CN117947138A - In-vitro cytotoxicity test method for oral cavity material - Google Patents
In-vitro cytotoxicity test method for oral cavity material Download PDFInfo
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- CN117947138A CN117947138A CN202410353615.6A CN202410353615A CN117947138A CN 117947138 A CN117947138 A CN 117947138A CN 202410353615 A CN202410353615 A CN 202410353615A CN 117947138 A CN117947138 A CN 117947138A
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- 238000000338 in vitro Methods 0.000 title claims abstract description 26
- 231100000263 cytotoxicity test Toxicity 0.000 title claims abstract description 17
- 238000010998 test method Methods 0.000 title claims description 12
- 239000011797 cavity material Substances 0.000 title description 16
- 210000000214 mouth Anatomy 0.000 title description 3
- 238000004113 cell culture Methods 0.000 claims abstract description 69
- 239000000463 material Substances 0.000 claims abstract description 57
- 238000000034 method Methods 0.000 claims abstract description 23
- 231100000135 cytotoxicity Toxicity 0.000 claims abstract description 21
- 230000003013 cytotoxicity Effects 0.000 claims abstract description 21
- 210000004268 dentin Anatomy 0.000 claims abstract description 18
- 238000012360 testing method Methods 0.000 claims abstract description 16
- 230000004888 barrier function Effects 0.000 claims abstract description 10
- 230000010261 cell growth Effects 0.000 claims abstract description 6
- 230000001678 irradiating effect Effects 0.000 claims abstract description 4
- 238000007789 sealing Methods 0.000 claims abstract description 4
- 238000011049 filling Methods 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 20
- 238000002386 leaching Methods 0.000 claims description 18
- 239000006285 cell suspension Substances 0.000 claims description 17
- 238000011081 inoculation Methods 0.000 claims description 17
- 238000001125 extrusion Methods 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 230000012010 growth Effects 0.000 claims description 9
- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 claims description 8
- 239000004568 cement Substances 0.000 claims description 6
- 238000003780 insertion Methods 0.000 claims description 5
- 230000037431 insertion Effects 0.000 claims description 5
- 239000010634 clove oil Substances 0.000 claims description 4
- 239000000805 composite resin Substances 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
- 150000002500 ions Chemical class 0.000 claims description 4
- 229920002379 silicone rubber Polymers 0.000 claims description 4
- 239000004945 silicone rubber Substances 0.000 claims description 4
- 239000011787 zinc oxide Substances 0.000 claims description 4
- LRXTYHSAJDENHV-UHFFFAOYSA-H zinc phosphate Chemical compound [Zn+2].[Zn+2].[Zn+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O LRXTYHSAJDENHV-UHFFFAOYSA-H 0.000 claims description 4
- 229910000165 zinc phosphate Inorganic materials 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 2
- IPCXNCATNBAPKW-UHFFFAOYSA-N zinc;hydrate Chemical compound O.[Zn] IPCXNCATNBAPKW-UHFFFAOYSA-N 0.000 claims description 2
- 238000002474 experimental method Methods 0.000 abstract description 2
- 230000001988 toxicity Effects 0.000 abstract description 2
- 231100000419 toxicity Toxicity 0.000 abstract description 2
- 238000010586 diagram Methods 0.000 description 5
- 231100000820 toxicity test Toxicity 0.000 description 4
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 3
- 235000017491 Bambusa tulda Nutrition 0.000 description 3
- 241001330002 Bambuseae Species 0.000 description 3
- 235000015334 Phyllostachys viridis Nutrition 0.000 description 3
- 239000011425 bamboo Substances 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 238000000016 photochemical curing Methods 0.000 description 3
- 239000000654 additive Substances 0.000 description 2
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- 241001465754 Metazoa Species 0.000 description 1
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- 238000004364 calculation method Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 210000003074 dental pulp Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000010440 gypsum Substances 0.000 description 1
- 229910052602 gypsum Inorganic materials 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 238000005498 polishing Methods 0.000 description 1
- -1 poly-zinc carboxylate Chemical class 0.000 description 1
- 239000000573 polycarboxylate cement Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
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- 239000002672 zinc phosphate cement Substances 0.000 description 1
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Abstract
The invention relates to the technical field of cell testing, and particularly discloses an in-vitro cytotoxicity testing method for oral materials, which comprises the following steps: preparing a dentin wafer and an embedded well, and sticking the dentin wafer to the bottom of the embedded well to serve as a barrier; assembling the culture device using an assembling device; irradiating from above the embedded well by adopting an LED light curing lamp, sealing the wellhead of the embedded well, and placing the culture device into an incubator for culture; the relative cell growth rate was determined and cytotoxicity was determined based on the relative cell growth rate values and pre-constructed cytotoxicity ranking criteria. According to the invention, the culture device is assembled by using the assembling device, so that the embedded well and the cell culture container in the culture device are quickly combined, the in-vitro cytotoxicity test difficulty is reduced, the method has the advantages of short time consumption, strong repeatability, capability of comparing the toxicity of different materials and the like, and can be widely applied to in-vitro cytotoxicity test experiments of oral materials.
Description
Technical Field
The invention particularly relates to the technical field of cell testing, in particular to an in vitro cytotoxicity testing method for oral materials.
Background
Along with the technological development of society, various novel tooth filling materials are continuously developed and clinically used, whether the materials are suitable for human oral cavities or not is judged by the biocompatibility of the materials, and the main modes for evaluating the biocompatibility of the materials are in vitro tests, animal tests and human body tests.
The dentin barrier method is characterized in that the dental filling material acts on the lower cells through the dentin barrier to measure cytotoxicity, the key point of the test is that dentin is used as the barrier, and the actual situation of the clinical dental filling material is better simulated, however, the toxicity test in the prior art has complicated steps, long time consumption and great difficulty, and has higher requirements on the experience of operators, so that the popularization rate is lower.
Disclosure of Invention
The invention aims to provide an in-vitro cytotoxicity test method for oral materials, which aims to solve the problems of complicated toxicity test experimental steps, long time consumption, high difficulty, high requirements on experience of operators and low popularization rate in the prior art.
In order to achieve the above purpose, the present invention provides the following technical solutions:
an in vitro cytotoxicity test method of oral materials, comprising the following steps:
Preparing a dentin wafer and an embedded well, and sticking the dentin wafer to the bottom of the embedded well to serve as a barrier;
Assembling a culture device using an assembly device, the culture device comprising an embedded well and a cell culture container; the assembly device comprises an assembly barrel, a feeding mechanism, a first container, an inoculation mechanism and a feeding mechanism, wherein:
an assembly barrel for loading the embedded well into the cell culture vessel;
The feeding mechanism is used for controlling the movement of the embedded well to enable the embedded well to reach the assembling position, and when the embedded well reaches the assembling position, the embedded well enters the inside of the assembling cylinder and is positioned in the center of the assembling cylinder;
a first container for storing a leaching solution of the filling material; and for adding a leaching solution to the interior of the embedded well when the embedded well reaches the assembly position;
an inoculation mechanism comprising an inoculator for inoculating a cell suspension into a cell culture vessel;
The feeding mechanism is used for controlling the cell culture container to move so as to enable the cell culture container to sequentially reach an inoculation position and an assembly position, when the cell culture container reaches the inoculation position, the cell culture container is positioned under the inoculator, and when the cell culture container reaches the assembly position, the cell culture container is positioned under the embedded well;
Irradiating from above the embedded well by adopting an LED light curing lamp, sealing the wellhead of the embedded well, and placing the culture device into an incubator for culture;
The relative cell growth rate was determined and cytotoxicity was determined based on the relative cell growth rate values and pre-constructed cytotoxicity ranking criteria.
As a further scheme of the invention: a method of assembling a culture device using an assembly device, comprising the steps of:
Adding a leaching solution of a filling material into the first container, adding a cell suspension into the inoculator, placing the cell culture container in a feeding mechanism, and placing an embedded well in the feeding mechanism;
Starting a feeding mechanism, wherein the feeding mechanism controls the cell culture container to reach an inoculation position, and the inoculator inoculates the cell suspension into the cell culture container; starting a feeding mechanism, wherein the feeding mechanism controls the embedded well to reach an assembling position, and the first container adds leaching liquid of filling materials into the embedded well;
The feeding mechanism controls the cell culture container to reach an assembling position, and the assembling cylinder is used for loading the embedded well into the cell culture container.
As still further aspects of the invention: the filling material is one of photo-curing composite resin, zinc oxide clove oil water heater, zinc phosphate water heater, glass ion water heater and polycarboxylic acid zinc water heater; an additive silicone rubber impression material was also selected as a blank for the filling material.
As still further aspects of the invention: the feeding mechanism is arranged below the assembly barrel, a cavity is arranged in the assembly barrel, an opening is formed in one side, facing the feeding mechanism, of the cavity, and the feeding mechanism is used for driving the cell culture container to move towards the assembly barrel; the feeding mechanism comprises a shell, a limiting frame and a second telescopic component, wherein the shell is communicated with the cavity, the telescopic end of the second telescopic component is connected with the limiting frame, the limiting frame is used for fixing an embedded well, and the limiting frame is driven to move into the cavity when the telescopic end of the second telescopic component stretches.
As still further aspects of the invention: the extrusion head and the first telescopic component for driving the extrusion head to move are arranged in the cavity.
As still further aspects of the invention: the feeding mechanism comprises a bottom plate, a base, a feeding table, a driving assembly for driving the feeding table to move along the base and a supporting frame for fixing the cell culture container; the base is fixedly arranged on the bottom plate, the feeding table is slidably arranged on the base, and a plurality of supporting frames are uniformly arranged on the feeding table; the driving assembly comprises a driving screw, a first gear, a second gear and a first motor, wherein the driving screw, the first gear, the second gear and the first motor are rotatably arranged on the base, the driving screw is rotatably arranged on the base and is in threaded connection with the feeding table, the second gear is fixedly arranged at one end of the driving screw, the first motor is arranged on the base, the first gear is fixedly arranged at the output end of the first motor, the first gear is meshed with the second gear, and the first gear is of a sector gear structure.
As still further aspects of the invention: the limiting frame comprises two limiting rings arranged up and down and a connecting plate for connecting the two limiting rings, and the connecting plate is connected with the telescopic end of the second telescopic component; a plurality of limiting strips are arranged below the inner side of the limiting ring, and the limiting strips are movably connected with the limiting ring through at least one swinging rod; the limiting ring is located at the rear side of the swinging rod and is further provided with a mounting plate, and the mounting plate is connected with the swinging rod through a spring.
As still further aspects of the invention: the utility model discloses an equipment section of thick bamboo cavity, including equipment section of thick bamboo cavity, extrusion head, the equipment section of thick bamboo cavity is inside to be provided with the honeycomb duct, the one end of honeycomb duct pass through the connecting pipe with the liquid outlet intercommunication, the other end of honeycomb duct with first container intercommunication, first container is used for storing the leaching liquor of filling material.
As still further aspects of the invention: the inoculation mechanism comprises an assembling cylinder, and is characterized in that inoculators are arranged on the front side and the rear side of the assembling cylinder, the inoculation mechanism further comprises a second container arranged on the assembling cylinder, an outlet of the second container is communicated with a liquid inlet of the inoculator through a liquid guide tube, the second container is used for storing cell suspension, and the inoculator is used for inoculating the cell suspension into the cell culture container before the cell culture container enters the assembling position.
As still further aspects of the invention: the calculation method of the relative growth rate of the cells comprises the following steps:；
Wherein, Is the relative growth rate of cells; /(I)For the OD value of the experimental group,/>OD values for the blank.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, the culture device is assembled by using the assembling device, so that the embedded well and the cell culture container in the culture device are quickly combined, the in-vitro cytotoxicity test difficulty is reduced, the method has the advantages of short time consumption, strong repeatability, capability of comparing the toxicity of different materials and the like, and can be widely applied to in-vitro cytotoxicity test experiments of oral materials.
Drawings
FIG. 1 is a flow chart of a method for testing cytotoxicity of oral materials in vitro.
Fig. 2 is a schematic structural diagram of an assembled device in an in vitro cytotoxicity test method of oral material.
Fig. 3 is a front view of the assembled device in an in vitro cytotoxicity test method of oral material.
Fig. 4 is a cross-sectional view of an assembled device in an in vitro cytotoxicity test method of oral material.
Fig. 5 is a schematic structural diagram of a feeding mechanism in an in vitro cytotoxicity test method of oral materials.
Fig. 6 is a schematic diagram of the structure of the limiting frame in the method for testing the cytotoxicity of oral materials in vitro.
Fig. 7 is a second schematic structural diagram of the limiting frame in the method for testing the cytotoxicity of oral materials in vitro.
FIG. 8 is a schematic diagram showing the structure of a culture apparatus in an in vitro cytotoxicity test method of oral material.
FIG. 9 is a cross-sectional view of a culture device in an in vitro cytotoxicity test method of oral material.
In the figure: 100-assembly device, 10-assembly cartridge, 11-draft tube, 12-connection tube, 13-first telescoping assembly, 14-extrusion head, 20-feed mechanism, 21-housing, 22-feed port, 23-stop, 231-stop collar, 232-swing lever, 233-stop bar, 235-connection plate, 236-mounting plate, 237-spring, 24-second telescoping assembly, 30-first container, 40-inoculation mechanism, 41-inoculator, 42-catheter, 43-second container, 50-feeding mechanism, 51-bottom plate, 52-base, 53-feeding table, 54-support frame, 55-first motor, 56-first gear, 57-second gear, 58-drive screw, 200-culture device, 60-embedding well, 70-cell culture container, 80-barrier, 90-filling material.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The dentin barrier method is characterized in that the dental filling material acts on the lower cells through the dentin barrier to measure cytotoxicity, the key point of the test is that dentin is used as the barrier, and the actual situation of the clinical dental filling material is better simulated, however, the toxicity test in the prior art has complicated steps, long time consumption and great difficulty, and has higher requirements on the experience of operators, so that the popularization rate is lower.
Referring to fig. 1-9, in an embodiment of the invention, an in vitro cytotoxicity test method for oral materials includes the following steps:
S101, preparing a dentin wafer and an embedded well 60, and adhering the dentin wafer to the bottom of the embedded well 60 to serve as a barrier 80;
In step S101 of the embodiment of the present application, the method for preparing the embedded well 60 is as follows: and cutting the 96-hole embedded well plate into a plurality of independent embedded wells 60 by adopting a micro saw, removing the filter membrane at the bottom of the embedded wells 60, and cleaning and drying by adopting an ultrasonic water bath to obtain the composite material.
In step S101 of the embodiment of the present application, the preparation method of the dentin wafer includes the following steps:
s201, selecting complete molar teeth of a dental crown, removing attachments on the surface of the teeth and sterilizing;
S202, embedding tooth roots with gypsum to form a base for fixing teeth, and slicing the teeth layer by layer in a direction perpendicular to the long axis of the teeth by using a saw blade until dental pulp is exposed;
s203, selecting a dentin dental film as a central area, polishing the dentin dental film into a circular sheet with the diameter of 4.0mm and the thickness of 0.5mm, cleaning by using an ultrasonic water bath, and drying the surface to obtain the dentin dental film.
S102, assembling a culture device 200 by using the assembling device 100, wherein the culture device 200 comprises an embedded well 60 and a cell culture container 70; the assembly device 100 comprises an assembly drum 10, a feeding mechanism 20, a first container 30, an inoculating mechanism 40 and a feeding mechanism 50, wherein:
An assembly cartridge 10 for loading an insertion well 60 into a cell culture vessel 70;
The feeding mechanism 20 is used for controlling the movement of the embedded well 60, so that the embedded well 60 reaches the assembling position, and when the embedded well 60 reaches the assembling position, the embedded well 60 enters the inside of the assembling cylinder 10 and is positioned in the center of the assembling cylinder 10;
a first container 30 for storing the leaching liquid of the filling material 90; and also for adding leaching liquid to the interior of the embedded well 60 when the embedded well 60 reaches the assembly position;
an inoculation mechanism 40 comprising an inoculator 41 for inoculating a cell suspension into the cell culture vessel 70;
And a feeding mechanism 50 for controlling the movement of the cell culture container 70 so that the cell culture container 70 sequentially reaches an inoculation position and an assembly position, wherein the cell culture container 70 is positioned directly under the inoculator 41 when the cell culture container 70 reaches the inoculation position, and the cell culture container 70 is positioned directly under the insertion well 60 when the cell culture container 70 reaches the assembly position.
In step S102 of the present application, a method of assembling the culture device 200 using the assembling device 100 includes the steps of:
S301, adding the leaching solution of the filling material 90 into the first container 30, adding the cell suspension into the inoculator 41, placing the cell culture container 70 into the feeding mechanism 50, and placing the embedded well 60 into the feeding mechanism 20;
In step S301, the filling material 90 is one of a photo-curing composite resin, a zinc oxide clove oil cement, a zinc phosphate cement, a glass ion cement and a zinc polycarboxylate cement, and in this embodiment, an addition type silicone rubber impression material is selected as a blank control group of the filling material 90;
S302, starting a feeding mechanism 50, wherein the feeding mechanism 50 controls the cell culture container 70 to reach an inoculation position, and the inoculator 41 inoculates the cell suspension into the cell culture container 70; starting the feeding mechanism 20, wherein the feeding mechanism 20 controls the embedded well 60 to reach the assembling position, and the first container 30 adds the leaching liquid of the filling material 90 into the embedded well 60;
S303, the feeding mechanism 50 controls the cell culture container 70 to reach an assembling position, the assembling cylinder 10 loads the embedded well 60 into the cell culture container 70, the assembling of the culture device 200 is completed, and after the assembling is completed, the feeding mechanism 50 drives the culture device 200 to leave the assembling cylinder 10.
S103, after the culture device 200 is completed, irradiating the embedded well 60 with an LED light curing lamp for 60S, sealing the wellhead of the embedded well 60, and placing the culture device 200 into a constant temperature box for culture, wherein in the step S103 of the embodiment, the internal temperature of the constant temperature box is 28-37 ℃, and the culture time is 24 hours;
s104, measuring the relative growth rate of cells, and judging cytotoxicity according to the relative growth rate value of the cells and a pre-constructed cytotoxicity grading standard;
in step S104 of the embodiment of the present application, the method for calculating the relative growth rate of cells includes: ；
Wherein, Is the relative growth rate of cells; /(I)For the OD value of the experimental group,/>OD values for the blank.
In this embodiment, the filling material 90 of the experimental group is one of a photo-curing composite resin, a zinc oxide clove oil water heater, a zinc phosphate water heater, a glass ion water heater and a poly-zinc carboxylate water heater; the filling material 90 of the blank set is an additive silicone rubber impression material.
In addition, in step S105 of the present embodiment, the pre-constructed cytotoxicity classification criteria are shown in table 1: TABLE 1
| Relative growth rate of cells | Cytotoxicity fractionation | Cytotoxicity of cells |
| ≥100 | 0 | Without any means for |
| 75-99 | 1 | Without any means for |
| 50-74 | 2 | Mild and mild |
| 25-49 | 3 | Poisoning of |
| 1-24 | 4 | Heavy weight |
| 0 | 5 | Heavy weight |
In the embodiment of the application, the feeding mechanism 50 is installed below the assembly barrel 10, a cavity is arranged inside the assembly barrel 10, an opening is arranged on one side of the cavity facing the feeding mechanism 50, the feeding mechanism 50 is used for driving the cell culture container 70 to move towards the assembly barrel 10, and when the cell culture container 70 is positioned right below the opening, the cell culture container 70 reaches the assembly position; the feeding mechanism 20 comprises a shell 21, a limiting frame 23 and a second telescopic assembly 24, wherein the shell 21 is communicated with the cavity, the telescopic end of the second telescopic assembly 24 is connected with the limiting frame 23, the limiting frame 23 is used for fixing the embedded well 60, the limiting frame 23 is driven to move into the cavity when the telescopic end of the second telescopic assembly 24 stretches, and when the embedded well 60 in the limiting frame 23 is positioned right above the cell culture container 70, the embedded well 60 reaches the assembling position;
The extrusion head 14 and the first telescopic component 13 driving the extrusion head 14 to move are arranged in the cavity, when the embedded well 60 and the cell culture container 70 reach the assembly position, the telescopic end of the first telescopic component 13 stretches, the extrusion head 14 is driven to move towards the embedded well 60, so that the embedded well 60 leaves the limiting frame 23 under the thrust action of the extrusion head 14, the embedded well 60 falls into the cell culture container 70, and the assembly of the embedded well 60 and the cell culture container 70 is completed.
In some embodiments of the application, the feeding mechanism 50 comprises a base plate 51, a base 52, a feeding stage 53, a drive assembly for driving the feeding stage 53 to move along the base 52, and a support frame 54 for holding the cell culture container 70; the base 52 is fixedly installed on the bottom plate 51, the feeding table 53 is slidably installed on the base 52, a plurality of supporting frames 54 are uniformly arranged on the feeding table 53, the plurality of supporting frames 54 can simultaneously accommodate a plurality of cell culture containers 70, the feeding mechanism 50 simultaneously controls the plurality of cell culture containers 70 to move, batch assembly of the cell culture containers 70 and the embedded wells 60 is realized, and therefore the assembly efficiency of the culture device 200 is improved;
Further, the driving assembly includes a driving screw 58 rotatably installed on the base 52, a first gear 56, a second gear 57, and a first motor 55, the driving screw 58 is rotatably installed on the base 52, the driving screw 58 is in threaded connection with the feeding table 53, one end of the driving screw 58 is fixedly installed with the second gear 57, the first motor 55 is installed on the base 52, the output end of the first motor 55 is fixedly installed with the first gear 56, the first gear 56 is meshed with the second gear 57, and the first gear 56 is in a sector gear structure, it is understood that the first gear 56 of the sector gear structure is intermittently meshed with the second gear 57, so that the second gear 57 and the driving screw 58 are driven to intermittently rotate, for keeping the feeding table 53 stationary when the embedded well 60 and the cell culture container 70 are assembled.
In the embodiment of the present application, the housing 21 is further provided with a feed port 22, and the feed port 22 is used for adding the embedded well 60 into the housing 21; the limiting frame 23 comprises two limiting rings 231 arranged up and down and a connecting plate 235 connected with the two limiting rings 231, and the connecting plate 235 is connected with the telescopic end of the second telescopic assembly 24; a plurality of limiting bars 233 are arranged below the inner side of the limiting ring 231, and the limiting bars 233 are movably connected with the limiting ring 231 through at least one swinging rod 232; the limit ring 231 is further provided with a mounting plate 236 at the rear side of the swing rod 232, the mounting plate 236 is connected with the swing rod 232 through a spring 237, it is to be noted that, in this embodiment, the opening of the embedded well 60 has a protruding edge, when the embedded well 60 falls into the inside of the limit frame 23, the limit bar 233 of the upper limit ring 231 presses the protruding edge under the pushing force of the spring 237 and the swing rod 232, so as to support the embedded well 60, when the embedded well 60 and the cell culture container 70 are assembled, the extrusion head 14 presses the embedded well 60 to move downward, so that the embedded well 60 supports the limit bar 233, and then falls onto the limit bar 233 of the lower limit ring 231, and as the extrusion head 14 continues to move downward, the extrusion head 14 presses the embedded well 60 to leave the lower limit ring 231 and fall into the cell culture container 70, and it is to be noted that, in this embodiment, the purpose of providing the two-stage limit ring 231 is to avoid the situation that the embedded well 60 collides with the cell suspension in the inside the cell culture container 70 rapidly due to the overlong dropping path of the embedded well 60, so as to cause damage to the cell suspension.
In the embodiment of the present application, a flow guiding tube 11 is further installed inside the cavity of the assembly barrel 10, a liquid outlet is provided on the extrusion head 14, one end of the flow guiding tube 11 is communicated with the liquid outlet through a connecting tube 12, the other end of the flow guiding tube 11 is communicated with the first container 30, it should be noted that, the first container 30 is used for storing leaching liquid of the filling material 90, and when the embedded well 60 on the limiting frame 23 reaches the assembly position, the first container 30 adds leaching liquid into the embedded well 60 through the flow guiding tube 11 and the connecting tube 12; in addition, since the extrusion head 14 moves up and down with the first telescopic assembly 13, the connection pipe 12 is provided in a hose structure;
It should be further noted that, the first container 30 may be provided with a plurality of storage chambers separately, so as to store the leaching solutions of the multiple filling materials 90, so that the multiple leaching solutions may be added into different embedded wells 60, thereby quickly grouping the leaching solutions in the toxicity test of the multiple filling materials 90 and improving the test efficiency.
In the embodiment of the present application, the inoculator 41 is disposed on both front and rear sides of the assembly barrel 10, the inoculation mechanism 40 further comprises a second container 43 mounted on the assembly barrel 10, an outlet of the second container 43 is communicated with a liquid inlet of the inoculator 41 through a liquid guide tube 42, the second container 43 is used for storing the cell suspension, and the inoculator 41 is used for inoculating the cell suspension into the cell culture container 70 before the cell culture container 70 enters the assembly position.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (10)
1. An in vitro cytotoxicity test method for oral materials, which is characterized by comprising the following steps:
Preparing a dentin wafer and an embedded well (60), and adhering the dentin wafer to the bottom of the embedded well (60) to serve as a barrier (80);
Assembling a culture device (200) using the assembly device (100), the culture device (200) comprising an embedded well (60) and a cell culture container (70); the assembly device (100) comprises an assembly drum (10), a feeding mechanism (20), a first container (30), an inoculating mechanism (40) and a feeding mechanism (50), wherein the method of assembling the culture device (200) using the assembly device (100) comprises:
The feeding mechanism (20) is used for controlling the movement of the embedded well (60) to enable the embedded well (60) to reach the assembling position, and when the embedded well (60) reaches the assembling position, the embedded well (60) enters the assembling cylinder (10) and is positioned in the center of the assembling cylinder (10);
adding leaching liquid to the interior of the insertion well (60) with the first container (30) when the insertion well (60) reaches the assembly position;
inoculating the cell suspension to a cell culture vessel (70) by an inoculating mechanism (40);
The cell culture container (70) is controlled to move through the feeding mechanism (50), so that the cell culture container (70) sequentially reaches an inoculation position and an assembly position, when the cell culture container (70) reaches the inoculation position, the cell culture container (70) is positioned under an inoculator (41) of the inoculation mechanism (40), and when the cell culture container (70) reaches the assembly position, the cell culture container (70) is positioned under the embedded well (60);
irradiating from above the embedded well (60) by adopting an LED light curing lamp, sealing the wellhead of the embedded well (60), and placing the culture device (200) into an incubator for culture;
The relative cell growth rate was determined and cytotoxicity was determined based on the relative cell growth rate values and pre-constructed cytotoxicity ranking criteria.
2. The method of in vitro cytotoxicity testing of oral material according to claim 1, characterized in that the method of assembling the culture device (200) using the assembly device (100) comprises the steps of:
Adding a leaching solution of a filling material (90) to the interior of the first container (30), adding a cell suspension to the interior of the inoculator (41), placing the cell culture container (70) in the feeding mechanism (50), and placing the insertion well (60) in the feeding mechanism (20);
Starting a feeding mechanism (50), wherein the feeding mechanism (50) controls the cell culture container (70) to reach an inoculation position, and the inoculator (41) inoculates the cell suspension into the cell culture container (70); starting a feeding mechanism (20), wherein the feeding mechanism (20) controls the embedded well (60) to reach an assembling position, and the first container (30) adds leaching liquid of the filling material (90) into the embedded well (60);
the feeding mechanism (50) controls the cell culture vessel (70) to reach an assembling position, and the assembling cylinder (10) loads the embedded well (60) into the cell culture vessel (70).
3. The method of claim 2, wherein the filling material (90) is one of a photo-setting composite resin, a zinc oxide clove oil water cement, a zinc phosphate water cement, a glass ion water cement, and a polycarboxylic acid zinc water cement; an addition type silicone rubber impression material was also selected as a blank for the filling material (90).
4. The method for testing the cytotoxicity of oral materials in vitro according to claim 1, wherein the feeding mechanism (50) is installed below the assembly barrel (10), a cavity is arranged inside the assembly barrel (10), an opening is arranged on one side of the cavity, facing the feeding mechanism (50), of the assembly barrel (10), and the feeding mechanism (50) is used for driving the cell culture container (70) to move towards the assembly barrel (10); the feeding mechanism (20) comprises a shell (21), a limiting frame (23) and a second telescopic component (24), wherein the shell (21) is communicated with the cavity, the telescopic end of the second telescopic component (24) is connected with the limiting frame (23), the limiting frame (23) is used for fixing an embedded well (60), and the limiting frame (23) is driven to move into the cavity when the telescopic end of the second telescopic component (24) stretches.
5. The method according to claim 4, characterized in that the cavity is internally provided with an extrusion head (14) and a first telescopic assembly (13) driving the extrusion head (14) to move.
6. The method according to claim 5, wherein the feeding mechanism (50) comprises a base plate (51), a base (52), a feeding table (53), a driving assembly for driving the feeding table (53) to move along the base (52), and a supporting frame (54) for fixing the cell culture container (70); the base (52) is fixedly arranged on the bottom plate (51), the feeding table (53) is slidably arranged on the base (52), and a plurality of supporting frames (54) are uniformly arranged on the feeding table (53); the driving assembly comprises a driving screw (58), a first gear (56), a second gear (57) and a first motor (55) which are rotatably arranged on the base (52), the driving screw (58) is in threaded connection with the feeding table (53), the second gear (57) is fixedly arranged at one end of the driving screw (58), the first motor (55) is arranged on the base (52), the first gear (56) is fixedly arranged at the output end of the first motor (55), the first gear (56) is meshed with the second gear (57), and the first gear (56) is of a sector gear structure.
7. The method for in vitro cytotoxicity testing of oral materials according to claim 4, wherein the limit frame (23) comprises two limit rings (231) arranged up and down and a connecting plate (235) connecting the two limit rings (231), the connecting plate (235) being connected with the telescopic end of the second telescopic assembly (24); a plurality of limit bars (233) are arranged below the inner side of the limit ring (231), and the limit bars (233) are movably connected with the limit ring (231) through at least one swing rod (232); the limiting ring (231) is located at the rear side of the swing rod (232) and is further provided with a mounting plate (236), and the mounting plate (236) is connected with the swing rod (232) through a spring (237).
8. The method according to claim 5, wherein a flow guide tube (11) is further installed in the cavity of the assembly tube (10), a liquid outlet is formed in the extrusion head (14), one end of the flow guide tube (11) is communicated with the liquid outlet through a connecting tube (12), the other end of the flow guide tube (11) is communicated with the first container (30), and the first container (30) is used for storing leaching liquid of the filling material (90).
9. The method according to claim 1, wherein the assembling cylinder (10) is provided with an inoculator (41) on both front and rear sides thereof, the inoculating mechanism (40) further comprises a second container (43) mounted on the assembling cylinder (10), an outlet of the second container (43) is communicated with a liquid inlet of the inoculator (41) through a liquid guide tube (42), the second container (43) is used for storing the cell suspension, and the inoculator (41) is used for inoculating the cell suspension into the cell culture container (70) before the cell culture container (70) enters the assembling position.
10. The method for in vitro cytotoxicity test of oral material according to claim 1, wherein the method for calculating the relative growth rate of cells comprises:；
Wherein, Is the relative growth rate of cells; /(I)For the OD value of the experimental group,/>OD values for the blank.
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