A light-cured resin with adjustable refractive index, its preparation method and application in tissue embedding

Abstract

The application provides a light-cured resin with adjustable refractive index, a preparation method of the light-cured resin and application of the light-cured resin in tissue embedding. Raw material components for preparing the light-cured resin include 0.5-5% of a photo initiator, 60-90% of polymerizable monomers, 1-20% of high refractive index monomers and 4-20% of diluents. The light-cured resin of the application does not contain toxic and harmful volatile organic solvents; the product has small viscosity, can penetrate into tissues as a mounting medium, and is fast and uniform; after curing, a semi-interpenetrating network structure is formed, which can effectively reduce volume shrinkage and improve mounting quality; the curing time can be shortened to 1 min, and the light-cured resin is expected to be applied to automatic mounting medium; and the cured mounting medium has high transparency and adjustable refractive index.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of tissue embedding technology, and particularly relates to a light refraction index adjustable photocuring resin, a preparation method thereof and application thereof in tissue embedding. BACKGROUND

[0002] In the disciplines of pathology and forensic science, when studying, observing and judging the morphological changes of cells and tissues, it is generally necessary to fix the tissues and slice them. In the slicing process, the tissue structure should not be damaged, and if there is an implant, the original tissue structure and morphology between the tissue and the implant should be maintained. Slicing includes paraffin sectioning, frozen sectioning, ultrathin sectioning and other technologies. In hard tissue sectioning, it is mainly aimed at bone tissue, bone tissue with implants, other tissue specimens with hard implants, or bone tissue with osteogenic fluorescent markers that cannot be decalcified during the animal experiment stage. Through dehydration, infiltration and embedding treatment. Fixing the tissue has always been a challenge in hard tissue sectioning. Hard tissue embedding and sectioning technology generally requires the following special processing steps: fixation, penetration dehydration, embedding, and precise slicing to obtain thin sections. In terms of the quality and quantity of regenerated bone tissue, hard tissue embedding technology can maintain the integrity of calcified deposits and the integrity of the original bone morphology.

[0003] Photocuring resin is a special class of high molecular weight material that can rapidly change from a liquid to a solid state under the irradiation of ultraviolet light or other light sources. This transformation is achieved through a photo-induced polymerization reaction, in which monomer or prepolymer molecules in the resin are chemically linked to form a three-dimensional network structure of polymer. Photocuring resin is widely used in 3D printing, printing photosensitive plates, microchip circuit diagrams, UV coatings, UV inks, UV adhesives and other fields. The advantages of using photocuring resin for hard tissue embedding mainly include:

[0004] (1) Rapid curing: Photocuring resin can rapidly solidify under ultraviolet light irradiation, greatly shortening the time of tissue embedding. Good preservation of tissue structure: Photocuring resin can well maintain the original structure and morphology of the tissue, which is very important for subsequent histological studies and analysis.

[0005] (2) Strong adaptability: Photocuring resin is suitable for various different hard tissue samples, including bone tissue, teeth and implanted non-metallic biomaterials, etc.

[0006] (3) Easy to operate: Compared with some traditional embedding materials, photocuring resin is easier and faster to operate, and is easy to master.

[0007] (4) High resolution: The tissue sections embedded with photocuring resin can achieve high resolution, which is beneficial for the observation of fine structures.

[0008] (5) Reduce sample damage: Since the curing process of light-cured resin does not require high temperature, it reduces the damage to heat-sensitive samples.

[0009] (6) Suitable for automated processing: The use of light-cured resin can be combined with automated sectioning and grinding equipment, improving the efficiency and repeatability of experiments.

[0010] (7) Good chemical stability: Light-cured resin has good chemical stability and is not easily affected by environmental factors, which is beneficial for long-term preservation of samples.

[0011] (8) Transparency: Light-cured resin has good transparency, which helps to observe the natural structure of the sample without staining.

[0012] (9) Multiple uses: Light-cured resin can not only be used for traditional histological research, but also be suitable for hard tissue samples that require special processing, such as research on teeth and bone tissue. High refractive index mounting agents can be directly applied to fluorescently labeled cells or tissue samples on microscope slides to improve the clarity and resolution of imaging. Used to mount stained tissue sections to obtain clearer microscopic effects and help long-term preservation of tissue specimens to prevent oxidation and discoloration.

[0013] In summary, high refractive index mounting agents are important in improving imaging quality, protecting fluorescently labeled samples, and specific industrial applications. Different application scenarios have different requirements for refractive index, and the refractive index of the resin needs to have simple and convenient adjustment capabilities.

[0014] The embedding agents in the prior art usually contain harmful volatile solvents, have large viscosity, uneven penetration, long curing time, usually up to 30 min, serious volume shrinkage, affect the mounting quality, need manual operation, low efficiency, and narrow adjustable range of refractive index.

[0015] Therefore, it is urgent to provide a light-cured resin with adjustable refractive index for tissue embedding. SUMMARY

[0016] Based on the defects of the prior art, the first purpose of the present application is to provide a light-cured resin with adjustable refractive index; the second purpose of the present application is to provide a preparation method of the light-cured resin with adjustable refractive index; the third purpose of the present application is to provide the application of the light-cured resin with adjustable refractive index in tissue embedding.

[0017] The purpose of the present application is achieved by the following technical solutions:

[0018] On the one hand, the present application provides a light-cured resin with adjustable refractive index, the raw material components for preparing the light-cured resin include, calculated as 100% by mass fraction:

[0019]

[0020] In the above light-cured resin, preferably, the photoinitiator is selected from 2,4,6 (trimethylbenzoyl) diphenyl phosphine oxide (TPO) and / or 2,4,6-trimethylbenzoyl ethyl phosphonate (TPO-L); but not limited thereto.

[0021] In the above light-cured resin, preferably, the polymerizable monomer is selected from methacrylate monomers.

[0022] In the above light-cured resin, preferably, the methacrylate monomer is selected from a combination of one or more of hydroxyethyl methacrylate, polyethylene glycol methacrylate, isobornyl methacrylate, methyl methacrylate, ethyl methacrylate, butyl methacrylate, isobutyl methacrylate, 2-ethylhexyl methacrylate, glycidyl methacrylate, phenoxyethyl methacrylate, dimethylaminoethyl methacrylate and trifluoroethyl methacrylate; but not limited thereto.

[0023] In the above light-cured resin, preferably, the polyethylene glycol methacrylate has a molecular weight of 200-2000.

[0024] In the above light-cured resin, preferably, the high refractive index monomer is selected from a combination of one or more of 2-methyl-2-propylene acid cyclo-mercapto-ethylmethyl ester, 2-(methylthio) ethyl methacrylate, 2-(methylseleno) ethyl methacrylate, 4-methylthiobenzyl methacrylate and 4-methylselenobenzyl methacrylate; but not limited thereto.

[0025] In the above light-cured resin, preferably, the diluent is selected from one or more of polyethylene glycol, polypropylene glycol and polyethylene imine; but not limited thereto.

[0026] In the above light-cured resin, preferably, the diluent has a molecular weight of 300-5000.

[0027] In another aspect, the present application also provides a preparation method of the above light-cured resin, which comprises:

[0028] The polymerizable monomer, the high refractive index monomer, the diluent and the photoinitiator are sequentially added to a stirred tank, and stirred uniformly to obtain the light-cured resin.

[0029] In still another aspect, the present application also provides the use of the above light-cured resin in tissue embedding.

[0030] The above description is only a summary of the technical scheme of the present application, in order to more clearly understand the technical means of the present application, and can be implemented according to the content of the specification, the following is the preferred embodiment of the present application and the detailed description of the drawings. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to more clearly illustrate the technical scheme of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or the prior art description, obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0032] Figure 1 The embedding effect comparison of the two groups of embedding agents for the conventional HE staining of the present application example 5 and the control group (low power objective), A is the present application example 5, B is the control group.

[0033] Figure 2 The embedding effect comparison of the two groups of embedding agents for the conventional HE staining of the present application example 5 and the control group (high power objective), A is the present application example 5, B is the control group.

[0034] The beneficial effects of the present application:

[0035] The photocuring resin of the present application:

[0036] (1) does not contain toxic and harmful volatile organic solvents, which can avoid the harm of harmful solvents to people and the corrosion of machines;

[0037] (2) the product viscosity is small, which can penetrate into the tissue as an embedding agent, and is fast and uniform;

[0038] (3) compared with the existing non-photocuring embedding agent, the photocuring agent of the present application forms a semi-interpenetrating network structure after curing, which can effectively reduce the volume shrinkage and improve the embedding quality;

[0039] (4) the embedding time is accelerated, compared with the existing non-photocuring embedding agent, the curing time of the photocuring agent of the present application can be shortened to 1 min, which is expected to be applied to automatic embedding agent;

[0040] (5) the refractive index can be accurately adjusted according to the content of sulfur-containing and selenium-containing monomers, which can effectively improve the refractive index of the resin, and the transparency of the cured embedding agent is high, and the refractive index is adjustable.

[0041] The above description is only a summary of the technical scheme of the present application, in order to more clearly understand the technical means of the present application, and can be implemented according to the content of the specification, the following is the preferred embodiment of the present application and the detailed description of the drawings. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of the present application. The process, conditions, reagents, experimental methods and the like for implementing the present application are common knowledge and common sense in the art, and the present application does not have special limitations.

[0043] Embodiment 1

[0044] The present embodiment provides a refractive index adjustable photocuring resin, and the raw material components for preparing the photocuring resin include, in mass percentage:

[0045] Polymerizable monomer (80%):

[0046] Hydroxyethyl methacrylate 30%

[0047]

[0048] The preparation method of the refractive index adjustable photocuring resin is as follows:

[0049] According to the mass ratio described above, hydroxyethyl methacrylate, polyethylene glycol methacrylate (molecular weight 400), isobornyl methacrylate, 2-(methylthio) ethyl methacrylate, polyethyleneimine (molecular weight 600) and 2,4,6 (trimethylbenzoyl) diphenyl phosphine oxide (TPO) are sequentially added to the stirred tank, and stirred uniformly to obtain an embedding resin, i.e. a refractive index adjustable photocuring resin.

[0050] The present embodiment also provides the application of the refractive index adjustable photocuring resin in tissue embedding.

[0051] Embodiment 2

[0052] The present embodiment provides a refractive index adjustable photocuring resin, and the raw material components for preparing the photocuring resin include, in mass percentage:

[0053]

[0054] The preparation method of the refractive index adjustable photocuring resin is as follows:

[0055] According to the mass ratio, hydroxyethyl methacrylate, polyethylene glycol methacrylate (molecular weight 400), isobornyl methacrylate, 2-(methylthio)ethyl methacrylate, polyethyleneimine (molecular weight 1000) and 2,4,6(trimethylbenzoyl) diphenyl phosphine oxide (TPO) are sequentially added to the stirred tank, and the embedding resin, i.e. the refractive index adjustable photocuring resin, is obtained by stirring uniformly.

[0056] The embodiment also provides application of the refractive index adjustable photocuring resin in tissue embedding.

[0057] Example 3:

[0058] The embodiment provides a refractive index adjustable photocuring resin. In mass percentage, raw material components for preparing the photocuring resin include:

[0059] Polymerizable monomer (80%):

[0060]

[0061] The preparation method of the refractive index adjustable photocuring resin is as follows:

[0062] According to the mass ratio, hydroxyethyl methacrylate, polyethylene glycol methacrylate (molecular weight 400), isobornyl methacrylate, 2-(methylthio)ethyl methacrylate, polyethyleneimine (molecular weight 2000) and 2,4,6(trimethylbenzoyl) diphenyl phosphine oxide (TPO) are sequentially added to the stirred tank, and the embedding resin, i.e. the refractive index adjustable photocuring resin, is obtained by stirring uniformly.

[0063] The embodiment also provides application of the refractive index adjustable photocuring resin in tissue embedding.

[0064] Example 4:

[0065] The embodiment provides a refractive index adjustable photocuring resin. In mass percentage, raw material components for preparing the photocuring resin include:

[0066] Polymerizable monomer (80%):

[0067] Hydroxyethyl methacrylate 40%

[0068] Isobornyl methacrylate 40%

[0069] High refractive index monomer: 2-(methylthio)ethyl methacrylate 10%

[0070] Diluent: polyethylene glycol (molecular weight 400) 9.5%

[0071] Photoinitiator: 2,4,6(trimethylbenzoyl)diphenyl phosphine oxide 0.5%

[0072] The preparation method of the refractive index adjustable photocuring resin is as follows:

[0073] According to the mass ratio, hydroxyethyl methacrylate, isobornyl methacrylate, 2-(methylseleno)ethyl methacrylate, polyethylene glycol (molecular weight 400) and 2,4,6(trimethylbenzoyl)diphenyl phosphine oxide (TPO) are sequentially added to the stirring kettle, and the embedding resin, i.e. the refractive index adjustable photocuring resin, is obtained after uniform stirring.

[0074] The embodiment also provides the application of the refractive index adjustable photocuring resin in tissue embedding.

[0075] Example 5:

[0076] The embodiment provides a refractive index adjustable photocuring resin. The raw material components for preparing the photocuring resin include, in mass percentage:

[0077] Polymerizable monomer (80%):

[0078] Hydroxyethyl methacrylate 40%

[0079] Isobornyl methacrylate 40%

[0080] High refractive index monomer: 2-(methylseleno)ethyl methacrylate 15%

[0081] Diluent: polyethylene glycol (molecular weight 2000) 4.5%

[0082] Photoinitiator: 2,4,6(trimethylbenzoyl)diphenyl phosphine oxide 0.5%

[0083] The preparation method of the refractive index adjustable photocuring resin is as follows:

[0084] According to the mass ratio, hydroxyethyl methacrylate, isobornyl methacrylate, 2-(methylseleno)ethyl methacrylate, polyethylene glycol (molecular weight 2000) and 2,4,6(trimethylbenzoyl)diphenyl phosphine oxide (TPO) are sequentially added to the stirring kettle, and the embedding resin, i.e. the refractive index adjustable photocuring resin, is obtained after uniform stirring.

[0085] The embodiment also provides the application of the refractive index adjustable photocuring resin in tissue embedding.

[0086] The viscosity before photocuring, photocuring time and refractive index of the photocuring resins prepared in the above Examples 1-5 are tested by using an M-2000V ellipsometer, and the test results are shown in Table 1 below.

[0087] Table 1:

[0088]

[0089] From the experimental data of Table 1, it can be seen that the viscosity of the photocuring agent can be adjusted by adding diluents, which is conducive to adapting to the operation of various automatic embedding machines. The refractive index of the embedding agent can be adjusted by adding high refractive index monomers. By adjusting the proportion, the refractive index of the embedding agent can be accurately adjusted between 1.51 and 1.68. Since it does not contain low-boiling toxic and harmful organic solvents, it can protect the health of experimental operators, and at the same time, it does not damage the automatic embedding machine.

[0090] Embedding experiment of tissue sections:

[0091] 400 pathological sections were randomly selected; commercial traditional neutral gum (20% xylene) and environmentally friendly photocuring agent prepared in Example 5 were used for embedding test.

[0092] The randomly taken pathological sections were divided into A and B groups, then placed in a 60°C oven for 30 min, then subjected to conventional HE staining, and after staining, placed in a fume hood for airing to semi-dry, and finally manually embedded by the same pathologist, wherein the A group of sections used traditional neutral gum for fixation, and the B group of sections used the environmentally friendly photocuring agent of Example 5 for fixation. The same pathologist embedded the A and B groups of sections, using a dropper to take an appropriate amount of reagent and drop it on the slide, appropriately cutting the angle and covering the cover glass, the A group of sections were placed in a fume hood at room temperature, and the B group of sections were placed under a UV lamp for irradiation to complete the embedding. The experimental results are shown in Table 1 below. Figure 1 and Figure 2 .

[0093] Table 1: Comparison of embedding effects of two groups of embedding agents

[0094] Features Group A (N=200) B (N=200) X 2 values P value No 148 131 Bubbles Few 37 31 56.214 <0.05 Many 15 38 Excess glue Yes 32 69 27.196 <0.05 No 168 131 Gum distribution Even 182 144 26.238 <0.05 Uneven 18 56 Clarity Clear 185 152 24.373 <0.05 Unclear 15 48 After drying Yellowing 11 15 9.313 <0.05 No 189 185

[0095] The results show that the A group is naturally dried and embedded, and the B group is completed within 1 min under the irradiation of the lightening gum under the UV light of 60W intensity. Through parallel observation and comparison between the two groups, it is found that there is no significant difference in the indicators of the pathological sections after embedding by the A and B groups of embedding agents. The overall section embedding agent is evenly distributed, with less overflow, good transparency, and a small number of yellowing after drying but does not affect the overall reading effect. Under the microscope, the tissue cells can present clear structure, and the conventional HE staining cells are clearly red and blue. Figure 1 、 Figure 2 The comparison of the effects of the two groups of sections is shown in Table 1. Both groups of sections can be stored for a long time after drying for pathological reading and archiving.

[0096] The application adopts an environment-friendly photocuring agent to perform ultraviolet curing after sealing, and proves that the environment-friendly sealing agent is a good choice for replacing the traditional neutral balsam sealing agent, and is a non-pollution safe reagent worthy of promotion without affecting pathological reading quality.

[0097] The principles and implementation manners of the present application are described by using specific examples, and the above examples are only used to help understand the method and core idea of the present application; meanwhile, for the general skilled person in the art, the specific implementation manners and application ranges will be changed according to the idea of the present application, and the above description should not be understood as limiting the present application.

Claims

1. A light-cured resin having an adjustable refractive index, characterized by, The raw material components for preparing the photocuring resin include, in terms of 100% by mass: a photoinitiator 0.5%~5%; a polymerizable monomer 60%~90%; a high refractive index monomer 1%~20%; a diluent 4%~20%; the polymerizable monomer is selected from methacrylate monomers; the methacrylate monomer is selected from a combination of one or more of hydroxyethyl methacrylate, polyethylene glycol methacrylate, isobornyl methacrylate, methyl methacrylate, ethyl methacrylate, butyl methacrylate, isobutyl methacrylate, 2-ethylhexyl methacrylate, glycidyl methacrylate, phenoxyethyl methacrylate, dimethylaminoethyl methacrylate and trifluoroethyl methacrylate; the high refractive index monomer is selected from a combination of one or more of 2-methyl-2-propenyl sulfidemethyl acrylate, 2-(methylthio)ethyl methacrylate, 2-(methylseleno)ethyl methacrylate, 4-methylthiobenzyl methacrylate and 4-methylselenobenzyl methacrylate; the diluent is selected from one or more of polyethylene glycol, polypropylene glycol and polyethyleneimine; the molecular weight of the diluent is 300~5000.

2. The photocurable resin according to claim 1, characterized in that: the photoinitiator is selected from 2,4,6(trimethylbenzoyl)diphenyl phosphine oxide and / or 2,4,6-trimethylbenzoyl ethyl phosphonate.

3. The light-cured resin of claim 1, wherein: the molecular weight of the polyethylene glycol methacrylate is 200~2000.

4. The method of producing a photocurable resin according to any one of claims 1 to 3, characterized by, The photocuring resin preparation method includes: adding the polymerizable monomer, the high refractive index monomer, the diluent and the photoinitiator into a stirred tank in sequence, stirring uniformly to obtain the photocuring resin.

5. Application of the photocuring resin of any one of claims 1~3 or prepared by the preparation method of claim 4 in tissue embedding.

Images