Construction method and application of high-precision controllable rat molar wear model

Abstract

The invention provides a construction method and application of a high-precision controllable rat molar wear model, and belongs to the technical field of oral medicine animal models. According to the method, after the rat is fed with hard food, anesthesia is performed, the oral cavity is opened, and the rat incisor is stimulated by selecting or not selecting any one or more modes of acid erosion and thermocycling stimulation, so that the high-precision controllable rat molar wear model is obtained. The invention develops an acid etching-hard food-cold and hot cycle three-factor time sequence controllable modeling system by simulating the diversity reasons of clinical wear, controlling the types of pathogenic factors and the model construction time to accurately control the severity degree of wear and simulating the progressive process of human functional occlusion wear. A wear model is established in the SD rat molar. The wear form of the model is a composite horseshoe-shaped wear pit which is highly matched with the morphology and biology of human pathological wear. As an experiment is carried out on the living medullary teeth, the pathological manifestation is consistent with the pathological manifestation of clinical progressive wear.

Description

Technical Field

[0001] This invention belongs to the field of animal model technology in oral medicine, and particularly relates to a method for constructing and applying a high-precision and controllable rat molar wear model. Background Technology

[0002] Human tooth wear is the result of long-term effects from multiple factors, including chemical (acid erosion), mechanical (chewing hard objects), and physical (temperature stress). Wear caused by a single factor as the main stimulus, such as wear caused by nocturnal bruxism or habitual hard clenching, differs greatly from wear caused by multiple factors in terms of appearance, pathological manifestations, and treatment plans.

[0003] Existing models of tooth wear rely on mechanical scraping and cutting, and lack standardized models, making it impossible to simulate the progressive tooth wear caused by the synergy of multiple clinical factors. There are problems such as uncontrollable wear degree, inaccurate location, and mismatch with the pathological wear process in humans, resulting in poor repeatability of experimental results and inability to accurately simulate the clinical wear mechanism. Existing animal models have the following defects: (1) Single-factor model: only acid etching leads to uniform demineralization (non-mechanical wear morphology); only mechanical cutting leads to surface scratches (lacking chemical corrosion characteristics), ignoring the reality that other stimulating factors besides the main stimulating factors at the natural wear site have not disappeared; (2) Lack of standardization: the temperature fluctuation of hot and cold cycles is uncertain, the composition of hard food is unstable, the type and acidity of acid etching are uncertain, and the time of various stimuli and the interval of each stimulation are not fixed, so the repeatability is poor; (3) The model is polished in a short time by a polishing device, and it is often an extracted tooth, which has lost the possibility of pathological changes. Therefore, it only simulates wear in appearance and has a narrow range of applications. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for constructing and applying a high-precision and controllable rat molar wear model, which overcomes the problems of existing extracted tooth wear models that rely on mechanical scraping and cutting, lack standardized models, cannot simulate the progressive tooth wear caused by multiple factors in clinical practice, have uncontrollable wear degree, inaccurate location, do not match the pathological wear process in humans, resulting in poor repeatability of experimental results and inability to accurately simulate clinical wear mechanisms.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for constructing a high-precision and controllable rat molar wear model, comprising the following steps:

[0007] After feeding rats hard food, they were anesthetized, their mouths were opened, and the rats' incisors were stimulated by one or more of the following methods, with or without acid erosion and hot-cold cycling stimulation, to obtain a high-precision and controllable rat molar wear model.

[0008] Preferably, the hardness of the hard food is 16-18 MPa.

[0009] Preferably, the acid erosion is performed by applying phosphate gel to both sides of the surface of the rat maxillary molars, the concentration of the phosphate gel is 35-40%, and the acid erosion time is 5-15 minutes.

[0010] Preferably, the hot and cold cycling stimulation is performed by alternating stimulation with a small popsicle stick and a dental gel tip, wherein the temperature of the small popsicle stick is 0°C and the temperature of the dental gel tip is 100-300°C.

[0011] Preferably, the stimulation time of the small popsicle and the dental gel tip is 20-40 seconds, and the total time of the hot and cold cycle stimulation is 4-14 minutes.

[0012] Preferably, the entire construction cycle of the rat is 2 to 6 weeks.

[0013] Preferably, a rat molar wear model with mild wear is obtained when one or more of the following stimulation methods are not selected: acid erosion and hot-cold cycle stimulation, and the construction period is 6 weeks.

[0014] A rat molar wear model with moderate wear was obtained by either acid erosion or hot-cold cycling stimulation for a period of 4 weeks, or by stimulating with both acid erosion and hot-cold cycling stimulation for a period of 2 weeks.

[0015] A rat molar wear model with severe wear was obtained by using either acid erosion or hot-cold cycling stimulation for a period of 6 weeks, or by using both acid erosion and hot-cold cycling stimulation for a period of 4 weeks.

[0016] This invention also provides the application of the high-precision and controllable rat molar wear model constructed by the above-described method in the study of tooth wear pathology.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention simulates the diverse causes of wear in clinical settings, precisely controlling the severity of wear by managing the types of pathogenic factors and the timing of model construction. It simulates the progressive process of functional occlusal wear in humans, developing a time-controllable modeling system based on three factors: acid etching (chemical), hard food (mechanical), and thermal cycling (physical). This system was used to establish a wear model in the molars of SD rats. The model boasts advantages such as high operability, controllable results, readily available equipment, rapid establishment, and low technical sensitivity. It fills a critical gap in the lack of standardized animal models for this common clinical condition, laying the foundation for subsequent in vivo experimental research.

[0019] Existing techniques, when used on extracted teeth, only show single scratches or uniform demineralization in terms of wear morphology. The rat molar wear model of this invention exhibits a complex horseshoe-shaped wear pattern, which highly matches the morphology and biology of pathological wear in humans. The microstructure of the wear area shows >90% similarity to human bruxism samples. Because experiments were conducted on vital teeth, periodontal manifestations such as gingival recession, alveolar bone resorption, and increased CEJ-AC (cementoenamel-alveolar crest) were observed, along with pathological manifestations such as pulpitis, pulp necrosis, reparative dentin formation, pulp calculi, and vacuolar degeneration of the pulp cavity, consistent with the pathological manifestations of clinical progressive wear.

[0020] This invention successfully constructed a rat molar wear model based on common clinical causes. Compared with extracted human teeth, this model is easier to obtain, and the construction tools and materials are inexpensive, raising fewer ethical concerns and making the model highly practical. Furthermore, the batch-to-batch wear depth variation is <5% (compared to >30% in traditional methods), successfully standardizing the rat molar wear model construction method. This model has high application value in studying the pathological changes of wear, clinical treatment plans, and the development of oral biological materials.

[0021] Human teeth are difficult to obtain and subject to strict ethical review. This invention uses rats as research subjects, which are readily available experimental animals. The procedure is simpler and more affordable compared to other large animals, causing less trauma to the experimental animals and will not violate the 3R principle or the Declaration of Helsinki. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the basic steps in developing a tooth wear model;

[0023] Figure 2 This is a quantitative analysis of feed hardness in two groups of rats;

[0024] Figure 3 The wear status of tooth tissue surface after different stimulation treatments is as follows: (wear status of maxillary molars of rats in the control group under normal diet (ad), mild wear of maxillary molars of rats in experimental group 1 under hard diet (eh), moderate wear of maxillary molars of rats in experimental group (il), wear status of maxillary molars of rats after 4 weeks of acid etching under hard food conditions (j), wear status of maxillary molars of rats after 4 weeks of heat cycling under hard food conditions (k), wear status of maxillary molars of rats after 2 weeks of heat cycling and acid etching under hard food conditions (l), severe wear of maxillary molars of rats in experimental group (mp), wear status of maxillary molars of rats after 6 weeks of acid etching under hard food conditions (n), wear status of maxillary molars of rats after 6 weeks of heat cycling under hard food conditions (o), wear status of maxillary molars of rats after 4 weeks of heat cycling and acid etching under hard food conditions (p), (bar: 2mm)).

[0025] Figure 4 The tooth wear grading standard ((A) is a macroscopic image of different degrees of wear on human molars, (B) is a macroscopic image of different degrees of wear on rat molars (bar=2mm));

[0026] Figure 5 It is a bar chart comparison of the distance from the cementoenamel junction (CEJ) to the alveolar ridge (AC) in four groups;

[0027] Figure 6 These are the H&E staining results in a rat tooth wear model (left: representative microscopic photograph of the severe wear group; right: corresponding specimen of the moderate wear group; blue arrow: alveolar bone resorption; yellow arrow: pulp necrosis; red arrow: pulp inflammation; green arrow: edema; black circle: pulp calculus; black arrow: pulp vacuoles; white arrow: repaired dentin (bar=0.5mm)). Detailed Implementation

[0028] This invention provides a method for constructing a high-precision and controllable rat molar wear model, comprising the following steps:

[0029] After feeding rats hard food, they were anesthetized, their mouths were opened, and the rats' incisors were stimulated by one or more of the following methods, with or without acid erosion and hot-cold cycling stimulation, to obtain a high-precision and controllable rat molar wear model.

[0030] In this invention, the hardness of the hard food is 16-18 MPa; the anesthesia uses aphthol solution, the concentration of which is preferably 0.2-0.3%, more preferably 0.25%, and the injection volume of which is preferably 200-400 mg / kg, more preferably 250-350 mg / kg, and even more preferably 300 mg / kg; before the acid erosion and hot-cold cycle stimulation treatment, the surface is disinfected with alcohol swabs, the concentration of which is preferably 90-98%, more preferably 95%; the acid erosion is performed by applying phosphate gel bilaterally to the surface of the rat's maxillary molars, the concentration of which is preferably 35-40%, more preferably 36-39%, and even more preferably 37%; the acid erosion time is preferably 5-15 min, more preferably 7-13 min, and even more preferably 10 min; the hot-cold cycle stimulation is performed by alternating stimulation with small popsicles and dental gel tips, the temperature of which is 0°C. The temperature of the dental gel tip is preferably 100–300℃, more preferably 150–250℃, and even more preferably 200℃; the stimulation time of the small popsicle and the dental gel tip is preferably 20–40 seconds, more preferably 30 seconds; the total time of the hot and cold cycle stimulation is preferably 4–14 minutes, more preferably 10 minutes; the entire construction period of the rat is 2–6 weeks; when no one or more of the acid erosion and hot and cold cycle stimulation is selected and the construction period is 6 weeks, a rat molar wear model with mild wear is obtained; when any one of the acid erosion and hot and cold cycle stimulation is selected and the construction period is 4 weeks, or when both acid erosion and hot and cold cycle stimulation are selected and the construction period is 2 weeks, a rat molar wear model with moderate wear is obtained; when any one of the acid erosion and hot and cold cycle stimulation is selected and the construction period is 6 weeks, or when both acid erosion and hot and cold cycle stimulation are selected and the construction period is 4 weeks, a rat molar wear model with severe wear is obtained.

[0031] This invention also provides the application of the high-precision and controllable rat molar wear model constructed by the above-described method in the study of tooth wear pathology.

[0032] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1

[0034] 1. Materials and Methods

[0035] Sprague-Dowley (SD) rats were obtained from the Animal Experiment Center of Peking University School of Medicine. Forty male SD rats (age: 6 weeks, weight: 150-200 g) were housed in an SPF-grade animal room with a 12-hour light-dark cycle, a controlled temperature of 25°C, and unlimited access to water and food. Upon arrival, the animals were housed at a rate of 4 rats per cage and underwent a 2-week acclimatization period. Experiments were conducted when the rats reached 8 weeks of age. This experimental protocol was approved by the Animal Ethics Committee (IACUC) of the Peking University School of Stomatology (Approval No.: BDKQ-202501210044).

[0036] The experimental animals were randomly divided into four groups (8 SD rats in each group): (1) Control group: normal diet; (2) Experimental group 1: hard food; (3) Experimental group 2: hard food + acid erosion; (4) Experimental group 3: hard food + hot and cold stimulation; (5) Experimental group 4: hard food + acid erosion + hot and cold stimulation.

[0037] 2.1 Construction of Tooth Wear Model

[0038] By controlling the hardness of food, the duration of hot and cold stimulation (homemade popsicles, teething toy fuses), and / or acid erosion stimulation ( The strength of Etch 35Gel (Kulzer, Germany) was used to simulate different degrees of wear on the maxillary molars of rats. Figure 1 ).

[0039] The control and experimental groups were fed two different rat diets, once daily, with each rat receiving 450g per feeding. The two diets were purchased from two different companies: (Diet HX-3: Beijing Vital River Laboratory Animal Technology Co., Ltd.) and (Diet MD 17121: Jiangsu Medison Biomedical Co., Ltd.). A representative sample of 100g was obtained from each feed bag using a cone and quartering method. From this sample, 20-25 intact grains of uniform size, length, and structural integrity were selected. Grain hardness was measured using a GWJ-2 grain hardness tester (Beijing Jinkelida Co., Ltd., China) by applying compressive force along the longitudinal axis of the grain. The hardness values ​​of all individual grains were recorded, and the average hardness was calculated.

[0040] Statistical analysis was performed using GraphPad Prism software (GraphPad Software Inc., San Diego, USA), version 9.0. Normally distributed data are expressed as mean ± standard deviation (mean ± SD). After comparing means using one-way ANOVA, Tukey's post-hoc test was used to determine statistical significance. A p-value < 0.05 was considered statistically significant.

[0041] Experimental results: such as Figure 2 As shown in the figure, we used a standardized indentation testing protocol to quantitatively characterize the mechanical properties of two commercial rodent feeds. The mean hardness values ​​of the two rodent feeds were 17.73 MPa (HX-3 feed) and 15.43 MPa (MD 17121 feed), respectively (p < 0.001). This validates feed hardness as a controlled experimental variable distinguishing the intervention group from the control group.

[0042] The acid etching process is as follows: Aphthyl (tribromoethanol + tert-butanol = 1:1) (Nanjing Aibei Biotechnology Co., Ltd., China) was diluted to 0.25% with physiological saline and used as a 300 mg / kg muscle anesthetic for rats. After anesthesia, the rat's mouth was opened using a rat mouth fixator, and the rat's incisors were fixed. The surface of the maxillary molars was wiped with cotton balls soaked in 95% alcohol, and after the surface dried, 37% phosphate gel ( Etch 35Gel (Kulzer, Germany) was applied bilaterally to the surface of the maxillary molars and left on for 10 minutes, during which time the gel covered the occlusal surface. The gel was removed with a cotton ball after treatment. The rats were then allowed to awaken naturally.

[0043] The hot and cold stimulation procedure was as follows: After anesthesia, the surface of the maxillary molars was wiped with cotton balls soaked in alcohol. After drying, clinical cold testing (small ice stick, 0°C) and dental gel tips heated to 200°C were used alternately for stimulation. Each temperature was maintained for 30 seconds, and the entire treatment lasted 10 minutes, divided into five groups. After treatment, the tooth surface was wiped with cotton balls, and the rats were allowed to wake up naturally.

[0044] Because the types and intensities of stimulation received by experimental group 4 were higher than those of other groups, the experimental endpoint for this group was ended two weeks earlier to avoid violating the 3R principle. 50% of the animals in experimental group 4 were euthanized two weeks after the intervention. Subsequently, 50% of the animals in each of experimental groups 2-4 were euthanized four weeks after the intervention. At the six-week endpoint, the euthanasia procedure included all remaining animals from the control group and experimental group 1, as well as 50% of the surviving animals from experimental groups 2 and 3. Euthanasia was performed via carbon dioxide inhalation, followed by confirmation using a confirmatory method (cervical dislocation / secondary thoracotomy), in accordance with the American Veterinary Medical Association (AVMA) guidelines. During the intervention period, animals were fed daily according to the above-mentioned feeding amounts, and acid etching and hot / cold stimulation were performed weekly.

[0045] Maxillary molar specimens were collected from SD rats after euthanasia and fixed with 4% formaldehyde solution (Seville, Wuhan, China) for 48 h.

[0046] Throughout the experiment, all groups of rats reached the pre-defined humane endpoint. Furthermore, regardless of treatment group, universally quantifiable molar wear was observed on the occlusal surfaces of all rats.

[0047] 2.2 Micro-CT Assessment

[0048] To assess differences in wear between groups, the maxillary alveolar bone and molars of SD rats were fixed and then subjected to micro-computed tomography (microCT) scanning and analysis using a Siemens (Germany) Inveon MM system. Scanning parameters were as follows: 80 kV, 500 mA, 360° rotation, exposure time 1500 ms, and reconstruction matrix 512×512. After scanning, the CT dataset was transferred to a workstation and further analyzed and reconstructed into three-dimensional images using analysis software (Inveon ResearchWorkplace, Siemens, Munich, Germany).

[0049] Wear grading assessment method

[0050] The assessment of rat tooth wear was based on the Smith grading system, the most commonly used method in clinical practice. This method classifies tooth wear according to the amount of tissue residue and surface morphology, based on clinical visual observation: Grade 0 indicates an intact enamel surface with visible developmental grooves or only superficial enamel wear. Grade 1 indicates a flat enamel surface with disappeared developmental grooves, but the damage is confined to the enamel layer and does not expose dentin. Grade 2 indicates localized dentin exposure (≤1 / 3 of the tooth surface area) without secondary dentin formation. Grade 3 indicates extensive dentin exposure (>1 / 3 of the tooth surface area) and / or the presence of secondary dentin. Grade 4 indicates pulp exposure or extensive secondary dentin exposure, accompanied by a decrease in occlusal vertical distance and / or pulp chamber perforation. Based on this, Grades 0 and 1, which are difficult to distinguish visually, were combined into a physiological wear control group. Correspondingly, Grade 2 corresponds to the mild wear group, Grade 3 to the moderate wear group, and Grade 4 to the severe wear group.

[0051] Experimental results: such as Figure 3 As shown, a molar wear model was successfully established by reconstructing the rat dentition using microCT, and the 3D rendering results confirmed the occlusal surface topography changes consistent with the clinical wear pattern.

[0052] The maxillary molars of the control group rats showed normal wear, characterized by a reduced cusp inclination on the occlusal surface. In contrast, the maxillary molars of the experimental group rats showed varying degrees of wear. Figure 3 (ad in the text)

[0053] In experimental group 1, rats showed enamel loss on the occlusal surface of their maxillary molars, but no significant changes in the occlusal morphology. This degree of wear was classified as mild wear. Figure 3 (eh in the middle).

[0054] Rats in experimental groups 2 and 3 received treatment for 4 weeks, while rats in experimental group 4 received stimulation for 2 weeks. During this period, their maxillary molars showed partial dentin loss. This degree of wear was classified as moderate wear. Figure 3 (il in the middle).

[0055] Experimental groups 2 and 3 received 6 weeks of treatment, while experimental group 4 received 4 weeks of stimulation. During this period, the amount of dentin loss significantly increased, and the pulp was exposed, which was classified as severe wear. Figure 3 (mp in the text).

[0056] These findings are consistent with natural wear and tear observed in human teeth. Figure 4 ).

[0057] 2.3 Pathological assessment

[0058] Periodontal health was assessed by measuring the distance from the cementoenamel junction (CEJ) to the alveolar ridge (AC). Statistical analysis was performed using GraphPad Prism software (GraphPad Software Inc., San Diego, USA), version 9.0. Normally distributed data are expressed as mean ± standard deviation (mean ± SD). Significance was determined using Tukey's post-hoc test after comparing means using one-way ANOVA. A p-value < 0.05 was considered statistically significant.

[0059] Tissue samples scanned by micro-CT were completely immersed in 0.5M EDTA decalcification solution (Seville, Wuhan, China) and agitated on a shaking platform. The decalcification solution was changed every other day until needle puncture was unimpeded. Tissue was sectioned into 5μm thick sections along the sagittal plane and stained with H&E. Histopathological structures were observed using a 3DHISTECH (Hungary) microscope after scanning.

[0060] Experimental results: such as Figure 5 As shown. In humans, a distance exceeding 2 mm indicates alveolar bone resorption. Compared with the control group (0.20±0.02 mm), the CEJ-AC distance in the rat model groups was significantly increased: mild group (0.40±0.04 mm), moderate group (0.68±0.06 mm), and severe group (0.82±0.12 mm) (P<0.001).

[0061] Histopathological results showed varying degrees of periodontal and pulp lesions in the rat molars, consistent with observations of human teeth. In the control group, the tooth surface and alveolar bone surface were smooth and intact, and the periodontal ligament space was uniform in width. Almost no inflammatory cell infiltration was observed in the periodontal and pulp tissues of the control rats. In contrast, the model group exhibited tooth surface destruction, reduced alveolar bone height, and pulp lesion characteristics, including inflammatory cell infiltration, vasodilation, and vacuolar lesions. Figure 6 As shown, the two images on the left illustrate the pathological features of the severely worn group, including pulp exposure, inflammatory cell infiltration, and alveolar bone resorption. The two images on the right show the characteristic pathological features of the moderately worn group, including pulp calculus and reparative dentin formation. These different pathological features correspond to different stages of wear-induced degeneration.

[0062] As demonstrated by the above embodiments, this invention simulates the diverse causes of wear in clinical settings, precisely controls the severity of wear by controlling the types of pathogenic factors and the timing of model construction, and simulates the progressive process of functional occlusal wear in humans. It develops a time-controllable modeling system for three factors: acid etching (chemical), hard food (mechanical), and thermal cycling (physical), and establishes a wear model in the molars of SD rats. The wear morphology of this rat molar wear model is a complex horseshoe-shaped pit, which highly matches the morphology and biology of pathological wear in humans. The microstructure of the wear area has a similarity of >90% to human bruxism samples. Because the experiment was conducted on vital teeth, the pathological manifestations are consistent with the pathological manifestations of progressive wear in clinical settings.

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a high-precision, controllable rat molar wear model, characterized in that, Includes the following steps: After feeding rats hard food, they were anesthetized, their mouths were opened, and the rats' incisors were stimulated by one or more of the following methods, with or without acid erosion and hot-cold cycling stimulation, to obtain a high-precision and controllable rat molar wear model.

2. The construction method according to claim 1, characterized in that, The hardness of the hard food is 16-18 MPa.

3. The construction method according to claim 1, characterized in that, The acid erosion was performed by applying phosphate gel to both sides of the surface of the maxillary molars of rats. The concentration of the phosphate gel was 35-40%, and the acid erosion time was 5-15 minutes.

4. The construction method according to claim 1, characterized in that, The hot and cold cycling stimulation is achieved by alternating stimulation with a small popsicle stick and a dental gel tip. The temperature of the small popsicle stick is 0°C, and the temperature of the dental gel tip is 100-300°C.

5. The construction method according to claim 4, characterized in that, The stimulation time for the small popsicle and the dental gel tip is 20-40 seconds, and the total time for the hot and cold cycle stimulation is 4-14 minutes.

6. The construction method according to claim 1, characterized in that, The entire construction period for the rats was 2 to 6 weeks.

7. The construction method according to claim 6, characterized in that, A rat molar wear model with mild wear was obtained when one or more of the acid erosion and hot-cold cycle stimulation methods were not selected and the construction period was 6 weeks. A rat molar wear model with moderate wear was obtained by either acid erosion or hot-cold cycling stimulation for a period of 4 weeks, or by stimulating with both acid erosion and hot-cold cycling stimulation for a period of 2 weeks. A rat molar wear model with severe wear was obtained by using either acid erosion or hot-cold cycling stimulation for a period of 6 weeks, or by using both acid erosion and hot-cold cycling stimulation for a period of 4 weeks.

8. The application of the high-precision controllable rat molar wear model constructed by the construction method according to any one of claims 1 to 7 in the pathological study of tooth wear.

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