CN103710256B - Root canal-periapical complex in vitro model - Google Patents

Abstract

The purpose of the present invention is to provide a root canal-periapical complex in vitro model. The isolated tooth of the composite in vitro model is consistent with the living situation, and the LB solid medium (various biologically relevant components can be added) simulates the periapical tissue. The complex in vitro model of the present invention has no source of infection outside the root canal; it can simulate the infection-infected root canal under the same natural state of the root canal as the living body; it can observe the situation that the infection invades the periapical region; it can observe the impact of clinical operations on the root canal. The influence of infection to the periapical invasion; the effect of periapical antibacterial substances (immune substances) on the invasion of infected root canals to the periapical can be observed.

Description

An in vitro model of root canal-periapical complex

technical field

The invention belongs to the field of root canal and periapical models; more specifically, the invention relates to a root canal-periapical complex in vitro model.

Background technique

Bacteria are the main pathogenic factors of periapical periodontitis. Some scholars believe that the bacteria in the root canal exist in the form of biofilm. Without treatment, the bacteria are not easy to penetrate the apical foramen, but the apical periodonitis caused by their virulence factors. Some scholars have also proposed that under the induction of environmental factors, some planktonic bacteria in the root canal biofilm can penetrate the apical foramen and dentinal tubules and invade the periapical tissue to form a root biofilm. In addition to direct bacterial invasion of the periapical tissue, bacterial virulence factors such as endotoxin in the root canal can also enter the periapical tissue and cause damage to the periapical tissue. Based on the current research, the microorganisms in the infected root canal can enter the root tip and form a root tip biofilm. However, it is still unclear whether the microorganisms that form the root tip biofilm enter the root tip randomly or after being stimulated by root canal treatment. In other words, in the absence of treatment, the infection of the root canal biofilm leading to periapical lesions is mainly caused by the bacteria entering the periapical tissue to secrete virulence factors, or the bacterial virulence factors in the root canal entering the periapical Mainly, or the joint effect of the two is still unclear, and there is no relevant literature report.

From a pathological point of view, the root canal and the periapical region can be regarded as a whole, that is, in the case of periodontal health, the periapical pathogenic stimuli mainly come from the root canal, and once the root canal is infected, it is almost inevitable can lead to periapical inflammation.

Many studies have involved the establishment of apical periodontitis models. Perters established an infected root canal model using bovine teeth [1]. Yang Weidong [2] established infected root canal models with Beagle canine anterior teeth, and observed the effects of ultrasound treatment and conventional treatment on bacteria in the root canal. Guo Huijie, Sun Tuoqi et al.[3,4] established an in vitro E. faecalis-infected root canal model with isolated bicuspid teeth to observe the colonization of E. faecalis in infected root canals and evaluate the effect of laser on biofilm. Zhuang Peilin et al [5] made dentin slices from isolated teeth, and established a root canal model infected by Enterococcus faecalis on it to study the effect of inorganic nano-antibacterial agents on biofilms. Liu Guixiang[6] and others tested the effect of ultrasonic irrigation on the inactivation and removal of root canal endotoxin with isolated maxillary premolars. The above research results show that artificial establishment of infected root canal model is established.

At present, the infected root canal and periapical infection (lesions) can only be studied separately. There are mainly the following methods for the research on infected root canals: (1) the classic method is to insert paper tip into the root canal to take materials, and to study the bacteria and their toxins; (2) to inoculate the bacteria in the isolated root canal for research; (3) ) making dentin into grinding slices and inoculating bacteria for research; (4) animal experiments: using dogs and other animals to make infected root canals. The study on periapical infection (lesion) was carried out by extracting affected teeth and collecting samples from the periapical region or by collecting samples through root apical surgery.

Based on this, the present invention proposes the concept of the root canal-periapical complex, and establishes an in vitro model of the root canal-pericapic complex.

Contents of the invention

Aiming at the deficiencies of the above-mentioned prior art, the purpose of the present invention is to provide an in vitro model of root canal-periapical complex. The in vitro model of the complex can be used to study the pathways and influencing factors of infected root canals forming apical periodontitis.

To achieve the above object, the present invention takes the following measures:

The root canal-periapical complex in vitro model of the present invention is established by the following steps:

(1) Take a single premolar extracted due to orthodontics, use a curette and an ultrasonic scaler to remove the soft tissue and calculus on the root surface, place it in 2.5-3.0mol/L formaldehyde solution, and store it at 20-30°C As a spare, isolated teeth are obtained;

(2) Take a glass bottle. The glass bottle is composed of a bottle body and a rubber stopper. According to the diameter of the neck of the isolated tooth, a hole is dug in the center of the rubber stopper, and the isolated tooth is inserted into the rubber stopper through the hole. The isolated tooth and the rubber stopper The interface of the bottle is sealed with light-cured flowing resin, and the bottle body and the rubber stopper with the isolated teeth are sterilized at a temperature of 110-125°C and a pressure of 10-20 MPa until the sterilization is qualified;

(3) In the ultra-clean workbench, inject aseptic LB solid medium into the bottle body qualified through sterilization; then the rubber stopper with the isolated teeth is closely connected with the bottle body with aseptic sealing glue, and the The LB solid medium just reached one-third of the root of the isolated tooth; two layers of nail polish were applied at the interface between the isolated tooth and the rubber stopper, and the root canal-periapical complex in vitro model was obtained.

In the establishment of the above-mentioned root canal-periapical complex in vitro model, the step (3) also includes after the LB solid medium is solidified, the part of the medium that is in contact with the isolated tooth root is excavated, Pass the needle with flexible tube through the rubber stopper fixed with isolated teeth, so that the needle reaches the part where the culture medium is excavated, and use a small rubber stopper to close the opening of the flexible tube, wherein the needle with flexible tube and the small rubber Before use, the plug is sterilized at a temperature of 110-125°C and a pressure of 10-20 MPa until the sterilization is qualified; two layers of nail polish are applied at the interface between the needle and the rubber stopper. The needle with a flexible tube can facilitate the addition of drugs to the LB solid medium by the outside world after the in vitro model of the complex is established, and observe the effect of periapical antibacterial substances (immune substances) on the invasion of infected root canals to the periapical region.

The present invention regards the root canal-periapical as a whole, establishes the in vitro model of the root canal-periapical complex, wherein the isolated tooth is consistent with the condition of the living body, and LB solid medium (various biologically related components can be added) Simulate the periapical tissue. The complex in vitro model can be used to study infection by performing various treatments on isolated teeth (such as pulp opening, pulp extraction, upper 2/3 root canal preparation, root canal preparation full length, and inoculation of various bacteria into the root canal). The way of root canal formation of apical periodontitis, the impact of clinical operation on the spread of infection to the periapical area, etc.

The beneficial effects of the present invention are:

(1) The in vitro model of the complex has no source of infection outside the root canal;

(2) The in vitro model of the complex can simulate the infection-infected root canal under the same root canal natural state as the living body;

(3) The in vitro model of the complex can observe the invasion of the infection to the periapical region;

(4) The in vitro model of the complex can perform the same operation on the isolated root canal as clinically, and observe the effect of the treatment on the invasion of the infection to the periapical region;

(5) The in vitro model of the complex can further observe the effect of periapical antibacterial substances (immune substances) on the invasion of infected root canals to the periapical region.

Description of drawings

Fig. 1 is a schematic diagram of the structure of the root canal-periapical complex in vitro model.

Fig. 2 is the electrophoresis diagram of the PCR amplification product of the root tip region sample in Example 3.

Fig. 3 is the electrophoresis diagram of the PCR amplification product of the sample in the tube of Example 4.

Fig. 4 is the electrophoresis diagram of the PCR amplification product of the root tip region sample in Example 5.

Fig. 5 is an electrophoresis diagram of the PCR amplification product of the apical region sample 1 day after the modeling in Example 6.

Fig. 6 is an electrophoresis diagram of the PCR amplification product of the apical region sample immediately after root canal preparation in Example 6.

Fig. 7 is an electrophoresis diagram of the PCR amplification product of the apical region sample 7 days after root canal preparation in Example 6.

Fig. 8 is an electrophoresis diagram of the PCR amplification product of the apical region sample 14 days after root canal preparation in Example 6.

Fig. 9 is an electrophoresis diagram of the PCR amplification product of the apical region sample 21 days after root canal preparation in Example 6.

Fig. 10 is an SEM image of the outer surface of the root apex of an isolated tooth 21 days after the full-length preparation of the infected root canal.

Fig. 11 is an SEM image of the outer surface of the root apex of the isolated tooth 21 days after the infected root canal was prepared 1 mm beyond the apical foramen.

Detailed ways

The content of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the embodiments of the present invention are only used to illustrate the present invention and not to limit the present invention. Without departing from the technical idea of the present invention, various replacements and changes can be made according to common technical knowledge and conventional means in the field. All should be included in the scope of the present invention.

The establishment of the root canal-periapical complex in vitro model of embodiment 1

(1) Take a single premolar extracted due to orthodontics, use a curette and an ultrasonic scaler to remove the soft tissue and calculus on the root surface, place it in 2.5-3.0mol/L formaldehyde solution, and store it at 20-30°C for later use , to obtain the isolated tooth 1;

(2) Take a plurality of penicillin bottles, the penicillin bottle is composed of the bottle body 2 and the rubber stopper 3, dig a hole in the center of the rubber stopper 3 according to the diameter of the neck of the isolated tooth, insert the isolated tooth 1 into the rubber stopper 3 through the hole The interface between the isolated tooth 1 and the rubber stopper 3 is sealed with light-cured flowable resin, and the bottle body 2, the rubber stopper 3 with the isolated tooth 1 fixed thereon, are extinguished at a temperature of 110-125°C and a pressure of 10-20 MPa. bacteria treatment;

Randomly select two rubber stoppers 3 with isolated teeth 1 and bottle body 2 that have been sterilized by high temperature and high pressure, place them in a test tube filled with sterile LB liquid medium, and culture them in a 37°C incubator for 48 hours. After 48 hours, if culture If the medium remains transparent, it proves that the sterilization is qualified. If the medium is turbid, it means that the sterilization is unqualified, and it needs to be sterilized again.

(3) In the ultra-clean workbench, inject sterile LB solid medium 4 into the bottle body 2 qualified through sterilization; connected, the LB solid medium 4 just reaches one-third of the root of the isolated tooth 1, and two layers of nail polish are applied at the interface between the isolated tooth 1 and the rubber stopper 3 to obtain the root canal-periapical Complex in vitro model.

Example 2 Establishment of root canal-periapical complex in vitro model

(1) Take a single premolar extracted due to orthodontics, use a curette and an ultrasonic scaler to remove the soft tissue and calculus on the root surface, place it in 2.5-3.0mol/L formaldehyde solution, and store it at 20-30°C for later use , to obtain the isolated tooth 1;

(2) Take a plurality of penicillin bottles, the penicillin bottle is composed of the bottle body 2 and the rubber stopper 3, dig a hole in the center of the rubber stopper 3 according to the diameter of the neck of the isolated tooth, insert the isolated tooth 1 into the rubber stopper 3 through the hole The interface between the isolated tooth 1 and the rubber stopper 3 is sealed with light-cured flowable resin, and the bottle body 2 and the rubber stopper 3 with the isolated tooth 1 fixed are sterilized at a temperature of 110-125°C and a pressure of 10-20 MPa. deal with;

Randomly select two rubber stoppers 3) with isolated teeth 1 fixed and bottle body 2 that have been sterilized by high temperature and high pressure, and place them in a test tube filled with sterile LB liquid medium, and culture them in a 37°C incubator for 48 hours. If the medium remains transparent, it proves that the sterilization is qualified. If the medium becomes turbid, it means that the sterilization is unqualified, and it needs to be sterilized again.

(3) In the ultra-clean workbench, inject sterile LB solid medium 4 into the bottle body 2 after the sterilization treatment; after the medium 4 is solidified, cultivate the surface that will be in contact with the root of the isolated tooth 1 Excavated part of the foundation; then the rubber stopper 3 fixed with the isolated tooth 1 is closely connected with the bottle body 2 with sterile sealing glue, and the LB solid medium 4 just reaches one-third of the root of the isolated tooth 1, Pass the needle 6 with the flexible tube 5 through the rubber stopper 3 fixed with the isolated tooth 1, so that the needle 6 reaches the excavated part of the culture medium 4, and use a small rubber stopper 7 to close the opening of the flexible tube 5; 1 and the interface of the rubber stopper 3, coated with two layers of nail polish, and coated with two layers of nail polish at the interface of the needle 6 and the rubber stopper 3, to obtain the root canal-periapical complex in vitro model; wherein the soft The needle 6 and the small rubber stopper 7 of the tube 5 are sterilized at a temperature of 110-125°C and a pressure of 10-20 MPa before use, and two needles 6 with the hose 5 and the small rubber stopper that have been sterilized by high temperature and high pressure are randomly selected 7 Place in a test tube containing sterile LB liquid medium, and incubate in a 37°C incubator for 48 hours. After 48 hours, if the medium remains transparent, it proves that the sterilization is qualified; Re-sterilize.

A schematic diagram of the structure of the in vitro model of the complex established in this example is shown in FIG. 1 .

The needle 6 with the hose 5 can facilitate the addition of drugs to the LB solid medium 4 after the in vitro model of the complex is established.

Embodiment 3 simulates no source of infection outside the root canal

Fifteen in vitro models of the complex established in Example 1 or 2 were used, and 5 models were randomly selected on the first day to detect bacteria in the periapical tissue. If bacteria were detected, it indicated that the model preparation was unsuccessful, and the model was remade; if no bacteria were detected, the remaining 10 models were naturally placed in the air without any treatment, and periapical bacteria were detected on the 21st day. At the same time, two positive control groups were set up: the penicillin vial containing the culture medium was directly exposed to the air. Using polymerase chain reaction (PCR, the primers are 16S rDNA universal primers shared by bacteria: upstream: 5'-AGAGTTTGATCCTGGCTCAG-3'; downstream: 5'-GGTTACCTTGTTACGACTT-3', the fragment length is about 1500bp, provided by Shanghai Sangong Bioengineering Company Synthetic.) Detect whether there are bacteria in each group, the results are as shown in Figure 2, and Figure 2 is the electrophoresis figure of the PCR amplification product of the root tip region sample (1.Marker; 2.Positive control group; 3.Double distilled water blank control group , double distilled water instead of the template; 4-7. Part of the apical sample of the model 1 day after modeling; 8-11. Part of the apical sample of the model 21 days after modeling). It can be seen from Fig. 2 that there is no bacteria in the LB solid medium 4 of the experimental group, indicating that the complex in vitro model established by the present invention has no source of infection outside the root canal around the apex.

Embodiment 4 simulates the infection under the natural state of the same root canal as the living body—infected root canal

With 10 in vitro models of the complex established in Example 1 or 2, the isolated tooth 1 in the model was pulped, the root canal of the isolated tooth 1 was opened, and exposed to the air for 21 days. As the experimental group, polymerase-linked Reaction (PCR) detects whether bacteria exists in the root canal, and the results are as shown in Figure 3, and Figure 3 is the electrophoresis figure of the sample PCR amplification product in the root canal (1.Marker; 2. positive control group, with embodiment 3; 3 . Double distilled water blank control group, double distilled water instead of template; 4-9. Parallel experimental group). It can be seen from Figure 3 that bacteria existed in the root canals of the experimental group, indicating that the infected root canals were established successfully.

Example 5 Observation of the situation where the infection invades the periapical region

With 25 in vitro models of the complex established in Example 1 or 2, 5 models of the control group were randomly selected on the first day to detect bacteria in the periapical tissue. If bacteria are detected, it means that the model preparation is unsuccessful, and the model is remade; if no bacteria are detected, the remaining 20 models are divided into control group and experimental group, with 10 models in each group, and the experimental group is treated with pulpectomy. The control group was left in the air without any treatment. On the 21st day, periapical bacteria (polymerase-linked reaction PCR) and endotoxin (end-point chromogenic method) were detected in the experimental group and the control group. Bacterial detection result is as shown in Figure 4, and Figure 4 is the electrophoresis figure of PCR amplification product of apical region sample (1.Marker; 2. positive control group, with embodiment 3; 3. double distilled water blank control group, double distilled water Instead of the template; 4-7. Part of the apical sample of the control group; 8-11 part of the apical sample of the experimental group). It can be seen from Figure 4 that there is no bacteria in the LB solid medium 4 of the experimental group, indicating that when the root canal is not treated, the bacteria in the root canal are not easy to invade from the infected root canal to the periapical area.

The detection results of endotoxin content around the root tip of the control group and the experimental group are shown in Table 1. The endotoxin content of the experimental group was 10.525 EU/mL, higher than the baseline level, indicating that in the complex in vitro model established by the present invention, the bacterial toxin can diffuse from the infected root canal to the periapical region. Endotoxin is the main virulence factor of Gram-negative bacteria and a recognized inflammatory initiator, which can lead to tissue dissolution and bone resorption, and plays an important role in the development of periapical disease. Studies have shown that endotoxin has strong permeability and can easily enter the periapical tissue through the apical foramen, and a small concentration of endotoxin can cause significant damage to the periapical tissue and lead to periapical bone destruction. Therefore, even if the bacteria do not enter the periapical area, infection of the root canal can also lead to the development of periapical tissue lesions.

Table 1 The periapical endotoxin content of Example 5 (EU·mL ^-1 , )

Embodiment 6 carries out the same operation as the clinic to the model root canal

With the 105 complex in vitro models established in Example 1, 5 of them were randomly selected 1 day after the modeling and detected whether there were bacteria in the LB solid medium 4 by polymerase chain reaction (PCR), and no bacteria were detected as a result. , as shown in Figure 5, Figure 5 is the electrophoresis of the PCR amplification product of the root tip region sample 1 day after modeling (1. Marker; 2. Positive control group; 3. Double distilled water blank control group, double distilled water instead of template ; 4-8. Parallel experimental groups).

The remaining 100 models were divided into 20 in the control group (without pulp opening) and 80 in the pulp-opening group. In the pulp-opening group, infected root canals were established according to Example 4. The pulp opening group was divided into 4 groups, and root canal preparation was performed on infected root canals. The first group was not treated, the upper 2/3 of the root canal was prepared in the second group, and the full length of the root canal was prepared in the third group (the end point was at the distance from the anatomical root apical foramen 1mm), and the preparation in group 4 exceeded the apical foramen by 1mm. Immediately after root canal preparation, 7 days, 14 days, and 21 days after the preparation, polymerase chain reaction (PCR) was used to detect whether there was presence or absence in LB solid medium 4 Bacteria, the test results are shown in Figures 6 to 9 respectively (1. Marker; 2. Positive control group, same as Example 3; 3. Double distilled water blank control group, double distilled water instead of template; 4. Control group; 5. Group 1; 6. Group 2; 7. Group 3; 8. Group 4). It can be seen from Figures 6 to 9 that bacteria were detected in the LB solid medium 4 21 days after the full-length preparation of the infected root canal and 21 days after the preparation beyond the apical foramen 1 mm. A scanning electron microscope (SEM) was used to observe the outer surface of the isolated tooth root tip, and a root tip biofilm composed of a large number of cocci can be seen. 21 days).

Immediately after root canal preparation, 7 days and 21 days after preparation, the endotoxin content in LB solid medium 4 was detected by end-point chromogenic method, and the results are shown in Table 2.

Table 2 Example 6 periapical endotoxin content

Compared with group 1 (only pulp opening treatment), there was no statistically significant difference in the periapical endotoxin in the upper 2/3 of the prepared root canal immediately after the preparation; while the total length of the prepared root canal and the preparation exceeded the apical foramen by 1mm , the periapical endotoxin content decreased, and the difference was statistically significant after statistical analysis; compared with the preparation beyond the apical foramen by 1mm and the prepared root canal, the periapical endotoxin content decreased more significantly, and the difference was statistically significant. At 7 days and 21 days after preparation, compared with group 1 (only pulp opening treatment), there was no statistically significant change in the endotoxin content in the upper 2/3 of the prepared root canal; The increase of periapical endotoxin content in the 1mm apical foramen was significant and statistically significant; the increase of periapical endotoxin content was more significant when the preparation exceeded the apical foramen by 1mm compared with the whole length of the prepared root canal, and the difference was statistically significant.

It can be seen from this example that, for different operations, the time points at which bacteria appear in LB solid medium 4 are different, and the endotoxin content is also different, indicating that for the complex in vitro model established by the present invention, different operations will cause infection to invade the periapical region. make an impact.

references

[1]Peters LB, Wesselink PR, Moorer WR.Penetration of bacteria in bovine rootdentine in vitro.Int Endond J,2000,33(1):28-36.

[2] Yang Weidong, Ge Jiuyu, Fu Yao. Experimental study on the effect of ultrasound on microorganisms in the root canal of Beagle canine anterior teeth. Journal of Dental Endodontics and Periodontology, 2007,17(11):618-621.

[3] Guo Huijie, Yue Lin. In vitro study on the colonization model of Enterococcus faecalis in root canals. Journal of Peking University (Medical Sciences), 2009, 41(6): 699-701.

[4] Sun Tuoqi, Wang Yao, Duan Xingyu. In vitro study on the bactericidal effect of Ka Vo KEY laser on infected root canals and root surfaces of Enterococcus faecalis. West China Journal of Stomatology, 2010, 28(4): 370-373.

[5] Zhuang Peilin, Gao Yan, Ling Junqi. Bactericidal effect of nano-silver on Enterococcus faecalis biofilm on dentin surface. Chinese Journal of Stomatological Research, 2011,5(5):463-469.

[6] Liu Guixiang, Wang Qing, Dou Huiqin. Inactivation and removal of endotoxin in infected root canals by ultrasonic irrigation. West China Journal of Stomatology, 2010, 28(2): 158-161.

Claims (2)

1. a root canal-periapical complex in vitro model is characterized in that, it is formed by the following steps: (1) Take a single premolar extracted due to orthodontics, use a curette and an ultrasonic scaler to remove the soft tissue and calculus on the root surface, place it in 2.5-3.0mol/L formaldehyde solution, and store it at 20-30°C As a spare, the isolated tooth (1) is obtained; (2) Take a glass bottle, the glass bottle is composed of a bottle body (2) and a rubber stopper (3), dig a hole in the center of the rubber stopper (3) according to the diameter of the neck of the isolated tooth, and insert the isolated tooth (1) through the hole Put it into the rubber stopper (3), seal the interface between the isolated tooth (1) and the rubber stopper (3) with light-cured flowing resin, and place the bottle body (2) and the rubber stopper with the isolated tooth (1) fixed (3) Sterilize at a temperature of 110-125°C and a pressure of 10-20 MPa until the sterilization is qualified; (3) In the ultra-clean workbench, inject sterile LB solid medium (4) into the bottle body (2) that has been sterilized and qualified; The aseptic sealing glue is closely connected with the bottle body (2), and the LB solid culture medium (4) just reaches one-third of the root of the isolated tooth (1); between the isolated tooth (1) and the rubber stopper (3) ) at the interface to apply two layers of nail polish to obtain the root canal-periapical complex in vitro model. 2. root canal-periapical complex in vitro model according to claim 1, is characterized in that: in described step (3), also comprise after treating that LB solid culture medium (4) is solidified, with isolated tooth ( 1) Excavate the part of the culture medium (4) that is in contact with the tooth root, pass the needle (6) with the hose (5) through the rubber stopper (3) fixed with the isolated tooth (1), and make the needle (6) ) to the excavated part of the culture medium (4), adopt a small rubber stopper (7) to close the opening of the hose (5), wherein the needle (6) with the hose (5) and the small rubber stopper (7 ) is sterilized at a temperature of 110-125° C. and a pressure of 10-20 MPa before use until the sterilization is qualified; two layers of nail polish are applied at the interface between the needle (6) and the rubber stopper (3).