Application status and research progress of animal models of knee osteoarthritis

Shi-Yao Zhang, Ning Li, Yong-Ju Yang, Shi-Wei Lv, Xue-Feng Guan✉

1. Liaoning University of Traditional Chinese Medicine, Shenyang 110847, China

2. The Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang 110032, China

Keywords:Knee osteoarthritis Animal model Traditional Chinese medicine Western medicine Review

ABSTRACT Appropriate animal model is the basis for the smooth development of scientific research, and also the guarantee of the experimental results. As a common clinical disease, the pathogenesis of KOA and the mechanism of action of various treatment methods need to be explored and verified by a large number of animal experiments. The selection and establishment of animal model is an important link, The selection of modeling methods in western medicine and the establishment of the model of combination of disease and syndrome in traditional Chinese medicine are the key points and difficulties. In this paper, the selection of KOA animal models was reviewed through literature retrieval, and the status quo of model establishment methods was summarized from the perspectives of western medicine and TCM. The main animal species include mice, chicken, rabbit, dog, sheep, horse, pig, monkey, emu and so on. The establishment methods of western medicine and TCM models are different. Western medicine models can be divided into spontaneous model induction model and transgenic model according to different establishment methods while TCM models are mainly established according to their syndromes.

Knee osteoarthritis (KOA) is a common clinical disease with pathological features of cartilage destruction, subchondral osteosclerosis, osteogenesis and synovial inflammatory lesions. Its clinical symptoms mainly include joint pain, swelling and limited activity. In severe cases, atrophy of surrounding muscles or even joint deformity affects daily life.According to the China Health and Pension Followup Survey [1], the prevalence of symptomatic KOA in China is 8.1%, and female is higher than male. The clinical treatment of KOA is mainly based on its imaging findings and the degree of lesions, conservative treatment or surgical treatment, but the fundamental pathogenesis has not been unified conclusion, so to explore and verify its pathogenesis and the mechanism of action of various treatment methods need to carry out a large number of animal experiments.Ideal animal models should be similar to human beings in anatomical structure, pathogenesis and disease progression, so the selection of animal models for different diseases should have different emphases.At present, the focus of KOA animal model research lies in the selection of modeling methods in Western medicine and the establishment of animal model combining disease and syndrome according to the characteristics of TCM syndromes.In order to provide some reference for colleagues who are doing or about to do related research work on KOA, the author summarized and summarized the articles related to KOA modeling retrieved in recent years.

1. Model selection

The selection of laboratory animals is the first and most important step in determining the success of basic research. There are many kinds of animals commonly used in scientific research. According to their size, they can be divided into small animals, medium animals and large animals.Small animals are convenient to raise, but their genes differ greatly from human beings.Large and medium sized animals, although genetically more similar to humans, are expensive to purchase and feed.They can be divided into rodents, mammals,poultry and primates.The selection of an animal for research often depends on a number of factors, such as genetic similarity to humans, purpose of research, procurement and feeding costs, ease of feeding, and ethical requirements.Due to the special physiological structure of KOA, the selection of experimental animals has its own particularity. The animals commonly used in KOA experimental studies and documented mainly include mice, rats, Hartley guinea pigs, C57 black rats, chickens, rabbits, dogs, goats, sheep, horses,pigs, macaque, macaque, crab-eating monkey, emu and so on.The most commonly studied are mice and rabbits.

1.1 Rat

Mice is one of the most common animal models in KOA experiments.Mice are small in size, cheap in price and low in feeding cost, and are often used in genetic related studies [2]. However, the small size of their knee joint and the small and thin cartilage make it inconvenient to collect materials for experiments.Compared with mice, rats have certain advantages, such as short feeding cycle,tenacious vitality, strong anti-infection ability, and relatively thick cartilage structure,which are favorable for experimental sampling.It is often used to study KOA pain and cartilage [3-4].Hartley guinea pig and C57 black mouse are often used as spontaneous KOA models due to their special physiological characteristics.Osteoarthritis may appear in the medial tibial plateau of guinea pigs about 3 months old,manifested in uneven cartilage surface and chondrocytopenia, etc[5].The incidence of KOA in C57 black mice was 20% after 2 months,and 80% after 16 months [6].Hartley guinea pigs are docile and sedentary, which can be used to study the degenerative mechanism caused by non-sports injury of KOA [7].However, due to the high price, long feeding cycle and high feeding cost of these two kinds of mice, their practical application is limited.

1.2 Rabbit

Rabbits are the second group of experimental animals most commonly used in KOA studies.They are gentle in character, simple in feeding, large in knee cavity, easy in sampling, and basically similar to human in knee structure except patella [8], so they are widely used in various KOA experimental studies.The disadvantage is that the knee joint is in flexion state for a long time, and the Angle of the knee joint is much different from that of human body, and the gait and biomechanics are obviously different from that of human body.

1.3 Other animals

Other animals documented for use in KOA studies include chickens, dogs, goats, sheep, horses, pigs, macaques, macaques,crab-eating monkeys, and emus.For example, dog knee joint has basically the same structure except one sesamoid bone and extensor tendon, which are different from human knee joint. Besides, dog gastrointestinal tract is similar to human body, so it can be used for some enteral therapy research [9].Catarino J et al. [10] found the applicability and safety of allograft PRP in dogs with osteoarthritis through a canine model study.The knee joints of goats and Mianyang are also similar to those of humans, so arthroscopic and MRI examinations can be performed like those of dogs for more accurate KOA studies.Howarth WR et al. [11] performed lateral or longitudinal meniscus tear at 1.0 cm in 40 castrated young adult male goats and modeling with or without microfracture. The study found that the healing rate of meniscus could be improved by releasing bone marrow elements to the knee through microfracture.But the joints of sheep are not easy to form arthritis, and the modeling time is longer.The genetic profile of pigs is similar to that of humans, and the degree of knee flexion during walking is similar to that of humans,so KOA pathology can be performed in pigs.Wu Qinghong et al. [12]found that resection of the lateral meniscus of the left knee joint and amputation of the anterior cruciate ligament was an ideal method to establish an animal model of early KOA in pigs, which could be applied in the in vivo functional MRI related studies of early KOA.The physiologic structure and degeneration mechanism of knee joint of macaques, macaques, and crab-eating monkeys, which belong to primates, are most similar to humans compared with other animals.Gang Liu et al. [13] studied cyb-eating monkeys of different ages and found that cyb-eating monkeys may be an animal model of agerelated changes in KOA, so it is believed that cyb-eating monkeys may be more suitable for the study of human degenerative knee osteoarthritis.Chickens, horses, and emus have been rarely used in KOA studies in recent years.Compared with rats and rabbits, the above animals are more similar to human beings in physiological structure in all aspects, but their application is limited to a large extent due to objective problems such as limited sources, high cost,difficulty in raising, and ethical difficulties.

2. Western medicine model

2.1 Model classification of pathogenesis in western medicine

The pathogenesis of KOA in Western medicine is mostly confined to cartilage, subchondral bone, synovium and other aspects, and its molecular biology level involves cytokines, inflammatory pathways,cartilage matrix degradation, oxygen free radical theory, etc. There are many opinions, but there is no unified conclusion.

Under the guidance of Western medicine theory, KOA animal models can be divided into spontaneous model, induced model and transgenic model.Spontaneous model refers to the animal model in which animals form certain pathological changes without any human intervention under natural conditions.Induction model refers to an animal model that artificially induces an animal to produce a disease similar to human.Induced in the mold was made in KOA induced and non-surgical induced model mainly includes surgery, surgery including induced from the cruciate ligament, methods of removal of meniscus, including diet induced, injection drug induced, nonsurgical induced joints brake induced, cold induced, bones, joints,stress induced in high pressure change induced, foreign bodies in induced and methods such as impact induced joints.Transgenic model refers to the animal model in which the desired physiological changes are obtained through gene knockout, insertion and transfection.

2.2 Spontaneous model

Hartley guinea pig and C57 black mouse are two common spontaneous models of KOA.Kang Xinyuan [14] studied 25 Dunkin-Hartley guinea pigs of different months of age, and the results confirmed that Dunkin-Hartley guinea pigs developed mild degeneration of articular cartilage when they were 3 months old in the natural growth state, and the disease gradually worsened with the increase of months of age.Yu PM et al.[15] established KOA models in C57 black mice and TNF-α knockout mice respectively and conducted whole-body vibration intervention.The relationship between TNF-α and candidate proteins was investigated by ELISA,histology, terminal deoxynucleotide transferase mediated notch end labeling, immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR). The results showed that a new mechanism of cartilage degeneration induced by systemic shock was found in KOA.The spontaneous model does not require human intervention, which is convenient for experimental operation,reduces the difficulty of the experiment, and avoids the influence of the experimental results caused by the mistakes in the modeling.However, the long modeling cycle leads to the slow down of the overall progress of the experiment and the consequent increase of various costs, which is the reason for the limited application of such models.

2.3 Induce model

2.3.1 Surgery induced

2.3.1.1 Transection of the anterior cruciate ligament

The primary functions of the ACL are to prevent the tibia from moving forward when the knee is bent and to prevent knee hyperextension when the knee is extended, and to assist in controlling the rotation of the knee.ACLT is formed by changing the stability of knee joint and accelerating cartilage wear. Due to its high repeatability and rapid OA progression, ACLT has become one of the most commonly used methods to induce KOA [16].Qian Jie et al.[17] found that the pathological changes of ACLT first occurred on the surface of cartilage, manifested in cartilage tarniness, softening and fibrosis, etc., but the modeling time of ACLT was longer, and 6 weeks was the early stage of KOA.Tawonsawatruk T et al. [18]compared the ACLT model with the cartilage injury model and found that the ACLT model presented progressive degenerative changes with persistent pain over time, indicating that ACLT was more in line with the characteristics of the development of KOA disease.

2.3.1.2 Meniscus-ectomyAs a joint filler, meniscus can reduce the friction of femur and tibia and buffer the impact of joint capsule and synovial membrane.Meniscectomy (MMT) results in the pathological appearance of KOA by destroying the joint structure and causing stress imbalance.Steinbruck et al. [19] observed that pathological changes at the 4th,6th and 10th week after surgery were consistent with typical KOA characteristics by removing the unilateral medial meniscus of rats and fixing the healthy limb to the abdomen.

2.3.1.3 Hulth

The Hulth method is divided into traditional and improved methods.The traditional Hulth method was proposed by Hulth et al. [20] in 1970 and is the most classic surgical modeling method of KOA.It causes joint instability by removing the anterior and posterior cruciate ligaments, the medial collateral ligaments and the medial meniscus.Phetsangouane S[21] found through experimental study that the pathological manifestations of the animal model established by traditional Hulth method were limited to the medial compartment of knee joint, which was close to the early OA manifestations of human beings.The knee joint injury and bleeding caused by this method are relatively large, and the progress of the experiment will be affected if the postoperative infection is not well controlled.Therefore, many researchers have designed a variety of improved HULTH methods through experiments, including simple resection of the medial meniscus and the anterior cruciate ligament, and resection of the medial meniscus, the anterior cruciate ligament and the medial collateral ligament.Wang Yunfeng et al. [22] modulated rabbits by removing the anterior cruciate ligament and medial meniscus, and found that synovial congestion and inflammatory exudation appeared at the early stage of KOA at 2 weeks, and at the late stage of KOA at 14 weeks, the cartilage loss and bone exposure of the medial tibial plateau of the femoral condyle and the surrounding obvious bone hyperplasia.

Compared with the traditional method, the improved HULTH method has the advantages of simpler operation, shorter modeling cycle and lower mortality rate.

2.3.1.4 Cartilage scratches

Cartilage scratch method is one of the least invasive methods of surgical induction, which can observe the development and repair process of KOA through cartilage defect.Marijnissen et al.[23] compared cartilage scratch induction with ACLT induction,and found that cartilage scratch induction could induce early KOA model, and biochemical analysis of cartilage showed that proteoglycan content and collagen damage were in line with KOA characteristics.The disadvantage is that the synovial tissue of the joint may be stimulated during induction, thus affecting the observation of cartilage lesions.

2.3.1.5 Ovariectomy

Ovariectomy induction is the only surgical induction method that does not model the knee joint itself, which induces KOA through ovariectomy to induce estrogen deficiency. Rat is the most commonly used animal model.The simulated objects are mainly postmenopausal women, who happen to be at this age group with high incidence of KOA, so it can be used as a special model for relevant research.At the same time, it can also be applied to the study of the mechanism of action of estrogen and its analogs on knee cartilage [24].Fontinele RG et al. [25] randomly divided 6-month-old female Wistar rats into 3 groups: control group without ovariectomies and exercise;The sedentary group underwent bilateral ovariectomy+ no exercise;The training group received bilateral ovariectomy+ exercise for 3 months.All rats were euthanized at 9 months of age, and histological sections were stained with hematoxylin and eosin.The results showed that the decrease of estrogen level caused important changes in the superficial layer of tibial cartilage in Wistar rats.The disadvantages of ovariectomy induction are the difficulty of operation and strict experimental conditions.

2.3.1.6 Blood cycle block

Good blood supply is the basis of maintaining the normal work of cartilage and synovial membrane of knee joint.Blood circulation occlusion induction is mainly through cutting or ligating the main blood supply vessels outside the joint, reducing the perfusion of the intraosseous artery, affecting venous return, resulting in increased pressure in the knee tissue, inducing cartilage degeneration, also known as intraosseous hypertension method.This method can be used to study the blood circulation and metabolomics of KOA.Wear seven class [26] under aseptic conditions ligation and cut off New Zealand rabbit on the right side of the buttocks of vein, lower right abdomen femoral vein and great saphenous vein, postoperative 8 weeks after put to death all the animals get right knee medial tibial plateau cartilage, through general observation and hematoxylin eosin staining, light microscope to observe the structural changes in the cartilage found form early metaphase KOA animal models.

2.3.1.7 Joint stress change

Knee stability need groups of osteogenesis, cartilage and subchondral bone, the meniscus, the lateral collateral ligament,before and after the cruciate ligament and the surrounding each muscle group synergy and antagonism effect together maintain,a link to appear problem, can cause knee joint stress change, thus under the effect of inflammatory factors and hormones such as common, the formation of KOA.

Therefore, the surgical methods, including the above ones by changing the anatomical structure of the knee joint, are all modeled by changing the stress of the knee joint.

Liu Jing et al. [27] concluded by cutting the anterior and posterior cruciate ligaments and medial collateral ligaments of Sprague-Dawley male rats that mechanical stress imbalance caused by joint instability was an important factor leading to traumatic KOA.

2.3.2 Nonsurgical induction 2.3.2.1 Diet

Epidemiological studies have shown that obesity is an important risk factor for the occurrence and progression of KOA.Therefore, an obesity model can be established through high-fat diet feeding and other methods to further study the relationship between obesity and KOA.Poulami Datta et al. [28] compared mice on high-fat diet with mice on low-fat diet, and found that mice on high-fat diet showed spontaneous age-related OA-like changes. Even when normal diet was restored, plasma metabolic indexes such as phosphatidylcholine and lysophosphatidylcholine were increased.

2.3.2.2 Injection of medicationDrug injection induction refers to the use of a variety of compounds injected into the knee joint, causing cartilage, synovial membrane,extracellular matrix and other inflammatory lesions, resulting in metabolic abnormalities, thus forming the KOA model.

2.3.2.2.1 Papain

Papain is a proteolytic enzyme that degrades proteoglycan in cartilage, thereby releasing chondroitin sulfate from the matrix.The intraarticular injection modeling is the most widely used modeling method besides surgical induction method, and it is also recognized as one of the safe and efficient methods.A study using Mankin score preliminarily confirmed that papain injection for 4 weeks could form an animal model of early knee osteoarthritis [29].Zhang et al.[30] injected a mixture of 2%, 5% and 10% papain and 0.03mol /L L-cysteine into the knee cavity of 72 female rats at a dose of 0.1ml/kg. Two weeks later, knee osteoarthritis models with different degrees of injury were obtained, in which the severe KOA model was obtained by injecting 10% papain.

2.3.2.2.2 Iodoacetate

Iodoacetate is a metabolic inhibitor that disrupts the cellular aerobic glycolysis pathway and induces cell death by inhibiting glyceraldehyde-3-phosphate dehydrogenase activity in chondrocytes.The intraarticular injection of MIA resulted in a decrease in the number of chondrocytes, and the histological and morphological changes of the joint were similar to those seen in human osteoarthritis.Ma Yy et al. [31] found that 1mg iodoacetate can induce stable pain behavior and progressive joint injury in rats, which is suitable for the evaluation of electroacupuncture treatment of KOA.Ming-jian wang etc. [32] by comparing the papain, iodine, sodium acetate and collagen type Ⅱ protease to build model of rat behavior,the influence of cytokines and pathology, the results found that there was no significant difference, iodine, sodium acetate model pathologies.

2.3.2.2.3 CollagenaseCollagenase destroys the joint structure containing type I collagen,destabilizes the knee joint, and ultimately leads to KOA.Adaes S et al. [33] observed the expression of transient receptor potential vanilate-1 in the ipsilateral dorsal root ganglia, and believed that 500 U collagenase injection in the knee joint of rats could be used as an alternative model to study the pain sensation associated with osteoarthritis.Utomo L et al. [34] induced knee joints of 33 male C57BL/6 mice with 10U collagenase and sacrificed at day 1, 3, 7,14, 28 and 56. The knee joints were taken for histological analysis.The results showed that meniscus compression occurred in the early stage of the mouse model, and meniscus injury and articular cartilage injury occurred simultaneously, indicating that collagenase induction model could be used to study the relationship between meniscus injury and KOA.

2.3.2.2.4 OthersUrokinase-type plasminogen activator is an important regulator of fibrinolysis in synovial fluid. Tao Jin et al. [35] injected Urokinasetype plasminogen activator directly into the knee cavity and found that it caused synovial inflammation and cartilage destruction through protein hydrolysis and cytokine triggering.Some studies[36] used the rat model of leptin induced osteoarthritis to detect its pathological characteristics, and found that some of the differentially expressed genes were related to leptin induced OA phenotype.These included MMPs related genes, inflammatory factors,growth factors, apoptotic genes and osteogenic genes.KOA model can also be induced by the injection of estradiol, philiphinin and adrenocorticosteroids into the knee joint [37-38], but the application has been less in recent years.

2.3.2.3 Arthrorisis

Joint immobilization is a common treatment in orthopedic department. It can relieve pain and accelerate tissue healing by restricting the active movement of the joint through external fixation materials and devices.Immobilization of a normal knee joint for a long period of time causes the surrounding muscles to atrophy and the ligaments to relax, thus creating an animal model with the same clinical manifestations as naturally occurring KOA.This method avoids limb damage caused by surgery or drug injections.Sun Yintie et al. [39] established an animal model by using the method of combining ordinary gypsum and polymer materials to fix the knee joint in extension position, and studied the relationship between the expression of NOD2 and NLRP3 and KOA, proving that this model establishment method is safe and stable.Yu Zhaoze [40] use Ilizarov circular external fixation support for drafting the rabbit knee joint, and USES the hinge joints keep knee a certain range, this method is a physiological environment can be simulated for cartilage regeneration and promote cartilage repair itself, the second is to guarantee the knee joint has certain range without cartilage injury because of the rigidity.

2.3.2.4 Cold stimulationCold stimulation induces KOA by affecting the internal environment of the knee joint, including changes in synovial tissue permeability,apoptosis, abnormal expression of inflammatory factors, degradation of cell matrix, etc. [41].At the same time, cold is one of the causes of KOA, so cold stimulation induction, like joint braking induction, can establish an animal model of KOA that is closer to human history and reflect the degenerative nature of KOA.Liu Yuan et al. [42] took the knee joint of healthy laying hens in ice bath for 2 h every day for 30 days. By scanning the cartilage surface with electron microscope,they found that the wave structure on the cartilage surface was low and flat, the arch structure disappeared, the cartilage surface was broken, and the collagen fiber and chondrocytes were exposed, and the structure was obviously loose

2.3.2.5 Joint impactDifferent from the natural degeneration induced by cold and joint braking, joint impingement simulates human knee lesions caused by trauma through external mechanical loading.At the same time, this method does not need to perform the ligament, meniscus, cartilage resection and other invasive operations, it is a non-invasive induction method, to avoid other problems caused by invasive injury.

In 2007, Furman et al. established the first mouse model of posttraumatic arthritis by inducing intraarticular fractures of the tibial plateau [43].They placed C57BL/6 mice in a customized scaffold,bent down to 90°, and applied a 55 N load to the proximal tibia at 20 N/s through a wedge-shaped pressure head on the device.Mrosek EH et al. [44] performed cross-joint impingement on patellar femoral joints of 12 adult beagles.

After 6 months, biopsies were taken and standard light microscopy and immunofluorescence tests of collagen I, collagen II, and proteoglycan were performed on fixed formalin sections.It was observed that joint impingence caused early osteoarthritis changes after 6 months, and that collagen network disruption resulted in proteoglycan loss.

2.4 Transgenic animal model

With the continuous progress of gene technology, the methods of gene knockout, insertion and transfection to obtain the desired specific gene defect and expression have also been applied to the basic research of KOA.Among them, gene knockout mainly leads to the formation of KOA through the regulation of excessive apoptosis of chondrocytes [45].Yu Fei et al. [46] successfully established knee osteoarthritis model by using Tamoxifen to induce SIRT1 gene knockout in cartilage tissue of 15-month-old mice.Studies [47] show Col11a1 gene knockout mice, but to the synthesis of collagen typeⅪ not normal collagen fiber, caused the mice achondroplasia, thus inducing KOA.It can also be achieved by knocking out IL-6 gene,ADAM-15 gene, A-1 integrin gene, MMP-14 gene, fibomodulin and basal membrane glycan gene double strand, fibomodulin, etc.The advantages of transgenic technology are that it avoids the possible operation errors of artificial modeling, eliminates the additional damage caused to animals by infection and trauma during modeling,and reduces the suffering of animals from the ethics.However, due to the incomplete maturity of transgenic technology at the present stage, there are still uncontrollable factors such as gene knockout and insertion, and it is difficult to determine whether it regulates the expression of some unknown proteins in the process of gene change and affects other physiological changes in vivo.Therefore, the application of transgenic animal models still needs the continuous development of technology.

3. TCM model

3.1 Classification of TCM pathogenesis and syndromes

KOA in traditional Chinese medicine is "Bi syndrome", "bone Bi"category, "Neijing" pointed out that "wind, cold and wet three Qi miscellany to, together for Bi.

"Zhang Yi Tong" : "Knee for the muscle of the house, knee pain is not due to deficiency of the liver and kidney, deficiency is the wind cold moisture attack.

"Miscellaneous diseases know heart record" : "the state of Bi closed also, is the pulse and astringent and less Xuan Tong of the machine,Qi and blood coagulation and less flow of potential.""Lingshu · God": "spleen is empty, limbs do not use."Thus it can be seen that the pathogenesis of KOA in Chinese medicine is marked by the evil of wind, cold and dampness, and is based on deficiency of liver, spleen and kidney, blood stasis and Qi stagnation.The Guided Principles for Clinical Research of New Traditional Chinese Medicine divides KOA into three syndrome types: deficiency of liver and kidney, stasis of muscles and veins, deficiency of spleen and kidney, deficiency of wet injection of bone joints, deficiency of liver and kidney, and obstruction of phlegm and stasis [48].In 2018, the Orthopedics and Traumatology Professional Committee of the Chinese Association of Integrated Traditional and Western Medicine issued a guideline for the diagnosis and treatment of knee osteoarthritis with integrated traditional Chinese and Western medicine [49], which divided it into four syndrome types: qi stagnation and blood stasis, wind cold and dampness arbidity, deficiency of liver and kidney, and dampnessheat accumulation syndrome.The guidelines for the diagnosis and treatment of knee osteoarthritis issued by the Orthopedics and Traumatology Branch of China Association for the Promotion of Traditional Chinese Medicine (2020 edition)[50] divided KOA into five syndrome types: qi stagnation and blood stasis, dampnessheat obstruction, cold and dampness obstruction, liver and kidney deficiency and qi and blood weakness.Thus it can be seen that there is no unified conclusion on the classification of TCM syndrome types of KOA, but its incidence involves nothing more than liver,spleen and kidney, and its pathogenesis mainly involves cold and dampness, blood stasis, Qi stagnation, phlegm turbidity and so on.

3.2 Model establishment based on TCM pathogenesis

The model building under the guidance of TCM theory has its particularity, but also its difficulties.The particularity lies in the fact that the clinical symptoms of TCM syndromes need to be reflected in animals, while the difficulty lies in the fact that in order to better reflect the particularity, many factors need to be considered in the model design, including animal constitution, the tightness of the combination of diseases and syndromes, environmental factors, etc.[51].

The author reviewed the literature and found that there were few experimental studies on the mechanism of TCM treatment of KOA based on the model of TCM syndrome type, and most of the retrieved articles centered on the type of kidney deficiency syndrome.

Liu Bo et al. [52] stimulated Japanese big-eared white rabbits with a high-current and low-current electrical stimulator, and established an animal model of exercise-induced fatigue "liver and kidney deficiency" by running, jumping and turning along the stimulation cage after stimulation.Liu Zhenfeng et al. [53] established a model of kidney deficiency through ovariectomization, based on the fact that ovariectomization can reduce the level of estrogen, which is the main characteristic of kidney deficiency.The team then used corticosteroids and epinephrine to establish a blood stasis model. The success of the model was determined by observing the changes of tongue cyanoplakia, ecchymosis, bleeding and hemodynamics.Jing Shenglong, Hou Lei, Li Ning et al. [54-56] all adopted the method of ovary removal to establish an animal model of kidney deficiency to explore the mechanism of action of traditional Chinese medicine on KOA, and good results were obtained in their experiments.It can be seen that ovary excision is the recognized method in the industry to establish the animal model of kidney deficiency syndrome.

4. Discussion

Through collecting a large number of relevant literatures on the selection and establishment of KOA animal models, the author found that there are many methods with advantages and disadvantages,so the author believes that there is only a suitable model but not an optimal one in animal modeling.An appropriate animal model is the basis for the smooth development of scientific research, and also the guarantee of the authenticity and effectiveness of experimental results.

Under the guidance of western medicine theory, there are many mature model building methods and their classification is relatively detailed.Natural induced model simulates the real process of KOA pathogenesis, and can study cartilage, synovial membrane,inflammatory factors and other aspects, but its experiment cycle is long and the cost is high.Operation-induced model has good stability and high success rate, and mature experimenter is required to participate in the whole process. Moreover, due to the large damage, the model induced by it is mostly manifested in middle and late KOA, so it is not suitable for the study of early KOA.There are many options of drug injection induction, which is simple and easy to operate, and the modeling time is short. It is suitable for the study of drug efficacy and screening, but it may cause the damage and destruction of other structures in the joint and affect the experimental results.Joint braking and cold stimulation induction are safe and simple to operate, which can better simulate the natural pathogenesis of KOA and conduct in-depth research on pharmacodynamics and mechanism of action. However, it also has the problems of long modeling time and high cost.Joint impingement can simulate joint inflammation caused by various injuries and sports injuries, which can avoid the high risk of infection in the operation. However, the stability of impingement device is a major technical problem in the modeling process, which requires delicate design to ensure its accuracy.Transgenic model is the product of scientific and technological progress, and also the direction of future development.To solve the stability of technology and control its cost is the key to whether the method can be widely used.

The author is mainly engaged in the basic and clinical work of TCM prevention and treatment of OA, and has always believed that TCM has many therapeutic methods with good clinical efficacy and worth promoting, such as internal administration of Chinese medicine, acupuncture, plaster external application, Chinese medicine fumigation and washing, etc.However, these treasures of traditional Chinese medicine need more modern scientific data to confirm their efficacy and mechanism of action, so the author pays special attention to the animal models constructed according to the basic theories of traditional Chinese medicine in experimental studies.According to literature statistics [57], there are more than 60 types of TCM syndrome models, involving more than 130 diseases,but these syndrome models still have many deficiencies.The author believes that due to the existence of the theories of "different treatment for the same disease" and "different treatment for different diseases", the highest level of constructing animal models based on TCM theories is to combine diseases and syndromes organically,that is, the model combining diseases and syndromes.The model of combination of disease and syndrome is the reflection of TCM syndrome differentiation in basic research. The establishment of such model can better demonstrate the pharmacological mechanism of TCM treatment of various diseases and conduct pharmacodynamics evaluation, and at the same time can help TCM promotion in the world, especially KOA.Although there are many modeling methods for various animal models such as spleen deficiency syndrome,heart-qi deficiency syndrome and blood deficiency syndrome,the author found through literature review that the establishment of these syndrome models is mainly applied in the study of other internal diseases, while few work has been done on the combination of KOA disease and model establishment.At the same time, among the methods described in this paper, except for three methods to establish the model of "deficiency of liver and kidney", "deficiency of kidney" and "blood stasis", only the method of cold stimulation induction is closer to the condition of simulating the onset of cold pathogenic factors induced by Bi syndrome, and the other methods of model establishment seem to have no correlation with the etiology and pathogenesis of KOA in Chinese medicine.Therefore,in order to further study the mechanism of TCM treatment of KOA,it is necessary to establish a recognized animal model for its basic research.

To sum up, Western medicine has many modeling methods, which increase the difficulty of its selection, while the establishment of TCM model combining disease and syndrome is a relatively lacking part of the current research on KOA.The author thinks that, to solve the experimental operation, budget control and stability of the model detail is the key to western medicine method modeling, and if it will be more surgery, drugs and other more advanced injection modeling method and syndrome of traditional Chinese medicine combined with western medicine, through the establishment of KOA ovaries removed kidney empty model, for example, might be a good way to promote Chinese medicine model combined disease.Of course, standardized scales and scoring systems are needed to evaluate various indicators of the model, whether it is based on the pathogenesis of Western medicine or the pathogenesis of Chinese medicine.For example, biochemical indicators and pathological examination that reflect Western medicine diagnostics, as well as defecation, urine, weight, hair, diet, sleep and mental state that reflect the information of the four diagnoses of Chinese medicine [58].This work is also the focus and difficulty of animal model selection and establishment in the future.We hope that more colleagues will join in the establishment of KOA animal model and evaluation system, and contribute to the overall development of KOA disease diagnosis and treatment.