Rat periodontitis model and construction method and application thereof

Abstract

The invention provides a rat periodontitis model and a construction method and application thereof, on the basis of ligating a tooth neck of a first molar of an SD (Sprague Dawley) rat by a traditional silk thread, light-cured composite resin is bonded in the occlusion direction of the silk thread to manufacture an appearance high point, the silk thread is fixed, a tartar is simulated, and an occlusion high point is formed; meanwhile, the gingival sulcus of the first molar of the upper jaw is cooperatively inoculated with human periodontitis pathogenic bacteria, the implementation method is simple, the repeatability is good, the stability is high, the silk thread loosening rate is reduced to 3.13% when modeling is carried out for 28 days, the loosening rate is greatly reduced compared with 34.38% of a traditional silk thread ligation model, subsequent experiment steps are simplified, lasting gingivitis cell infiltration is caused, and the method is suitable for popularization and application. The number of inflammatory cells is 63.27 + / -7.07% when modeling is carried out for 28 days, experimental periodontitis models of different degrees can be constructed within 2-4 weeks, compared with a traditional model, forming is faster, the model is closer to periodontitis clinical characteristics, and a model basis is provided for researching intervention strategies of different stages.

Description

Technical Field

[0001] This invention belongs to the field of animal model making technology, specifically relating to a rat periodontitis model, its construction method, and its application. Background Technology

[0002] Periodontitis is a chronic inflammatory disease initiated by plaque microorganisms and involving the host immune response. It is characterized by progressive destruction of periodontal supporting tissues (gingiva, alveolar bone, and periodontal ligament). Its pathogenesis involves the synergistic effects of mechanical stimulation such as plaque / calculus accumulation, microbial infection, and the host immune response. However, existing animal models struggle to simultaneously simulate this multi-factor interaction in the clinicopathological process, primarily for the following reasons:

[0003] I. Single-factor models, such as ligation (e.g., suture / wire ligation), can promote plaque accumulation through physical stimulation, but they only simulate mechanical factors and ignore the role of microbial factors in disease progression; bacterial inoculation mainly involves direct injection or application of pathogenic bacteria or their virulence factors. Although this method can induce inflammation, it ignores the influence of mechanical stimulation factors such as tartar on the occurrence and development of periodontitis; high-sugar diets promote plaque accumulation through sticky foods, but when used alone, it takes more than 8 weeks to see slight bone resorption, which is a long cycle and results in mild lesions.

[0004] Second, in order to approximate clinical realities, researchers explored various combined approaches, but none of them fundamentally overcame the aforementioned shortcomings. For example, the combined use of ligation and bacterial inoculation, while increasing infection efficiency through high-frequency inoculation, did not solve the problem of plaque retention failure caused by loosening of the ligation suture, and the timing was not standardized, resulting in asynchronous mechanical stimulation and infection. The pathological manifestations were mainly acute inflammation, lacking the chronic progressive characteristics of human periodontitis. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing a rat periodontitis model. The method involves using light-cured composite resin to form high points on the teeth, increasing local tissue stress through occlusal trauma to promote the release of inflammatory factors, and using fixed sutures to stably maintain mechanical stimulation. The method also simulates the microbial infection process by frequently inoculating core pathogenic bacteria. The construction method shortens the modeling cycle and closely matches clinical pathological features through the synergistic effect of mechanical and microbial factors.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for constructing a rat periodontitis model, characterized by comprising the following steps:

[0008] (1) Feed 8-week-old specific pathogen-free (SPF) grade male S-Prague-Dawley (SD) rats weighing 280-300g with water containing antibiotics for 3 days.

[0009] (2) Anesthetize rats by intraperitoneal injection, and clean the rat's oral cavity and the buccal surfaces of the bilateral maxillary molars;

[0010] (3) Using 3-0 sterile surgical sutures, ligate the neck of the bilateral maxillary first molars of the rats, and then press the surgical sutures into the gingival sulcus.

[0011] (4) Apply light-cured composite resin to the mesial and / or distal surfaces of the rat's maxillary first molar and shape it into a high point in the oral cavity. The resin at the lower end of the high point covers the space between the mesial and / or distal surfaces and the surgical sutures, and the upper end of the high point protrudes above the tooth. noodle;

[0012] (5) Inject 3×10⁻⁶ doses into the buccal and palatal gingival sulci of the bilateral maxillary first molars of rats every 3 days. 9 CFU / mL of Porphyromonas gingivalis bacterial suspension.

[0013] As a preferred technical solution of the present invention, the antibiotic in step (1) is ampicillin.

[0014] As a preferred technical solution of the present invention, the 3-0 sterile surgical suture described in step (3) is sterilized by high pressure for 30 minutes before use, and then placed in an oven at 37°C for complete drying.

[0015] As a preferred technical solution of the present invention, the drug used for intraperitoneal injection anesthesia of rats in step (2) is sodium pentobarbital with a mass concentration of 3%.

[0016] As a preferred technical solution of the present invention, the preservation number of Porphyromonas gingivalis in step (5) is ATCC33277.

[0017] As a more preferred technical solution of the present invention, the upper end of the high point of the outer shape in step (4) is higher than the teeth. Surface thickness 0.5-5mm.

[0018] As a preferred technical solution of the present invention, the photocurable composite resin in step (4) is 3M. TM Filtek TM P60Posterior Restorative light-cured composite resin.

[0019] Another objective of this invention is to provide a rat periodontitis model obtained using the above-described construction method.

[0020] Another objective of this invention is to provide the application of the rat periodontitis model in the study of the pathogenesis of periodontitis, drug efficacy evaluation, and periodontal tissue regeneration mechanism.

[0021] The beneficial effects are as follows:

[0022] The construction method provided by this invention first involves ligating the teeth with silk thread, then fixing the silk thread with resin to form a protrusion on the mesial and / or distal surfaces that is higher than the teeth. The high points of the surface are used to simulate calculus and occlusal trauma, change the occlusal direction, and achieve the temporal synergy of mechanical stimulation and microbial infection by ligating sutures, using high points of the surface, and inoculating Porphyromonas gingivalis. This solves the shortcomings of traditional models, such as easy suture loosening, slow pathological progress, and low clinical fit, speeds up the modeling process, is closer to clinical practice, and provides a model basis for studying intervention strategies at different stages. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the ligation process for the periodontitis model in Embodiment 1 of the present invention.

[0024] Figure 2 This is a comparison of the loosening of the threads in the model of this invention and the traditional model.

[0025] Figure 3 HE staining comparison of rat periodontal tissue pathological sections to show the degree of gingival inflammation between the model of this invention and the traditional model.

[0026] Figure 4 This image shows a comparison of the proportion of inflammatory cells in the periodontal tissue of rats, comparing the degree of gingival inflammation between the model of this invention and the traditional model.

[0027] Figure 5 This is a schematic diagram of the model structure of the present invention. Detailed Implementation

[0028] To more clearly demonstrate the technical solution and effects of the present invention, a detailed description is provided below through specific embodiments. The present invention is further illustrated below with reference to specific embodiments; unless otherwise specified, all raw materials are commercially available.

[0029] Example 1

[0030] A method for preparing a rat periodontitis model is as follows: Figure 1 As shown, the specific steps are as follows:

[0031] (1) Eight-week-old SPF-grade male SD rats were selected and housed in a standard animal room with free access to food and water. After acclimatizing to the environment for one week, ampicillin (50 mg / kg / day) was added to their drinking water three days before the operation.

[0032] (2) *Porphyromonas gingivalis* strain with accession number ATCC33277 was inoculated into the culture medium and cultured anaerobicly at 37°C for 48 h. After collecting the bacterial culture, it was washed three times with sterile PBS solution, and the bacterial concentration was adjusted to 3 × 10⁻⁶. 9 The concentration of CFU / mL requires an anaerobic environment during preparation to prevent the inactivation of pathogenic bacteria.

[0033] (3) After anesthetizing the rats by intraperitoneal injection, the rats' oral cavity was cleaned. Sterile cotton balls soaked in physiological saline were used to wipe the buccal and lingual surfaces of the maxillary molars on both sides to remove food residues. The anesthetic drug was 3% sodium pentobarbital.

[0034] (4) Using sterile surgical sutures, ligate the necks of the bilateral maxillary first molars of the rats, and then gently press them into the gingival sulcus. The sterile surgical sutures are of specification 3-0, and must be autoclaved for 30 minutes before use, and then completely dried in a 37°C oven before use.

[0035] (5) Apply 3M to both the mesial surface 5 and distal surface 6 of the maxillary first molar. TM Filtek TM P60 Posterior Restorative light-cured composite resin is used to shape a first contour point 1 on the mesial surface and a second contour point 7 on the distal surface within the oral cavity. The resin at the proximal end of the first contour point 1 bonds the surgical suture 2 to the mesial surface 5, and the resin at the proximal end of the second contour point 7 bonds the surgical suture 2 to the distal surface 6. The resin at the proximal end of the first contour point 1... Face 8 compared to teeth The height a of the edge line 3 of surface 4 is 1mm, and the second outer contour height point 7 is near Face 9 compared to teeth The height a of the edge line 3 of surface 4 is 1mm, and the structure is as follows: Figure 5 As shown.

[0036] (6) Inject 50 μL of pathogenic bacterial solution into the buccal and palatal gingival sulci of the first maxillary molars of rats using a microsyringe, once every 3 days.

[0037] (7) Periodontal tissue lesions were assessed by histopathology at 7, 14, 21 and 28 days after modeling.

[0038] Example 2

[0039] The following animal experiments will illustrate the method and effects of this invention.

[0040] Experimental Groups:

[0041] The study included: blank control group (NC group), silk ligation group (S group), silk ligation + high-profile group (SR group), high-profile group (R group), Porphyromonas gingivalis injection group (P group), silk ligation + Porphyromonas gingivalis injection group (SP group), and silk ligation + high-profile group + Porphyromonas gingivalis injection group (SRP group). Each group consisted of 16 male SD rats aged 8 weeks and weighing 280-300 grams.

[0042] Experimental methods:

[0043] Group S was established according to (1), (3), and (4) of Example 1; Group R was established according to (1), (3), and step "3M was applied to both the mesial surface 5 and distal surface 6 of the maxillary first molar" in Example 1. TM Filtek TM P60 Posterior Restorative light-cured composite resin is used to shape a first contour point 1 on the mesial surface and a second contour point 7 on the distal surface within the oral cavity. The proximal ends of the first contour point 1 and the second contour point 7 are both located at the positions corresponding to the upper edge of the surgical suture 2 in step (4) of Example 1. The proximal ends of the first contour point 1 are located at the positions corresponding to the upper edge of the surgical suture 2 in step (4) of Example 1. Face 8 compared to teeth The edge line 3 of surface 4 is 1mm high, and the second outer shape high point 7 is near Face 9 compared to teeth The edge line 3 of surface 4 is established with a height of 1mm; the SR group is established according to (1), (3), (4), (5) of the present invention; the P group is established according to (1), (2), (3), (6) of Example 1; the SP group is established according to (1), (2), (3), (4), (6) of the present invention; the SRP group is established according to the method provided in Example 1.

[0044] In Example 2, the ligatures of the rats in the experimental groups were examined for loosening on days 7, 14, 21, and 28 after modeling. The ligature status of the SR group and the S group was compared as follows: Figure 2 As shown, the filament loosening rate in group S increased with the increase of modeling time, reaching 6.25%, 12.50%, 21.88%, and 34.38% on days 7, 14, 21, and 28, respectively. In group SR, filament loosening only began to occur on day 28, with a loosening rate of 3.13%.

[0045] For the experimental groups formed in Example 2, rats were sacrificed by overdose with 1% sodium pentobarbital on days 7, 14, 21, and 28 after modeling. The rat maxillae were isolated, fixed with paraformaldehyde for 48 hours, decalcified for 4 weeks, and then sectioned and stained with hematoxylin and eosin (HE). The degree of gingival inflammation in the rats was compared to that in Example 2. Figure 3 and 4As shown, the inflammatory cell count at day 28 was 4.91% ± 0.50% in the NC group; 12.64% ± 1.00% in the S group; 26.27% ± 2.70% in the SR group; 13.32% ± 2.49% in the R group; 12.94% ± 2.33% in the P group; and 27.87% ± 5.57% in the SP group. In the SRP group, inflammatory cell expression decreased slightly at day 14 and then continued to increase, reaching 63.27% ± 7.07% at day 28. In contrast, the other groups only showed a significant increase at day 7, followed by a gradual decrease.

[0046] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for constructing a rat periodontitis model, characterized in that, Includes the following steps: (1) Feed 8-week-old SPF-grade male SD rats weighing 280-300g with water containing antibiotics for 3 days. (2) Anesthetize rats by intraperitoneal injection, and clean the rat's oral cavity and the buccal surfaces of the bilateral maxillary molars; (3) Using 3-0 sterile surgical sutures, ligate the neck of the bilateral maxillary first molars of the rats, and then press the surgical sutures into the gingival sulcus. (4) Apply light-cured composite resin to the mesial and / or distal surfaces of the rat's maxillary first molar and shape it into a high point in the oral cavity. The resin at the lower end of the high point covers the space between the mesial and / or distal surfaces and the surgical sutures, and the upper end protrudes above the tooth. noodle; (5) Inject 3×10⁻⁶ doses into the buccal and palatal gingival sulci of the bilateral maxillary first molars of rats every 3 days. 9 CFU / mL of Porphyromonas gingivalis bacterial suspension.

2. The construction method according to claim 1, characterized in that, The preservation number of Porphyromonas gingivalis mentioned in step (5) is ATCC33277.

3. The construction method according to claim 1, characterized in that, The highest point of the tooth is higher than the upper first molar. Surface thickness 0.5-5mm.

4. The construction method according to claim 1, characterized in that, The drug used to anesthetize rats via intraperitoneal injection in step (2) is sodium pentobarbital with a mass concentration of 3%.

5. The construction method according to claim 1, characterized in that, The photocurable composite resin mentioned in step (4) is 3M. TM Filtek TM P60 Posterior Restorative UV-curable composite resin.

6. The construction method according to claim 1, characterized in that, The 3-0 sterile surgical sutures described in step (3) are sterilized by high pressure for 30 minutes before use, and then placed in a 37°C oven to dry completely.

7. The construction method according to claim 1, characterized in that, The antibiotic mentioned in step (1) is ampicillin.

8. A rat periodontitis model, characterized in that, Obtained by the construction method according to any one of claims 1-7.

9. The application of the rat periodontitis model of claim 8 in the study of the pathogenesis of periodontitis, drug efficacy evaluation, and periodontal tissue regeneration mechanism.

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