CN107384788B

CN107384788B - Experimental device for in vitro dentin barrier cytotoxicity experiment

Info

Publication number: CN107384788B
Application number: CN201710584208.6A
Authority: CN
Inventors: 郑刚; 林红; 胡孟龙
Original assignee: Peking University School of Stomatology
Current assignee: Peking University School of Stomatology
Priority date: 2017-07-18
Filing date: 2017-07-18
Publication date: 2023-05-12
Anticipated expiration: 2037-07-18
Also published as: CN107384788A

Abstract

An experimental device for an in vitro dentin barrier cytotoxicity experiment comprises a culture dish and a material fixing ring, wherein a cell culture solution is contained in a culture well on the culture dish, a platform is arranged on the culture well and used for placing dentin sheets or other substitute materials, the material fixing ring is arranged on the culture well, dental restoration filling materials are filled in an inner ring of the fixing ring, and a protective cover is arranged on the culture dish. The cell nutrient solution in the culture well of the culture dish permeates into the material fixing ring through the micro-gap of the dentin sheet or the substitute material placed on the culture well and contacts with the dental restoration filling material in the material fixing ring, so that the proliferation rate of target cells in the cell culture solution in the culture well can be measured, the cell morphology can be observed, the cytotoxicity of the dental restoration filling material to be measured can be judged, the in vitro cytotoxicity experiment has better correlation with the in-vivo experiment, the biocompatibility of the dental restoration material can be measured better, the mesh structure is simple, the use is convenient, the manufacturing cost is low, and the experimental result repeatability is good.

Description

Experimental device for in vitro dentin barrier cytotoxicity experiment

Technical Field

The invention relates to an experimental device for in vitro dentin barrier cytotoxicity experiment

Background

With the progress of the scientific development technology, more and more novel dental filling and repairing materials are appeared. A clinically acceptable prerequisite for these dental filling restorative materials is that safety and efficacy tests and evaluations must be performed, and that determination of biocompatibility is an important prerequisite to ensure safe application of the materials to the human body. Biocompatibility refers to a property of living tissue that reacts to inactive materials, generally refers to the compatibility between the material and the host. After the biological material is implanted into a human body, the biological material can be influenced and acted on a specific biological tissue environment, the biological tissue can also be influenced and acted on the biological material, and the circulation action of the biological material and the biological material can be continued until the balance is achieved or the implant is removed. Biocompatibility is a subject of constant penetration in the study of biological materials.

The main means for evaluating the biocompatibility of oral materials are classified into in vitro tests, animal tests and clinical tests. Although the clinical relevance of in vitro tests is slightly less than that of the other two methods, the method has the advantages of simplicity, rapidness, capability of avoiding damage to animals and human bodies, and the like, so that the practical application is more common. In vitro cytotoxicity test methods of oral materials are classified into three types, i.e., leaching liquor test, direct contact test and indirect contact test.

Dental filling materials have been mainly used for the leaching solution test and the indirect contact test. The method comprises the steps of taking a leaching solution from a leaching solution sample, wherein the cytotoxicity can be quantitatively determined by the MTT method, the XTT method, the neutral red pickup method and other tests in the leaching solution test, and the agar diffusion method and the filter membrane diffusion method which are commonly used in indirect contact tests belong to the qualitative evaluation of the cytotoxicity of the material.

Although the above method has been commonly used for the detection of oral material biocompatibility, many studies have shown that the correlation between the response of cells in conventional in vitro tests and the response of pulp cells in vivo tests is low, and this difference is related to the structure of dentin and the specific growth environment of pulp cells in vivo. Because of the special contact mode of the dental filling material and the human body, namely dentin separation exists between the material and dental pulp cells, a scholars in the eighty th century put forward an in vitro cytotoxicity test which is specially applicable to the dental filling material and uses dentin as a barrier, and the result is found to have good correlation with the clinical practical situation.

Through long-term experiments and researches of different students, the test method is greatly improved, and the test device is also developed into a fixed model. The dentin barrier method applied at present simulates the process of contacting dentin after filling the dentin with a dental filling material, the material and filterable components thereof penetrate into cells below the dentin through dentinal tubules and contact with the cells, and the cytotoxicity of the material can be judged by quantitatively analyzing the survival of the cells.

Compared with other in vitro cytotoxicity tests, the method better simulates the clinical actual situation, has the possibility of replacing animal tests (such as dental pulp dentin test), and is more suitable for evaluating the cytotoxicity of dental filling materials and materials in direct contact with dentin. This method was listed in the data appendix B of the international standard for evaluating biocompatibility of dental medical instruments by the international standard organization ISO in 2008, and has been widely used for toxicity studies of related materials.

Disclosure of Invention

The invention aims to provide an experimental device for in vitro dentin barrier cytotoxicity experiment, which can be used for measuring cytotoxicity of a dental filling restoration material directly contacted with dentin and further evaluating biocompatibility of the dental filling restoration material. The device can simulate the contact mode of the dental filling repair material and dental pulp cells, better simulate the in-vivo situation and solve the problem that in the prior art, the in-vivo and in-vitro biocompatibility experiment results of part of dental filling repair materials are inconsistent.

The invention aims to realize the experimental device for the in-vitro dentin barrier cytotoxicity experiment, which comprises a culture well and a corresponding material fixing ring, wherein the culture well is used for accommodating cell culture solution, a protective cover is arranged above the culture well, tooth filling repair materials to be detected are placed in the material fixing ring, the material fixing ring is placed on a barrier sheet, and the barrier sheet is placed on an inner annular platform of the culture well in the culture well.

The depth of the culture well in the culture dish can be adjusted according to the requirement.

The barrier sheet is a dentin sheet of isolated human teeth or bovine teeth, or a porous ceramic sheet, or other alternative materials, and the thickness can be adjusted according to the requirements.

The culture dish, the material fixing ring and the protective cover are made of optical PMMA or polystyrene resin materials or other materials which can be used for culturing cells.

The invention has the following beneficial effects that the cell culture solution is placed in the culture well in the culture dish, the barrier sheet is placed on the annular platform above the culture well, and the cell culture solution is contacted with the barrier sheet; a material retainer ring is disposed on the barrier sheet, and material is disposed therein. The chemical components separated out of the material to be tested can contact with the cell culture solution through the barrier sheet, so that the growth of cells is influenced. The cytotoxicity of the test material can be determined by measuring the proliferation rate of cells in the culture solution and observing the morphological structure of the cells. The experimental device has better correlation with in-vivo experiments, can better simulate the clinical actual state, can better determine the biocompatibility of the dental filling repair material, and has the advantages of simple structure, low manufacturing cost, convenient use and good repeatability of experimental results.

Drawings

FIG. 1 is a schematic view of the structure of a culture dish, a material retainer ring and a protective cover above the culture dish according to the present invention;

FIG. 2 is a schematic diagram of the structure of the independent unit in the invention;

FIG. 3 is a schematic cross-sectional view of the present invention;

in the figure, 1, a base of a culture dish, 2, the culture dish, 3, a culture well in the culture dish, 4, a material fixing ring, 5, a protective cover of the culture dish, 6, a barrier sheet and 7, a dental restoration filling material to be measured.

Detailed Description

Example 1 an experimental set-up for in vitro dentin barrier cytotoxicity experiments comprising a culture well 3 for culturing cells, a material retainer 4, a culture dish protecting cover 5, and a culture dish 2. The culture dish 2 and the culture dish base 1 are integrated, 15 culture wells 3 are arranged in three rows of 5 culture wells, and the depth in the culture wells can be adjusted. The barrier sheet 6 is placed on the annular platform 3 on the culture well, and the barrier sheet can be an isolated human dentin sheet or a barrier sheet with a similar permeability substitute material, and the thickness of the barrier sheet can be adjusted according to experimental requirements. The number of material fixing rings 4 is 15, and the material fixing rings are respectively arranged on the barrier sheets 6 on the platform of each culture well 3.

The diameter of the barrier sheet 6 is larger than the inner diameter of the culture well 3, and the barrier sheet is directly placed on an annular platform on the culture well without any bonding. The operation of the step is simple and stable.

A plurality of culture wells 3 can be manufactured on the culture dish 2 according to experimental requirements, and the culture wells 3 are connected with the base 1 into a whole.

The barrier sheet 6 is placed on the annular platform on the culture well 3, then is contacted with the cell culture liquid in the culture well 3, and cells are inoculated in the culture well 3. The material fixing ring 4 is arranged on the barrier sheet 6, and the tooth filling repair material 7 to be measured is filled in the material fixing ring 4, and the operation sequence completely simulates the actual clinical operation process. The cell culture fluid in the culture well 3 can permeate through the barrier sheet 6 and contact the material 7 to be tested in the material fixing ring 4. Meanwhile, the chemical components of the material 7 to be tested also contact with the cell culture liquid in the culture well 3 through the barrier sheet 6, so that the cells in the culture well 3 are affected. After a period of action, the material 7 to be tested, the material fixing ring 4 and the barrier sheet 6 are removed, and the cell proliferation rate in the culture well 3 is measured and the morphological structure of the cells is observed. And judging the cytotoxicity of the dental restoration filling material to be tested according to the proliferation rate of the cells and the morphological structure of the cells.

All the devices described above are made of optical-grade PMMA or polystyrene resin or other materials that can be used to culture cells.

Claims

1. An experimental device for in vitro dentin barrier cytotoxicity experiments, characterized in that: the cell culture medium comprises a culture dish 2, a culture well 3 in the culture dish, a cell culture medium is contained in the culture well 3, the diameter of a barrier sheet 6 is larger than the inner diameter of the culture well 3, the barrier sheet 6 is directly arranged on an annular platform on the culture well 3, no adhesive is needed, a material fixing ring 4 is arranged on the barrier sheet 6, a material 7 to be tested is applied in the material fixing ring 4, and a protective cover 5 is arranged on the whole culture dish 2; a plurality of culture wells 3 are manufactured on the culture dish 2 according to experimental requirements, the culture wells 3 are connected with the base 1 into a whole, the barrier sheet 6 is arranged on an upper annular platform of the culture wells 3, the barrier sheet 6 is an isolated human dentin sheet or a barrier sheet with similar permeability and alternative materials, and the thickness of the barrier sheet 6 is adjusted according to experimental requirements; the material fixing ring 4 is used for fixing the material 7 to be tested, so that the size is uniform, and meanwhile, the diameter and the depth of the inner ring of the material fixing ring 4 are manufactured into other sizes according to experimental requirements.

2. An experimental set-up for in vitro dentin barrier cytotoxicity experiments according to claim 1, wherein: the depth and diameter of the culture well 3 are designed to be of other dimensions according to experimental requirements.