintaining the integrity of cell structures. EDTA decalcification has the characteristics of mildness and stable effects, but the process is relatively slow.

[0004] Bone tissue decalcification involves more complex structures, including osteoblasts, bone matrix, and minerals. A rapid and effective decalcification method is crucial for bone histology, bone pathology, and bone biology research. Hydrochloric acid and formic acid are commonly used in bone tissues due to their rapid decalcification ability, but these acidic decalcifying solutions may damage the microstructure of tissues and destroy antigen components. To balance the decalcification speed and the quality of tissue preservation, researchers have explored improved acidic and non-acidic decalcifying solutions. Currently, EDTA is widely used in the hard tissue decalcification process because it can effectively bind to calcium ions to achieve the purpose of decalcification. Compared with strong acid decalcification, such as formic acid and hydrochloric acid, EDTA can well preserve tissue structures, the antigen substances in tissues, and the activities of certain enzymes, ensuring the accuracy of the final research results. However, the decalcification time of the EDTA decalcifying solution is too long. Typically, the decalcification of bone samples takes 4 to 5 weeks. This long decalcification process not only reduces the research efficiency but also affects the staining results for chromogenic studies. For example, in fluorescence-labeled staining, the fluorescence label will quench over time.

[0005] Currently, an improved EDTA formulation is used to shorten the decalcification time. For example, 40% formaldehyde is added to EDTA to obtain an improved EDTA decalcifying solution. However, it can only eliminate the specimen fixation time and cannot effectively shorten the specimen decalcification time. For tissues with high calcification levels such as teeth, the decalcification time is much longer than the specimen fixation time, and an overly long decalcification time will damage the antigen and affect subsequent histological studies. Summary of the Invention

[0006] To solve the problems of long decalcification time of the existing EDTA decalcifying solution, affecting tissue structure details, and antigen loss, the present invention provides an improved hard tissue decalcifying solution and its application.

[0007] To achieve the above object, the specific technical solutions of the present invention are as follows.

[0008] In the first aspect of the present invention, an improved hard tissue decalcifying solution is provided. The improved hard tissue decalcifying solution is obtained by mixing EDTA, ammonia water, and water, and using ammonia water and EDTA to chelate Ca 2+ ions in an aqueous environment; The mass-volume ratio of EDTA, ammonia water, and water is 140 g - 280 g: 20 mL - 400 mL: 1000 mL - 2000 mL; wherein, the ammonia water is ammonia water with a mass percentage concentration of 28%; The pH value of the improved hard tissue decalcifying solution is 6.5 - 7.5.

[0009] In the present invention, the mass-volume ratio of EDTA, ammonia water, and water is 140 g - 280 g: 20 mL - 400 mL: 1000 mL - 2000 mL. By controlling the amount of saturated ammonia water used, it can not only prevent the ability of EDTA to chelate calcium ions from being affected due to excessive addition amount, but also avoid the solubility of EDTA from being affected due to too low addition amount, thereby affecting the decalcification efficiency. If the pH value is too acidic or too alkaline, the effective chelating ability for calcium ions will be reduced, and the stability and effect of the improved hard tissue decalcifying solution during use will be affected. When the pH value is 6.5 - 7.5, 90% of the EDTA molecules lose 3 protons from their acetic acid parts, so the dominant ion in the solution is [HEDTA] 3- . The fourth acetic acid proton is transferred to a nitrogen atom. When [HEDTA] 3- undergoes chelation, the last acetic acid proton is lost, forming a molecule dominated by [EDTA] 4- , thus quickly achieving hard tissue decalcification.

[0010] The improved hard tissue decalcifying solution in the present invention can significantly shorten the decalcification time required and prevent antigen loss without damaging the tissue structure details, effectively solving the problems of long time and possible affecting tissue detail preservation and antigen loss in traditional decalcification methods.

[0011] In another preferred embodiment, the pH value is adjusted using a hydrochloric acid solution.

[0012] In another preferred embodiment, the mass percentage of the hydrochloric acid solution is 36% - 38%.

[0013] The second aspect of the present invention provides the application of the improved hard tissue decalcifying solution in hard tissue decalcification.

[0014] The third aspect of the present invention provides a method for hard tissue decalcification, using the improved hard tissue decalcifying solution provided by the first aspect of the present invention to decalcify hard tissue.

[0015] In another preferred embodiment, the hard tissue is completely immersed in the improved hard tissue decalcifying solution, and the improved hard tissue decalcifying solution is replaced every 2 to 3 days until a pin can penetrate the hard tissue, at which point decalcification is completed.

[0016] In another preferred embodiment, the volume ratio of the improved hard tissue decalcifying solution to the hard tissue is 10 - 50:1.

[0017] In another preferred embodiment, the hard tissue is bone tissue or dental tissue.

[0018] Compared with the prior art, the present invention has the following beneficial effects.

[0019] (1) In the present invention, the improved hard tissue decalcifying solution uses ammonia water solution and EDTA to jointly chelate Ca 2+ ions, effectively improving the decalcification effect. By controlling the addition amount of saturated ammonia water, while ensuring the ability of EDTA to chelate calcium ions, it can also increase the solubility of EDTA. Compared with traditional formic acid and strong acid decalcifying solutions, it can effectively solve the problems of tissue microstructure damage and antigen component destruction caused by acidic decalcifying solutions.

[0020] By controlling the pH value of the improved hard tissue decalcifying solution of the present invention at 6.5 - 7.5, the decalcification speed can be further accelerated, ensuring the effective chelating ability of calcium ions and the stability of the improved hard tissue decalcifying solution during the decalcification process.

[0021] (2) The improved hard tissue decalcifying solution in the present invention can not only shorten the decalcification time, but also maintain the microstructure and antigen epitopes of the tissue after decalcification, achieving mild decalcification, effectively solving the problems of existing acidic decalcification damaging the tissue microstructure and destroying antigen components. Moreover, it has a wide application range and is applicable to various types of hard tissue samples, including bone tissue, teeth, etc.

[0022] (3) The improved hard tissue decalcifying solution in the present invention can shorten the decalcifying time of the traditional EDTA decalcifying solution by about 50% or more. For example, the decalcification of human teeth and bone tissues is shortened from 4 - 8 weeks to 2 - 4 weeks, greatly improving the efficiency of tissue processing. Ammonia in the improved hard tissue decalcifying solution can chelate Ca 2+ ions, thus more effectively removing calcium from bone. By precisely controlling the pH value, not only is the decalcifying speed accelerated, but also it is ensured that antigen-antibody binding is not affected after decalcification, providing high-quality samples for subsequent histological research. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It shows the decalcifying principle of the improved hard tissue decalcifying solution in the present invention.

[0024] Figure 2 It is a typical cross-sectional Micro-CT image of the decalcification of canine teeth and jawbone tissues by the traditional decalcifying solution EDTA; among them, A is a typical cross-sectional Micro-CT image of canine teeth and jawbone tissues without decalcification, and B is a typical cross-sectional Micro-CT image of canine teeth and jawbone tissues decalcified by the traditional decalcifying solution EDTA for 1 month.

[0025] Figure 3 It is a typical posterior tooth coronal Micro-CT image of the decalcification of canine teeth and jawbone tissues by the traditional decalcifying solution EDTA; among them, A is a typical posterior tooth coronal Micro-CT image of canine teeth and jawbone tissues without decalcification, and B is a typical posterior tooth coronal Micro-CT image of canine teeth and jawbone tissues decalcified by the traditional decalcifying solution EDTA for 1 month.

[0026] Figure 4 It is a typical anterior tooth coronal Micro-CT image of the decalcification of canine teeth and jawbone tissues by the traditional decalcifying solution EDTA; among them, A is a typical anterior tooth coronal Micro-CT image of canine teeth and jawbone tissues without decalcification, and B is a typical anterior tooth coronal Micro-CT image of canine teeth and jawbone tissues decalcified by the traditional decalcifying solution EDTA for 1 month.

[0027] Figure 5 It is a typical cross-sectional Micro-CT image of the decalcification of canine teeth and jawbone tissues by the improved hard tissue decalcifying solution of Example 1 of the present invention; among them, A is a typical cross-sectional Micro-CT image of canine teeth and jawbone tissues without decalcification, and B is a typical cross-sectional Micro-CT image of canine teeth and jawbone tissues decalcified by the improved hard tissue decalcifying solution in Example 1 for 1 month.

[0028] Figure 6Typical posterior tooth coronal Micro-CT images of the decalcified canine teeth and jaw tissues with the improved hard tissue decalcifying solution of Example 1 of the present invention; among them, A is the typical posterior tooth coronal Micro-CT image of the canine teeth and jaw tissues without decalcification, and B is the typical posterior tooth coronal Micro-CT image of the canine teeth and jaw tissues decalcified with the improved hard tissue decalcifying solution of Example 1 of the present invention for 1 month.

[0029] Figure 7 Typical anterior tooth coronal Micro-CT images of the decalcified canine teeth and jaw tissues with the improved hard tissue decalcifying solution of Example 1 of the present invention; among them, A is the typical anterior tooth coronal Micro-CT image of the canine teeth and jaw tissues without decalcification, and B is the typical anterior tooth coronal Micro-CT image of the canine teeth and jaw tissues decalcified with the improved hard tissue decalcifying solution of Example 1 of the present invention for 1 month.

[0030] Figure 8 Statistical chart of density values after decalcifying canine teeth and jaw tissues with different decalcifying solutions.

[0031] Figure 9 HE staining results of tissues after decalcification with the improved hard tissue decalcifying solution of Example 1 of the present invention; A is HE staining of mouse jaw bone and dental follicle sections, and B is HE staining of mouse palatal mucosa tissue sections.

[0032] Figure 10 Microscopic image of COL-1 immunohistochemical staining of mouse jaw bone and dental follicle sections in the present invention.

[0033] Figure 11 Microscopic images of immunofluorescence staining of mouse jaw bone and dental follicle sections after decalcification with the improved hard tissue decalcifying solution of Example 1 of the present invention; A shows the expression of Axin2 in the 488 channel with Axin2-GFP; B shows the cell nucleus in the 405 channel with DAPI; C shows the expression of RUNX2 in the 596 channel with RUNX2; D shows the expression of CD31 in the 647 channel with CD31; E shows the merged display of multiple channels with Merge. Detailed implementation manners

[0034] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods; unless otherwise specified, the reagents and materials can all be obtained in the market.

[0036] In order to improve the decalcification efficiency of EDTA, the prior art has the following improvement methods for EDTA.

[0037] Add 40% formaldehyde to EDTA and adjust the pH to 7.0. The specimen is decalcified while being fixed, which improves the decalcification efficiency. However, usually the specimen fixation time is 4 - 24 hours. Adding 40% formaldehyde to EDTA can only save the specimen fixation time. For tissues with a high degree of calcification such as teeth, the decalcification time is much longer than the specimen fixation time. This method has limited improvement in decalcification efficiency, and the excessive decalcification time will damage the antigen. Or add physical stirring during EDTA decalcification and heat during EDTA decalcification. The above methods have limited improvement in decalcification efficiency and will also cause damage to tissue antigens.

[0038] Based on this, after the prior art improves EDTA, it cannot achieve rapid and gentle decalcification of hard tissues, namely bone tissues or tooth tissues.

[0039] The embodiment of the present invention provides an improved hard tissue decalcifying solution and its application. By mixing EDTA with an ammonia water solution and precisely controlling the pH value, the decalcification time can be effectively shortened. Among them, ammonia water can chelate Ca 2+ ions, thereby more effectively removing calcium from bone. Under the precise control of the pH value, the decalcification speed is further accelerated. At the same time, it also ensures that the antigen-antibody binding is not affected after decalcification, realizing gentle decalcification and providing high-quality samples for subsequent histological research. The main component of dental and skeletal calcifications is Ca3(PO4)2. The working principle of the improved hard tissue decalcifying solution in the present invention is as Figure 1 shown.

[0040] The following specifically describes an improved hard tissue decalcifying solution and its application.

[0041] In the following examples, COL-1 was purchased from Proteintech, USA, with the model number 66761-1-Ig, 1:500.

[0042] The 10wt% neutral EDTA decalcifying solution was purchased from Solarbio E1171, China.

[0043] Example 1: An improved hard tissue decalcifying solution, which is prepared by the following method.

[0044] Add 100 mL of ammonia water with a mass percentage concentration of 28% to 1000 mL of distilled water and mix well, then slowly add 150 g of EDTA, and adjust the pH value to 7.0 with hydrochloric acid with a mass percentage of 36% to obtain the improved hard tissue decalcifying solution.

[0045] Example 2: An improved hard tissue decalcifying solution, which is prepared by the following method.

[0046] After adding 20 mL of ammonia water with a mass percentage concentration of 28% to 1000 mL of distilled water and mixing well, 140 g of EDTA was slowly added, and the pH value was adjusted to 6.5 with hydrochloric acid with a mass percentage of 37% to obtain a modified hard tissue decalcifying solution.

[0047] Example 3: A modified hard tissue decalcifying solution, which is prepared by the following method.

[0048] After adding 50 mL of ammonia water with a mass percentage concentration of 28% to 1000 mL of distilled water and mixing well, 145 g of EDTA was slowly added, and the pH value was adjusted to 6.8 with hydrochloric acid with a mass percentage of 38% to obtain a modified hard tissue decalcifying solution.

[0049] Example 4: A modified hard tissue decalcifying solution, which is prepared by the following method.

[0050] After adding 200 mL of ammonia water with a mass percentage concentration of 28% to 1600 mL of distilled water and mixing well, 200 g of EDTA was slowly added, and the pH value was adjusted with hydrochloric acid with a mass percentage of 37% to make the pH value reach 7.2 to obtain a modified hard tissue decalcifying solution.

[0051] Example 5: A modified hard tissue decalcifying solution, which is prepared by the following method.

[0052] After adding 300 mL of ammonia water with a mass percentage concentration of 28% to 1800 mL of distilled water and mixing well, 240 g of EDTA was slowly added, and the pH value was adjusted with hydrochloric acid with a mass percentage of 38% to make the pH value reach 7.4 to obtain a modified hard tissue decalcifying solution.

[0053] Example 6: A modified hard tissue decalcifying solution, which is prepared by the following method.

[0054] After adding 400 mL of ammonia water with a mass percentage concentration of 28% to 2000 mL of distilled water and mixing well, 280 g of EDTA was slowly added, and the pH value was adjusted with hydrochloric acid with a mass percentage of 37% to make the pH value reach 7.5 to obtain a modified hard tissue decalcifying solution.

[0055] In the above Examples 1 to 6, a modified hard tissue decalcifying solution was prepared, and the effects were comparable. Taking the modified hard tissue decalcifying solution in Example 1 as an example, the description is as follows.

[0056] 1. Decalcification of canine teeth and jawbone tissues.

[0057] The improved hard tissue decalcifying solution in Example 1 and the traditional EDTA decalcifying solution were used to decalcify hard tissue canine teeth and jaw tissues respectively, and the results are as Figures 2 to 7 shown. Among them, the volume ratios of the improved hard tissue decalcifying solution in Example 1 and the traditional EDTA decalcifying solution to the canine teeth and jaw tissues are both 50:1.

[0058] As can be seen from Figures 2 to 7 using the traditional EDTA decalcifying solution and the improved hard tissue decalcifying solution in Example 1 of the present invention to decalcify canine teeth and jaw tissues, Materialise Mimics 21.0 medical imaging analysis software was used for analysis before decalcification and 1 month after decalcification. After 1 month of decalcification with the improved hard tissue decalcifying solution in Example 1 of the present invention, the sample density value was significantly lower than that of the decalcification group with the traditional EDTA decalcifying solution.

[0059] Figure 8 It is a statistical chart of the density values of canine teeth and jaw tissues after decalcification with different decalcifying solutions. As can be seen from Figure 8 the bone density after 1 month of decalcification with the traditional EDTA decalcifying solution is significantly higher than that after 1 month of decalcification with the improved hard tissue decalcifying solution in Example 1 of the present invention. It shows that the improved hard tissue decalcifying solution in Example 1 of the present invention has a good decalcification effect.

[0060] 2. Decalcification of mouse mandibles.

[0061] Fresh mandibles of 3-month-old adult mice with mandibular molars were taken, and the taken mandibles were fixed in 4wt% paraformaldehyde at 4°C for 12 h, and the soft tissues were removed to complete the fixation.

[0062] Traditional decalcification method: After fixation, it was immersed in 10wt% neutral EDTA decalcifying solution and decalcified with shaking at room temperature. The volume ratio of the EDTA decalcifying solution to the mandible was 10:1.

[0063] Decalcification method with the improved hard tissue decalcifying solution in Example 1 of the present invention: After fixation, it was immersed in the improved hard tissue decalcifying solution and decalcified with shaking at room temperature. Among them, the volume ratio of the improved hard tissue decalcifying solution to the mandible was 10:1.

[0064] The improved hard tissue decalcifying solution was changed every 3 days. When a pin could easily penetrate the tissue, it indicated that decalcification was completed, and the time was recorded; if there was resistance when piercing, decalcification continued. After decalcification was completed, it was thoroughly washed with distilled water to remove the residual improved hard tissue decalcifying solution.

[0065] HE staining: For routine sections, dewax, soak in hematoxylin staining solution for 10 min, wash with water for 1 min, differentiate with 1 wt% hydrochloric acid ethanol for 20 s, wash with water for 2 min, blue with ammonia water for 1 min, wash with water for 2 min, stain with eosin for 30 s, wash with water for 1 min, dehydrate with gradient ethanol, clear with xylene, and mount with neutral gum.

[0066] The quality of HE section staining was evaluated and scored according to the section quality scoring standard in the "Clinical Technical Operation Specification - Pathology Volume", and the results are shown in Table 1.

[0067] Table 1 Average decalcification time of mouse molars and HE section staining quality scores with two decalcifying solutions

[0068] Note: n is the sample size, a and b indicate significant differences, P < 0.05.

[0069] It can be seen from the results in Table 1 that compared with the EDTA decalcifying solution, the improved hard tissue decalcifying solution in Example 1 of the present invention can effectively shorten the decalcification time, and from the perspective of the HE section staining quality score, when decalcifying with two different decalcifying solutions, the HE section staining quality scores are the same. It can be seen that the improved hard tissue decalcifying solution in the present invention does not affect the subsequent HE section staining research.

[0070] Figure 9 The following is the HE staining result using the improved hard tissue decalcifying solution in Example 1 of the present invention. Figure 9 It can be seen that the tissue sections are complete, the decalcification is uniform, the thickness is uniform, there are no knife marks, wrinkles, folds, no pollution, good transparency, the contrast between the nucleus and cytoplasm staining is clear, and the sections are not loose and are neat.

[0071] Figure 10This is the immunohistochemical staining result after decalcification sectioning with the improved hard tissue decalcifying solution in Example 1 of the present invention. Fresh mandibles with mandibular molars from 1-month-old adult mice were taken, and the obtained mandibles were fixed in 4 wt% paraformaldehyde at 4 °C for 12 h to remove soft tissues. After fixation, they were placed in the improved hard tissue decalcifying solution of Example 1 and decalcified by oscillation at room temperature. The improved hard tissue decalcifying solution was changed every 3 days. After 4 days of decalcification, a pin could easily penetrate the tissue, and it was paraffin-embedded and sectioned. The tissue sections were routinely dewaxed, hydrated, and soaked in distilled water through xylene, xylene, 100 wt% ethanol, 100 wt% ethanol, 95 wt% ethanol, and 75 wt% ethanol. High-temperature and high-pressure repair was adopted, and it was repaired with a citric acid repair solution for 2.5 min. After natural cooling, it was placed in 3 wt% H2O2 for 10 min and rinsed 3 times with PBS, 3 min each time. The anti-COL-1 primary antibody was added and incubated overnight at 4 °C, and then rinsed 3 times with PBS, 3 min each time; the secondary antibody was added and incubated at 37 °C for 30 min, and then rinsed 3 times with PBS, 3 min each time. DAB was used for color development, and hematoxylin was used for counterstaining for 3 min. After acidification, it was blued, dehydrated, cleared, and sealed. The results are as Figure 5 shown. It can be seen from Figure 5 the tissue sections that they are complete, the decalcification is uniform, the thickness is uniform, there are no knife marks, wrinkles, or folds, there is no pollution, the transparency is good, the contrast between the nucleus and cytoplasm staining is clear, the sections are not loose, neat, and the DAB color development is good.

[0072] Figure 11This is the immunofluorescence staining result after decalcification sectioning with the improved hard tissue decalcifying solution in Example 1 of the present invention. Fresh mandibles of 1-month-old adult mice with mandibular molars were taken. The obtained mandibles were fixed in 4 wt% paraformaldehyde at 4 °C for 12 h to remove soft tissues. After fixation, they were placed into the improved hard tissue decalcifying solution and decalcified by oscillation at room temperature. The improved hard tissue decalcifying solution was changed every 3 days. After 4 days of decalcification, a pin could easily penetrate the tissue, and it was cryoembedded and sectioned. The sections were fixed in 4 wt% paraformaldehyde at room temperature for 15 minutes and permeated with PBS solution containing 0.25 wt% Triton X-100 for 10 minutes. To reduce non-specific binding, they were blocked with PBS solution containing 5 wt% normal goat serum for 30 minutes to 1 hour. Subsequently, the sections were covered with corresponding primary antibodies, such as antibodies against Axin2, RUNX2, CD31, purchased from Abcam, USA, and incubated overnight at 4 °C. The next day, the sections were washed 3 times with PBS for 5 minutes each time, and then incubated with corresponding fluorescently labeled secondary antibodies, in 488, 596, 647 channels, for 1 hour under dark conditions at room temperature. The sections were washed again with PBS, and nuclear staining was performed with DAPI for 10 minutes. Finally, the sections were sealed in an anti-fluorescence quenching mounting medium and sealed with a coverslip. A fluorescence microscope was used to observe and capture the expression of each marker. The expression of Axin2 was observed through the 488 channel, the nuclear DAPI staining was observed through the 405 channel, the expression of RUNX2 was observed through the 596 channel, and the expression of CD31 was observed through the 647 channel. At the same time, the overall expression was shown by multi-channel merging. From Figure 11 It can be seen that the tissue sections are complete, the decalcification is uniform, the thickness is uniform, there are no knife marks, wrinkles, folds, no pollution, the transparency is good, the contrast between nuclear and cytoplasmic staining is clear, the sections are not loose, neat, the multi-channel colors of the triple immunofluorescence are clear, and the color development is good.

[0073] From the above experimental results, it can be seen that the improved hard tissue decalcifying solution of the present invention can not only decalcify quickly, shortening the decalcification time of the traditional EDTA decalcification method by about 50% or more, but also will not damage the microstructure of the tissue and destroy the antigen components after decalcification, and can continue to be used for subsequent histological research.

[0074] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An improved decalcifying solution for hard tissues, characterized in that, The improved hard tissue decalcifying solution is obtained by mixing EDTA, ammonia water and water, and uses ammonia water and EDTA to jointly chelate Ca 2+ ions in an aqueous environment; The mass-volume ratio of EDTA, ammonia water and water is 140 g to 280 g: 20 mL to 400 mL: 1000 mL to 2000 mL; The pH value of the improved hard tissue decalcifying solution is 6.5 to 7.

5.

2. The improved hard tissue decalcifying solution according to claim 1, wherein The pH value is adjusted with hydrochloric acid solution.

3. The improved hard tissue decalcifying solution according to claim 2, wherein, The mass percentage concentration of the hydrochloric acid solution is 36% to 38%.

4. Application of the improved hard tissue decalcifying solution according to any one of claims 1 to 3 in hard tissue decalcification.

5. A method for decalcifying hard tissue, characterized in that, The hard tissue is decalcified with the improved hard tissue decalcifying solution according to claim 3.

6. The method for decalcifying hard tissue according to claim 5, characterized in that The hard tissue is completely immersed in the improved hard tissue decalcifying solution, and the improved hard tissue decalcifying solution is replaced every 2 to 3 days until the needle can penetrate the hard tissue, and the decalcification is completed.

7. The method for decalcifying hard tissues according to claim 5, characterized in that, The volume ratio of the improved hard tissue decalcifying solution to the hard tissue is 10 to 50:

1.

8. The method for decalcifying hard tissue according to claim 5, characterized in that, The hard tissue is teeth and bones.

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