Selection and application of experimental animals in stomatological research
I. Oral anatomy and physiological characteristics of commonly used experimental animals
Stomatology, like other medicine, has developed rapidly. New treatments, drugs, orthopedic materials, etc. They are constantly being discovered, some of which can be directly used in clinic, while others need to be verified by animals before they can be used in clinic. Therefore, experimental animals play an increasingly important role in stomatological research. Experimental animals can be selected to study the congenital malformation, defect, acquired trauma, inflammation and tumor of oral organs. Such as caries or non-caries dental diseases, pulp diseases, periapical diseases, periodontal diseases, oral mucosal diseases, tumors, salivary glands, joint and jaw diseases and malocclusion, etc., including etiology, pathology, course of disease, diagnosis, prevention and treatment (including inlay and restoration techniques). When selecting experimental animals for various studies of stomatology, we should first be familiar with the anatomical and physiological characteristics of the oral cavity of these experimental animals.
(1) Number and growth characteristics of teeth in commonly used experimental animals
1. Tooth type and number (based on permanent teeth)
Monkey 2(2 123/2 123)=32 Dog 2 (3142/3143) = 42;
Cat 2 (3131/3121) = 30 rabbit 2 (2033/1033) = 28;
Guinea pig 2 (1013/13) = 20 hamster 2 (1003/1003) =16;
Rat 2 (1003/1003) =16; Mouse 2 (1003/1003) =16.
See table 10-34 for the number of teeth of commonly used experimental animals.
Table 10-34 Number of Permanent Teeth in Human and Common Laboratory Animals
The number of premolars, molars and permanent teeth of human and animal incisors (canine teeth)
Person 8 4 8 12 32
Monkey 8 4 8 12 32
Dog 12 4 16 10 42
Cat 12 4 10 4 30
Rabbit 6 0 10 12 28
Dolphin and mouse 4 0 4 12 20
Gopher 4 0 0 12 16
Rats 4 0 0 12 16
Mouse 4 0 0 12 16
2. Tooth growth characteristics The growth and shedding of monkey teeth have certain rules. Newborn monkeys generally have no teeth and occasionally grow two front teeth. Within 6 months, 20 deciduous teeth grew, and stopped growing until 14 ~ 16 months. Then begin to grow and replace deciduous teeth at a constant age. The order of tooth growth in rhesus monkeys is as follows: deciduous teeth: middle and lower door → upper middle door → lower side door → upper dog → lower dog → upper jaw 2→ lower jaw 3→ upper jaw 3; Permanent teeth; Lower mortar 1→ upper mortar 1→ upper middle door or upper side door (replacement) → lower middle door (replacement) → upper side door (replacement) → upper mortar 2→ lower mortar 2→ front mortar 1 (replacement) → front mortar 2 (replacement) →
The age and tooth growth of rhesus monkeys are shown in table 10-35.
Table 10-35 tooth growth of rhesus monkeys
Age (month) tooth growth
Newborn monkeys generally have no teeth, and a few occasionally grow two front teeth.
1.0 Four central incisors grow in the upper and lower jaws at the same time.
1.5 can grow 8 front teeth in 36 days.
2.0 Maxillary canine teeth just appeared.
3.0 The maxillary canine and the first premolar rise simultaneously.
4.0 Maxillary and mandibular first premolars grow out
4.5 The mandibular second premolars are elevated or 1 premolars are all aged.
5 ~ 6 deciduous teeth are all flush.
14 ~ 16 The first molar on the right side of mandible grows first, and the left side of mandible 1 molar grows later.
The first molar grows from 17 to 18, and the situation is different.
19 ~ 3 1 The maxillary and mandibular first molars are full.
32 ~ 40 to change the front teeth and lateral teeth, the order changes greatly. Change canine teeth and grow a second molar.
42 ~ 43 change mandibular lateral incisors and central incisors. The second molars have all grown out and the first premolars have been replaced.
44 ~ 56 are second premolars and canine teeth.
65 ~ 78 mandibular third molars grow.
72 ~ 82 maxillary third molars have grown, so far all permanent teeth are flush.
Dogs have 42 permanent teeth, the front teeth gradually increase from 1 to the third, and the lower front teeth are smaller than the upper front teeth. Canine teeth are well developed, large, sharp and curved. The number of molars varies with species, and the general tooth type is 6/7. However, in short-headed dogs, the molar is usually 5/7, and the molar size is also very different. The fourth tooth of the upper molar and the lower molar 1 teeth are the largest, and the front and rear teeth gradually become smaller. Puppies give birth to deciduous teeth ten days after birth. After two months, the front teeth, canine teeth and molars gradually changed into permanent teeth, and the teeth were replaced in 8 ~ 10 months, but the canine teeth took10.5 years to grow. See table 10-36 for the age, tooth growth, replacement and wear of dogs.
Table 10-36 Dog Age and Teeth
Age-related dental condition
Only deciduous teeth (white, thin and pointed) under 2 months old.
Replace the front teeth in 2 ~ 4 months.
Change canine teeth (white teeth and blunt teeth) every 4 to 6 months.
Replace molars at 6 ~ 10 months.
1 year-old teeth are long, white and bright, and the front teeth have sharp protrusions.
The tip of the 2-year-old lower incisor was ground flat.
At the age of 3, the tips of the upper and lower incisors were ground flat.
At the age of 4 ~ 5, the upper and lower incisors began to wear and turn slightly yellow.
6 ~ 8 years old, the front teeth are worn to the roots, the canine teeth are yellow and wear the lips, and the beard is white.
10 years old or older, with worn front teeth, incomplete canine teeth, yellow roots and white lips and beard.
(2) Some anatomical and physiological features in the oral cavity of commonly used experimental animals.
Dogs, cats and other carnivores have well-developed canine teeth. The cuticle of filiform papillae on the cat's tongue is thick and hooked, which is convenient for scraping the meat on the bone. Rabbits are herbivores, with well-developed incisors for cutting grass, no canine teeth, smooth mucosa on the hard palate of the top wall of the mouth, and many transverse folds on the mucosa. The upper lip of sheep and goats is sensitive and flexible.
The salivary glands of dogs are well developed, including parotid gland, submandibular gland, sublingual gland and orbital gland. Some people think that dog's saliva does not contain amylase, but contains lysozyme, which can kill bacteria, so it is very common for dogs to clean wounds with their tongues and disinfect them. Because dogs have no sweat glands, they can secrete a lot of saliva to dissipate heat in hot weather.
Cats have five pairs of salivary glands, namely, subauricular gland, submandibular gland, sublingual gland, molar gland and infraorbital gland, which all open in the mouth to form a mixed secretion-saliva.
The salivary glands of rabbits are well developed. Besides the three pairs of salivary glands (parotid gland, submandibular gland and sublingual gland) common in mammals, there are also a pair of suborbital glands and some scattered small glands. Rabbit saliva contains amylase, but its ability to decompose starch is very weak.
Guinea pigs have five pairs of salivary gland, parotid gland, submandibular gland, zygomatic gland, sublingual gland and sublingual gland. In addition, there are labial glands near the lip corner, and buccal glands on the buccal side wall of the mouth.
Rats and mice have three pairs of salivary glands, namely, subauricular gland, submandibular gland and sublingual gland.
There are many nipples on the surface of rabbit tongue, which are taste receptors, and the tip and sides of the tongue are the most densely distributed. A dog's tongue is wide and thin in front and thick in the back. There is a longitudinal groove in the center of the back of the tongue, and the surface is covered with a dense filiform nipple. Mushroom papillae are distributed on both sides of the tongue, and at the back of the tongue, besides filamentous papillae, there are also conical papillae, all of which are taste receptors. There are 1 pairs of small salivary gland papillae near the posterior midline of rat incisors. There are wavy nipples near the base of the tongue, conical nipples and filiform nipples on the back of the tongue.
The shape and size of a dog's mouth are closely related to the formation of its skull. Dogs with long heads have long and narrow mouths, while dogs with short heads have short and wide mouths. The dog's mouth is very cracked, and the corner of his mouth is probably facing the third or fourth molar. The lips are thin and flexible, with touching hairs on the surface. There is a small area in the center of the upper lip that does not touch the hair, but there is a central groove (human), and the side edge of the lower lip has serrated protrusions.
Second, the choice and application of animals
(1) Macaques and Marmosets
Macaque is the first choice for experimental research in stomatology. In particular, orthodontics and stomatology are commonly used, such as the observation of the effect of replanting teeth; Histopathological changes; Study on histopathological changes of dry socket: and explore the influence of various treatment methods and materials on tissue healing, so as to obtain the best clinical treatment effect.
The number of teeth of macaques is the same as that of humans, and the arrangement of teeth is similar to that of humans. Many microorganisms in the mouth are the same as those in the human mouth, such as adding sugar to the food of macaques and feeding them to animals. It can induce dental caries of deciduous teeth and permanent teeth, and the changes of dental caries are similar to those of human beings. Macaca mulatta can be selected to study the etiology, incidence and treatment of dental caries.
Periodontal tissues of marmosets are extremely sensitive to general metabolic changes. The changes of periodontal ligament in elderly marmosets are very similar to those in the elderly. In addition, the occurrence process and histopathological changes of periodontitis in marmosets are similar to those in humans, so marmosets are ideal animals for periodontal disease research.
The macaque was also used to study the way, degree, treatment time, treatment method and histopathological changes of tooth fracture in order to explore the treatment effect. It is also often used to study the toxicity and carcinogenicity of filling materials and dental materials to dental pulp, periodontal tissue and bone tissue.
(2) rabbits
The distribution of blood vessels in the neck and maxillofacial region of rabbits is similar to that of humans. Ligation of external carotid artery is often needed in human neck and face surgery. In view of a series of problems caused by ligation, such as blood circulation disorder and blood circulation recovery time, adult rabbits can be selected to ligate unilateral external carotid artery and observe the changes of vascular image in its distribution area, so as to explore the recovery of vascular image and the development of collateral circulation channels.
Rabbit lips are commonly known as rabbit lips. Rabbits are excellent animals to study the etiological relationship of cleft lip and palate (such as heredity, drugs, environment, etc.). ) and other congenital defects (such as brain edema, spina bifida, dementia, chondrodysplasia, etc. ).
Mandibular protrusion in rabbits has been reported, and the reason is similar to that in humans. It can be used to study the causes of mandibular protrusion (such as heredity, malocclusion, acromegaly, etc.) and explore the measures of mandibular protrusion to guide clinical practice.
Rabbits are sensitive animals to observe Arthus's inflammatory reaction in dental pulp. Arthus reaction is an inflammatory reaction, which is characterized by edema, erythema, induration and necrosis. Within a few hours after injecting the same antigen into the skin of an animal exposed to the antigen. Its reaction mechanism belongs to type ⅲ allergic reaction, which is often used to detect whether there are specific circulating antibodies in the body. In the experiment, 2.5kg male rabbits were sensitized with O hemolysin until the antibody titer rose and Arthus skin reaction appeared, and then the pulpotomy was locally attacked with the same antigen. After that, the dental pulp was taken out at different times and observed by histological method and immunofluorescence technique. Light microscope confirmed that there was acute inflammation in dental pulp, immunofluorescence confirmed that there was immune complex in dental pulp tissue, and the specific fluorescence was mainly in the blood vessel wall. It is proved that Arthur inflammatory reaction exists in dental pulp. With this reaction, it shows that if the body comes into contact with the same antigen again through the pulp, the bone marrow tissue will become inflamed, which is the result of the local reaction of antigen and antibody.
Establishment of rabbit model of oral mucosal ulcer. Oral mucosal ulcer is very common, which is generally considered to be caused by autoimmune or immune function damage. The antigen is a normal stillborn baby (the death time is not more than 2-3 hours). Oral mucosa was collected under aseptic conditions, washed with physiological saline and put into 0. 1MpH7.4 phosphate buffer. Cut the mucosa, grind it into tissue pulp, add the same amount of Freund's complete adjuvant or incomplete adjuvant, and mix well to form emulsion. The solution was injected intradermally on both sides of rabbit spine, and the number of injections was 8- 10. Oral mucosal ulcer began to appear on the third day after the fourth injection, and then appeared repeatedly until the fourth month of observation.
Experimental study on bone marrow transplantation of rabbit cancellous bone. Bone marrow and bone marrow (PCBM) transplantation is to repair maxillofacial bone defects with bone fragments, periosteum and bone marrow excavated from iliac crest as grafts. After anesthesia, healthy rabbits are often selected, and iliac bone blocks of 1.5×0.5cm2 are cut from the right iliac crest, and appropriate PCBM is dug out, and then repaired.
Rabbits are also suitable for toxicity experiments of oral plastic materials. Polytetrafluoroethylene composite is an ideal and widely used artificial bone material in maxillofacial surgery and plastic surgery, which can replace bone, cartilage and soft tissue. White-eared rabbits are often selected for toxicity experiments.
Rabbits are also used to study the etiology and pathological changes of oral mucosal diseases and periodontal diseases. For example, the injury of maxillary or mandibular branches of trigeminal nerve in rabbits can lead to experimental nutritional ulcer of rabbit lips. It can also be used in orthopedic experimental research, such as pad experiment, to explore the mechanism of tooth function regulation.
(3) dogs
Dogs are widely used in stomatological research. For example, after the second, third and fourth premolars of dogs are extracted, such as removing bone septa, which is similar to human tooth extraction wounds, it is used to study the animal model of Sjogren's disease. After dog teeth are extracted, the periodontal ligament is tough, the pulp cavity is large, and tooth fracture or alveolar fracture often occurs, but the periodontal ligament is still not easy to break. During tooth extraction, the periodontal ligament should be chiseled off repeatedly with narrow moth eyebrows along the periodontal space, and then the periodontal ligament should be chiseled off as much as possible, and then the teeth should be loosened with narrow teeth. Finally, clamp the teeth with tweezers and tap the tweezers along the long axis of the teeth with a hammer to rinse the teeth, for example, with teeth.
Histology of periodontal ligament, histopathology of periodontitis and epidemiology of periodontal disease in dogs are similar to those in humans, so it is very ideal to study dogs as animal models of periodontal disease.
Dogs are commonly used in the research of autologous tooth transplantation and radiotherapy. Some congenital diseases of dogs, such as cleft lip, cleft palate and mandibular protrusion, have certain genetic factors. The way of dog mandibular protrusion is similar to that of human mandibular ilium protrusion, so dogs can also be used as animal models of maxillofacial deformities.
(4) Golden Hamster
Six weeks later, hamster cheek pouch was smeared with carcinogen DMBA, and all animals induced epithelial dysplasia leukoplakia, which was similar to oral leukoplakia in clinical patients. Therefore, hamster is a suitable animal to study epithelial dysplasia leukoplakia, and it is easy to succeed to choose 60g golden hamster.
Chronic mechanical injury tobacco and alcohol can stimulate the back of hard palate and the front of soft palate in golden hamsters or rats, which can successfully induce the same oral mucosal leukoplakia as humans. The clinical and pathological features of animal leukoplakia and human oral leukoplakia are basically similar, and their constant period is longer. During the experimental period, the lesion did not become malignant, and it will not subside in a short time after stopping stimulation.
Golden hamster is also used in the study of tongue cancer. 1973, Fujita et al. scraped the tongue with a root canal pulpotomy needle, and then applied acetone solution containing DMBA. After 13 ~ 25 weeks, 100% of the animals developed tongue cancer.
(5) Rats and mice
The rapidly proliferating epithelial and mesenchymal odontoblasts at the base of rat incisors are the most sensitive to cyclophosphamide, and their enamel epithelium can tolerate the cytotoxicity of 40mg/kg cyclophosphamide, which can be used to explore the effect of cyclophosphamide cytotoxicity on the growth of incisors.
There are a large number of palatal glands under the rat palatal mucosa, and the catheter opens on the mucosal surface. This structure can be used as an excellent portal for carcinogen invasion. Implanting carcinogens into rat mandible by surgery can successfully induce mandibular bone cancer, which can be used as an animal model for bone cancer research.
The latent period of tongue white shift induced by DMBA in rats is short and the proportion is high. 100% animals can produce leukoplakia, but it is difficult to induce abnormal epithelial hyperplastic leukoplakia, and the lesions gradually fade after formation, which may be related to the strong disease resistance of rats, saliva secretion in the mouth and the cleaning effect of tongue movement on the drugs used. Different parts of the experiment have different results. However, artificial methods can be used to reduce the cleaning effect of drugs used in rats and improve the carcinogenicity rate. For example, designing an artificial pouch lined with lower lip mucosa epithelium and using carcinogens to prolong the duration of carcinogens on mucosa can successfully induce oral mucosal cancer in rats.
Rats are suitable animals for candidal leukoplakia. Candida infection is related to some types of leukoplakia, especially granular leukoplakia. Six-month-old mice are usually selected. After inoculation with Candida albicans on the back of tongue, the normal nipple structure of tongue disappeared, and the hyphae of Candida albicans invaded the stratum corneum of normal keratinized epithelium, causing proliferation and inflammatory changes, and the epithelium gradually keratinized or incompletely keratinized. Finally, the appearance is similar to that of human candidal leukoplakia, but there is an incomplete keratinization layer at the end of lip mucosa.
Cleft lips and cleft palate in mice are similar to those in humans. It is reported that the genetic situation is similar, so mice are very suitable for making animal models of cleft lip and palate.
The enamel thickness of rat teeth is thinner than that of human teeth, and rats have no cariogenic function, so once dental caries occur, it develops rapidly and is seriously damaged. Therefore, it is necessary to inject this characteristic into the experimental analysis. In addition, the front teeth of mice are constantly growing, so their front teeth are not suitable for the study of dental caries.
Different species, strains and ages of rodents have different susceptibility to dental caries. For example, the sensitivity of young NIH rats is significantly lower than that of Sprague-Dawley rats; The range of dental caries caused by Streptococcus mutans in mice is much smaller than that in hamsters and rats, and the dental caries caused by Streptococcus mutans in hamsters is more extensive than that in rats. With the increase of age, rats become insensitive to dental caries, which may be due to the maturity of enamel. So most dental caries experiments need to start from 17 ~ 24 days.
Different strains of mice have different sensitivities to periodontal disease. For example, STR/N mice are prone to periodontal disease, while DBA/2A mice are resistant to periodontal disease.
The salivary glands of mice and rats are well developed and can be used to replicate animal models of salivary gland diseases.
Croton oil can promote the lip cancer induced by herpes simplex virus in mice, and 2-month-old mice are often selected for experiments.
There are pits and grooves on the molars of Sprague-Dawley rats, Charles COBC rats, Fisher rats, Osborn-Mendel rats, Wistar rats, golden hamsters, rhesus monkeys, pigs and other animals, and their anatomical morphology is similar to that of human molars. If pathogenic bacteria and cariogenic food are given, the same caries damage as human teeth can be produced in macroscopic and histopathological aspects, and animal models for studying dental caries can be established by using these animals.
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