阅读说明：本技术 模拟大鼠骨折内固定合并低毒感染的动物模型构建方法 (Animal model construction method for simulating rat fracture internal fixation and low-toxicity infection ) 是由 朱晓彬 肖凌飞 肖潇 王嘉俊 姬文晨 于 2020-04-02 设计创作，主要内容包括：本发明公开了模拟大鼠骨折内固定合并低毒感染的动物模型构建方法,该方法包括如下步骤：S1、动物的选择及饲养条件；S2、菌种的选择及培养；S3、动物麻醉和大鼠股骨骨折内固定模型构建；S4、低毒感染的模拟；S5、术后动物观察；S6、动物染菌模型评价,本发明涉及医学动物模型研究技术领域。该模拟大鼠骨折内固定合并低毒感染的动物模型构建方法,通过构建一种实用、可行的大鼠骨折内固定合并低毒感染模型,为人们研究骨折内固定合并低毒感染提供动物实验模型以及检测指标,该实验方法设计严谨,极大程度地模拟骨折内固定合并低毒感染,造模后大鼠适合长期饲养,能够减少因为实验过程中动物死亡造成的经济和时间上的损失。(The invention discloses a method for constructing an animal model for simulating fracture internal fixation and low-toxicity infection of a rat, which comprises the following steps: s1, selecting animals and raising conditions; s2, selecting and culturing strains; s3, constructing an animal anesthesia and rat femoral fracture internal fixation model; s4, simulating low-toxicity infection; s5, observing the animals after the operation; s6, animal contamination model evaluation, and relates to the technical field of medical animal model research. According to the animal model construction method for simulating the internal fixation and low-toxicity infection of the rat fracture, a practical and feasible model for the internal fixation and low-toxicity infection of the rat fracture is constructed, an animal experiment model and detection indexes are provided for people to research the internal fixation and low-toxicity infection of the fracture, the experiment method is rigorous in design, the internal fixation and low-toxicity infection of the fracture is simulated to a great extent, the rat after the model is constructed is suitable for long-term feeding, and the economic and time losses caused by animal death in the experiment process can be reduced.)

1. The method for constructing the animal model for simulating the fracture internal fixation and low-toxicity infection of the rat is characterized by comprising the following steps of: the method specifically comprises the following steps:

s1, selection of animals and feeding conditions: selecting rats to be raised and tested in an SPF animal house;

s2, selecting and culturing strains: firstly, using an inoculating loop to lightly pick inclined surface staphylococcus epidermidis and add the inclined surface staphylococcus epidermidis into a nutrient broth culture medium for culture until the bacteria reach a medium concentration, then inoculating the bacteria on a nutrient agar plate by a scribing method under an aseptic condition, then using the inoculating loop to pick a single bacterial colony, inoculating the bacterial colony into a new nutrient broth culture medium, putting a bacterial tube into a constant temperature shaking table for culture, and adopting a Mach turbidimetry method to regulate the bacteria concentration by using a fresh nutrient broth culture medium the next day when the bacteria are in a stable growth period;

s3, establishing an animal anesthesia and rat fracture internal fixation model: 1-4% isoflurane is used for sucking the isoflurane into a rat, the continuous administration is carried out during the operation, and then the skin preparation, the disinfection, the towel paving, the exposure of the femur of the rat, the installation of an internal fixing instrument and the wire saw bone cutting operation are carried out in sequence;

s4, simulating low-toxicity infection, namely injecting 30m L of staphylococcus epidermidis liquid into a marrow cavity, a fracture broken end and an internal fixing instrument by using an injector, closing a wound layer by using silk threads, and disinfecting the epidermis by adopting povidone iodine;

s5, post-operative animal observation: after the experimental rat is anesthetized and conscious, the experimental rat is fed in a single cage, the free movement space in the cage is appropriate, the feeding standards are uniform, and no antibacterial agent is used after the operation;

s6, evaluation of animal contamination model: and observing the physical sign condition of the rat, and respectively carrying out blood conventional index detection, microbiological detection, imaging detection and pathological tissue observation.

2. The method for constructing an animal model simulating fracture internal fixation of rats combined with low-toxicity infection as claimed in claim 1, wherein: the rats in step S1 are SD rats and male rats, and have a body weight of 300 + -20 g.

3. The method for constructing an animal model simulating fracture internal fixation of rats combined with low-toxicity infection as claimed in claim 1, wherein: the Staphylococcus epidermidis strain in the step S2 is RP62A, purchased from American model culture Collection, and cultured under conditions of constant temperature shaking table at 100-300rpm at 32-37 ℃.

4. The method for constructing an animal model simulating fracture internal fixation of rats combined with low-toxicity infection as claimed in claim 1, wherein: the time for culturing the fungus tube in the step S2 in the constant temperature shaking table is 1-12h, and the time for inoculating the fungus tube on the nutrient agar plate is 15-24 h.

5. The method for constructing an animal model simulating fracture internal fixation of rats combined with low-toxicity infection as claimed in claim 1, wherein: the specific operation of step S3 is as follows:

1, 1-4% of isoflurane is inhaled into a rat, continuous administration is carried out during the operation, the anesthetic dose needs to be adjusted according to the operation time, the condition in the operation and the individual difference of animals, after the animal anesthesia takes effect, a shaver is used for skin preparation of an operation part, after povidone iodine is used for skin disinfection, a sterilizing glove is arranged, a sterile hole towel is laid, the position of the femur is determined by hand touch, and an outer side incision with the length of about 2-4cm is made at the middle section of the femur along the femoral shaft direction;

a2, separating subcutaneous tissues and superficial fascia to obtain an obvious white line which is a muscle space formed by the muscle fascia, carrying out blunt separation along the muscle space to directly expose to the femoral shaft, carrying out blunt separation on muscle tissues around the femoral shaft, turning the shank inwards to drive the femur to rotate outwards, and then fully exposing the front of the femoral shaft in the surgical field;

a3, bending the titanium alloy steel plate appropriately according to the angle of the femoral shaft front arch, washing the pre-bent titanium alloy steel plate for 3 times by PBS (phosphate buffer solution), then placing the titanium alloy steel plate in a smooth position in front of the femoral shaft, if the titanium alloy steel plate is attached to the bone surface, tapping by using a 1mm drill bit, and then screwing 4 screws with the diameter of 1.1mm for fixing;

a4, after the titanium alloy steel plate is firmly fixed, a fretsaw with the diameter of 1mm passes through the rear side of the femur by using a bending forceps, the position is adjusted to cut the bone in the middle part of the second and third screws to cause transverse fracture, and the fretsaw is separated from the surrounding muscle tissue by using cotton balls or gauze when the fretsaw cuts the bone so as to avoid the soft tissue damage caused by high heat generated by operation;

a5, checking the stability of the titanium alloy steel plate screw again after osteotomy, then flushing with normal saline, and sucking liquid in the operation area by gauze to dry the operation area.

6. The method for constructing an animal model simulating fracture internal fixation and low-toxicity infection in rats according to claim 1, wherein the concentration of the bacterial liquid in step S4 is 1 × 10⁹CFU/mL。

7. The method for constructing an animal model simulating fracture internal fixation of rats combined with low-toxicity infection as claimed in claim 1, wherein: the evaluation of the animal contamination model in the step S6 specifically includes:

t1, animal signs: including rat diet, body weight, activity, degree of limb swelling, and wound healing;

t2, blood index detection, namely taking blood from tail veins of rats and detecting indexes such as blood routine, liver and kidney functions, I L-6 and the like;

t3, microbiological detection: the experimental rats are used for bacterial culture of soft tissues around the fracture, the rats are killed, 0.8-1.2g of soft tissues around the fracture of the femoral specimen are taken under the aseptic condition, then the tissues are placed in aseptic normal saline for 10-time concentration gradient dilution, stock solution and diluent are taken for blood plate coating, bacteria are counted the next day, bacterial colonies growing on the blood plate are picked out for gram staining and plasma coagulase experiments, and the bacterial species and pathogenicity are respectively identified;

t4, imaging detection: taking an X-ray positive lateral photograph of the removed femur, carrying out Micro-CT scanning reconstruction, and observing periosteal reaction, callus formation, and cortical bone destruction and absorption conditions;

t5, pathological tissue observation: randomly selecting animals which are successfully molded, taking a tibial metaphysis specimen after sacrifice, placing the tibial metaphysis specimen in a paraformaldehyde solution with the volume concentration of 4% for tissue fixation, taking out the specimen after a period of fixation, washing the specimen by running water, and sequentially decalcifying, paraffin embedding, dyeing, observing and scoring the specimen.

8. The method for constructing an animal model simulating fracture internal fixation of rats combined with low-toxicity infection according to claim 7, wherein: the step T5 of sequentially decalcifying, paraffin embedding, staining and observing and scoring the specimen specifically comprises the following steps:

b1, firstly, processing a sample by EDTA decalcification solution, and embedding the decalcified sample by using paraffin;

b2, roughly trimming the embedded sample, and then preparing a tissue slice on a microtome;

b3, drying the tissue slice patch, and finally performing HE staining and Masson staining;

b4, observation under light microscope, histological scoring was performed.

Technical Field

The invention relates to the technical field of medical animal model research, in particular to a method for constructing an animal model for simulating fracture internal fixation and low-toxicity infection of rats.

Background

Post-traumatic fractures require restoration of the anatomical location of the fracture and fixation using internal fixation instruments to aid in fracture healing. However, when such internal fixation devices are implanted, due to improper surgical operation or incomplete sterilization of the devices, bacteria are easily introduced, thereby causing infection. Such infections are particularly difficult to control and ultimately lead to bacterial implant infections, which can lead to implant failure. A more specific category of these is low toxicity infections, which, although not clinically evident, may cause non-union or long-term pain at the fracture site. Low-toxicity infections are mostly caused by low-virulence pathogens, such as certain coagulase-negative staphylococci.

How to simulate fracture internal fixation and low-toxicity infection is a difficult point and a key point of the current research in the medical field. Rats are used as experimental animals to simulate the infection and research the clinical symptoms of animals caused by the diseases, and animal experimental models and detection indexes are provided for the internal fixation and low-toxicity infection of later-stage human fracture.

The invention takes the rat internal fixed animal model as a template, and explores a practical and feasible animal model of low-toxicity rat infection caused by the contamination of the operation part. The experimental operation roughly comprises the following steps: (1) animal selection and feeding conditions; 2) selecting and culturing strains; (3) constructing an animal anesthesia and rat femoral fracture internal fixation model; (4) establishing an animal contamination model; (5) observing the animals after the operation; (6) the invention provides an animal experimental model and detection indexes for researching fracture internal fixation and low-toxicity infection by constructing a practical and feasible rat fracture internal fixation and low-toxicity infection model.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides the animal model construction method for simulating the internal fixation and low-toxicity infection of the rat fracture, and provides an animal experiment model and detection indexes for the research of the internal fixation and low-toxicity infection of the fracture by constructing a practical and feasible model for the internal fixation and low-toxicity infection of the rat fracture.

(II) technical scheme

In order to achieve the purpose, the invention is realized by the following technical scheme: the method for constructing the animal model for simulating the fracture internal fixation and low-toxicity infection of the rat specifically comprises the following steps:

s1, selection of animals and feeding conditions: selecting rats to be raised and tested under the condition of room temperature ventilation;

s2, selecting and culturing strains: firstly, using an inoculating loop to lightly pick inclined surface staphylococcus epidermidis and add the inclined surface staphylococcus epidermidis into a nutrient broth culture medium for culture until the bacteria reach a medium concentration, then inoculating the bacteria on a nutrient agar plate by a scribing method under an aseptic condition, then using the inoculating loop to pick a single bacterial colony, inoculating the bacterial colony into a new nutrient broth culture medium, putting a bacterial tube into a constant temperature shaking table for culture, and adopting a Mach turbidimetry method to regulate the bacteria concentration by using a fresh nutrient broth culture medium the next day when the bacteria are in a stable growth period;

s3, establishing an animal anesthesia and rat fracture internal fixation model: 1-4% isoflurane is used for sucking the isoflurane into a rat, the continuous administration is carried out during the operation, and then the skin preparation, the disinfection, the towel paving, the exposure of the femur of the rat, the installation of an internal fixing instrument and the wire saw bone cutting operation are carried out in sequence;

s4, simulating low-toxicity infection, namely injecting 30m L of epidermal staphylococcus liquid into a marrow cavity, a fracture broken end and an internal fixing instrument by using an injector, closing a wound layer by using silk threads, and disinfecting the epidermis by using povidone iodine (iodine);

s5, post-operative animal observation: after the experimental rat is anesthetized and conscious, the experimental rat is fed in a single cage, the free movement space in the cage is appropriate, the feeding standards are uniform, and no antibacterial agent is used after the operation;

s6, evaluation of animal contamination model: and observing the physical sign condition of the rat, and respectively carrying out blood conventional index detection, microbiological detection, imaging detection and pathological tissue observation.

Preferably, the rat in step S1 is an SD rat, male, and has a body weight of 300 ± 20 g.

Preferably, the Staphylococcus epidermidis strain in the step S2 is RP62A, purchased from American Type Culture Collection (ATCC), and cultured under the conditions of being placed in a constant temperature shaker at 100-300rpm between 32-37 ℃.

Preferably, the time for culturing the fungus tube in the step S2 in the constant temperature shaking table is 1-12h, and the time for inoculating the fungus tube on the nutrient agar plate is 15-24 h.

Preferably, the specific operation of step S3 is as follows:

1, 1-4% of isoflurane is inhaled into a rat, continuous administration is carried out during the operation, the anesthetic dose needs to be adjusted according to the operation time, the condition in the operation and the individual difference of animals, after the animal anesthesia takes effect, a shaver is used for skin preparation of an operation part, after povidone iodine (aniodide) is used for skin disinfection, a sterile glove is arranged, a sterile hole towel is laid, the position of a femur is determined by hand touch, and an external incision with the length of about 2-4cm is made at the middle section of the femur along the femoral shaft direction;

a2, separating subcutaneous tissues and superficial fascia to obtain an obvious white line which is a muscle space formed by the muscle fascia, carrying out blunt separation along the muscle space to directly expose to the femoral shaft, carrying out blunt separation on muscle tissues around the femoral shaft, turning the shank inwards to drive the femur to rotate outwards, and then fully exposing the front of the femoral shaft in the surgical field;

a3, bending the titanium alloy steel plate appropriately according to the angle of the femoral shaft front arch, washing the pre-bent titanium alloy steel plate for 3 times by PBS (phosphate buffer solution), then placing the titanium alloy steel plate in a smooth position in front of the femoral shaft, if the titanium alloy steel plate is attached to the bone surface, tapping by using a 1mm drill bit, and then screwing 4 screws with the diameter of 1.1mm for fixing;

a4, after the titanium alloy steel plate is firmly fixed, a fretsaw with the diameter of 1mm passes through the rear side of the femur by using a bending forceps, the position is adjusted to cut the bone in the middle part of the second and third screws to cause transverse fracture, and the fretsaw is separated from the surrounding muscle tissue by using cotton balls or gauze when the fretsaw cuts the bone so as to avoid the soft tissue damage caused by high heat generated by operation;

a5, checking the stability of the titanium alloy steel plate screw again after osteotomy, then flushing with normal saline, and sucking liquid in the operation area by gauze to dry the operation area.

Preferably, the bacterial liquid in the step S4Concentration of 1 × 10⁹CFU/mL。

Preferably, the evaluating of the animal contamination model in step S6 specifically includes:

t1, animal signs: including rat diet, body weight, activity, degree of limb swelling, and wound healing;

t2, blood index detection, namely taking blood from tail veins of rats and detecting indexes such as blood routine, liver and kidney functions, I L-6 and the like;

t3, microbiological detection: the experimental rats are used for bacterial culture of soft tissues around the fracture, the rats are killed, 0.8-1.2g of soft tissues around the fracture of the femoral specimen are taken under the aseptic condition, then the tissues are placed in aseptic normal saline for 10-time concentration gradient dilution, stock solution and diluent are taken for blood plate coating, bacteria are counted the next day, bacterial colonies growing on the blood plate are picked out for gram staining and plasma coagulase experiments, and the bacterial species and pathogenicity are respectively identified;

t4, imaging detection: taking an X-ray positive lateral photograph of the removed femur, carrying out Micro-CT scanning reconstruction, and observing periosteal reaction, callus formation, and cortical bone destruction and absorption conditions;

t5, pathological tissue observation: randomly selecting animals which are successfully molded, taking a tibial metaphysis specimen after sacrifice, placing the tibial metaphysis specimen in a paraformaldehyde solution with the volume concentration of 4% for tissue fixation, taking out the specimen after a period of fixation, washing the specimen by running water, and sequentially decalcifying, paraffin embedding, dyeing, observing and scoring the specimen.

Preferably, the step T5 sequentially decalcifies, paraffin embedding, staining and observation scoring of the specimen specifically includes the following steps:

b1, firstly, processing a sample by EDTA decalcification solution, and embedding the decalcified sample by using paraffin;

b2, roughly trimming the embedded sample, and then preparing a tissue slice on a microtome;

b3, drying the tissue slice patch, and finally performing HE staining and Masson staining;

b4, observation under light microscope, histological scoring was performed.

(III) advantageous effects

The invention provides a method for constructing an animal model for simulating fracture internal fixation and low-toxicity infection of rats. Compared with the prior art, the method has the following beneficial effects:

(1) the used fracture internal fixation scheme is stable in effect, the fracture of the rat in the control group can be normally healed in one month, the internal fixation is loosened in one month at the fracture end of the infected group, the fracture is healed badly or not healed, and the disease model with poor bone healing caused by human body infection is well simulated.

(2) The selected staphylococcus epidermidis (RP62A) strain is locally planted to manufacture an infection focus, and the experimental results of imaging, pathological detection and the like show that the local infection effect of the manufactured internal plant is definite, fracture healing is obviously influenced, healing of fracture ends is delayed or not healed, and loosening of internal fixation appears, and the infection focus is caused by the planted staphylococcus epidermidis (RP62A) strain due to the positive bacterial culture result of local tissues of the rat.

(3) The animal model construction method for simulating rat fracture internal fixation and low-toxicity infection has the advantages that the whole body infection symptom is light due to the used local planting bacteria, the whole body conditions of animals such as body temperature, body weight, diet and activity and the like are not obviously changed after the selected staphylococcus epidermidis (RP62A) strain is locally planted, the indexes of whole body inflammation detected by a laboratory are slightly high, the clinical manifestations of plant low-toxicity infection in human bodies are met, the occurrence of local and even whole body acute suppurative infection caused by the use of staphylococcus aureus is avoided, and the animal model construction method can be well used for simulating in-vivo chronic and low-toxicity internal plant infection.

(4) The method for constructing the animal model for simulating the fracture internal fixation and low-toxicity infection of the rat provides a fracture internal fixation infection drug-resistant model. The formation of a biological film is one of important mechanisms for generating drug resistance characteristics by bacteria fixedly planted on the surface of a prosthesis, and in-vitro experimental results show that a staphylococcus epidermidis (RP62A) strain used by the invention has strong capability of generating the biological film, and animal experiments detect a titanium alloy steel plate polluted by bacteria, and the bacterial biological film is found on the surface of the titanium alloy steel plate, which shows that the internal fixed infection model manufactured by the invention generates the effect of resisting the antibiotic drug resistance through the mechanism of the bacterial biological film.

(5) The animal model construction method for simulating the internal fixation and low-toxicity infection of the rat fracture provides an animal model for the internal fixation and low-toxicity infection of the fracture, is not reported in domestic patents at present, is rigorous in design, and can be used for simulating the internal fixation and low-toxicity infection of the fracture to a great extent.

Drawings

FIG. 1 is a flow chart of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1, an embodiment of the present invention provides a technical solution: the method for constructing the animal model for simulating the fracture internal fixation and low-toxicity infection of the rat specifically comprises the following steps:

s1, selection of animals and feeding conditions: selecting rats, breeding and testing under room temperature ventilation condition, wherein the rats are SD rats and male rats, and the weight of the rats is 300 +/-20 g;

s2, selecting and culturing strains: firstly, using an inoculating loop to lightly pick inclined surface staphylococcus epidermidis to be added into a nutrient broth culture medium for culture until the bacteria reach a medium concentration, then inoculating the bacteria on a nutrient agar plate by a streaking method under an aseptic condition, then using the inoculating loop to pick a single bacterial colony to be inoculated into a new nutrient broth culture medium, putting a bacterial tube into a constant temperature shaking table for culture, wherein the bacteria are in a stable growth period, and adopting a Mach's turbidimetry method the next day, using a fresh nutrient broth culture medium to adjust the bacterial concentration, wherein the staphylococcus epidermidis is RP62A and is purchased from an American model culture collection and storage library (ATCC), the culture condition is that the bacterial tube is placed in the constant temperature shaking table with the rotation speed of 100 plus 300rpm between 32 ℃ and 37 ℃ for culture, the time of putting the bacterial tube into the constant temperature shaking table for culture is 1-12 hours, and the time of inoculating on the nutrient agar plate is 15-;

s3, establishing an animal anesthesia and rat fracture internal fixation model: 1-4% isoflurane is used for sucking the isoflurane into a rat, the continuous administration is carried out during the operation, and then the skin preparation, the disinfection, the towel paving, the exposure of the femur of the rat, the installation of an internal fixing instrument and the wire saw bone cutting operation are carried out in sequence;

the specific operation is as follows:

1, 1-4% of isoflurane is inhaled into a rat, continuous administration is carried out during the operation, the anesthetic dose needs to be adjusted according to the operation time, the condition in the operation and the individual difference of animals, after the animal anesthesia takes effect, a shaver is used for skin preparation of an operation part, after povidone iodine (aniodide) is used for skin disinfection, a sterile glove is arranged, a sterile hole towel is laid, the position of a femur is determined by hand touch, and an external incision with the length of about 2-4cm is made at the middle section of the femur along the femoral shaft direction;

a2, separating subcutaneous tissues and superficial fascia to obtain an obvious white line which is a muscle space formed by the muscle fascia, carrying out blunt separation along the muscle space to directly expose to the femoral shaft, carrying out blunt separation on muscle tissues around the femoral shaft, turning the shank inwards to drive the femur to rotate outwards, and then fully exposing the front of the femoral shaft in the surgical field;

a3, bending the titanium alloy steel plate appropriately according to the angle of the femoral shaft front arch, washing the pre-bent titanium alloy steel plate for 3 times by PBS (phosphate buffer solution), then placing the titanium alloy steel plate in a smooth position in front of the femoral shaft, if the titanium alloy steel plate is attached to the bone surface, tapping by using a 1mm drill bit, and then screwing 4 screws with the diameter of 1.1mm for fixing;

a4, after the titanium alloy steel plate is firmly fixed, a fretsaw with the diameter of 1mm passes through the rear side of the femur by using a bending forceps, the position is adjusted to cut the bone in the middle part of the second and third screws to cause transverse fracture, and the fretsaw is separated from the surrounding muscle tissue by using cotton balls or gauze when the fretsaw cuts the bone so as to avoid the soft tissue damage caused by high heat generated by operation;

a5, after the osteotomy operation is performed on the titanium alloy steel plate, the situation that the fractured end is difficult to reset due to the fact that the fractured end is pulled and displaced by muscles around the femur under the action of traction is avoided, the operation difficulty and the operation time are increased, the stability of the titanium alloy steel plate screw is checked again after the osteotomy operation, then the titanium alloy steel plate screw is washed by normal saline, and liquid in an operation area is absorbed by gauze to dry the operation area;

s4, simulating low-toxicity infection, namely injecting 30m L of staphylococcus epidermidis bacterial liquid into a marrow cavity, a fracture broken end and an internal fixing device by using an injector, then closing a wound layer by using silk threads, and disinfecting the epidermis by adopting povidone iodine (iodine), wherein the concentration of the bacterial liquid is 1 × 10⁹CFU/mL；

S5, post-operative animal observation: after the experimental rat is anesthetized and conscious, the experimental rat is fed in a single cage, the free movement space in the cage is appropriate, the feeding standards are uniform, and no antibacterial agent is used after the operation;

s6, evaluation of animal contamination model: and observing the physical sign condition of the rat, and respectively carrying out blood conventional index detection, microbiological detection, imaging detection and pathological tissue observation.

The evaluation of the animal contamination model specifically comprises the following steps:

t1, animal signs: including rat diet, body weight, activity, degree of limb swelling, and wound healing;

t2, blood index detection, namely taking blood from tail veins of rats and detecting indexes such as blood routine, liver and kidney functions, I L-6 and the like;

t3, microbiological detection: the experimental rats are used for bacterial culture of soft tissues around the fracture, the rats are killed, 0.8-1.2g of soft tissues around the fracture of the femoral specimen are taken under the aseptic condition, then the tissues are placed in aseptic normal saline for 10-time concentration gradient dilution, stock solution and diluent are taken for blood plate coating, bacteria are counted the next day, bacterial colonies growing on the blood plate are picked out for gram staining and plasma coagulase experiments, and the bacterial species and pathogenicity are respectively identified;

t4, imaging detection: taking an X-ray positive lateral photograph of the removed femur, carrying out Micro-CT scanning reconstruction, and observing periosteal reaction, callus formation, and cortical bone destruction and absorption conditions;

t5, pathological tissue observation: randomly selecting animals which are successfully molded, taking a tibial metaphysis specimen after sacrifice, placing the tibial metaphysis specimen in a paraformaldehyde solution with 4% volume concentration for tissue fixation, taking out the specimen after a period of fixation, washing the specimen by running water, and sequentially decalcifying, paraffin embedding, dyeing, observing and scoring the specimen, wherein the method specifically comprises the following steps of:

b1, firstly, processing a sample by EDTA decalcification solution, and embedding the decalcified sample by using paraffin;

b2, roughly trimming the embedded sample, and then preparing a tissue slice on a microtome;

b3, drying the tissue slice patch, and finally performing HE staining and Masson staining;

b4, observation under light microscope, histological scoring was performed.

The experimental results show that: from the peripheral connective tissue of the bone marrow and the bone tissue to the bone marrow, a large number of neutral eosinophilic cells are dispersedly infiltrated, both osteoblasts and osteoclasts are distributed in a large number, a large number of dark purple inflammatory cells are arranged around the osteoclasts, trabeculae are obviously reduced, and dead bones are formed.

In conclusion, the fracture internal fixation scheme used by the invention has stable effect, the fracture of the rat in the control group can be normally healed in one month, the internal fixation is loosened, the fracture is healed badly or not healed in one month at the fracture end of the infected group, and a disease model of the bone healing badly caused by human body infection is well simulated.

The bacterial infection local effect used by the invention is exact, the selected staphylococcus epidermidis (RP62A) strain adopts a method for manufacturing infection focuses by local planting, and the experimental results of imaging, pathological detection and the like show that the local infection effect of the manufactured internal implant is definite, the fracture healing is obviously influenced, the fracture end is delayed to heal or is not healed, the internal fixation shows loose, and the positive culture result of the local tissue bacteria of the rat confirms that the infection focus is caused by the planted staphylococcus epidermidis (RP62A) strain.

The local planting of bacteria used in the invention causes light systemic infection symptoms, after the selected staphylococcus epidermidis (RP62A) strain is locally planted, the systemic conditions of animals, such as body temperature, body weight, diet, activity and the like, are not obviously changed, the laboratory detection shows that the systemic inflammation index is slightly high, the clinical manifestations of low-toxicity plant infection in human bodies are met, the occurrence of local and even systemic acute pyogenic infection caused by the use of staphylococcus aureus is avoided, and the invention can be well used for simulating in-vivo acute and severe internal plant infection.

The invention provides a fracture internal fixation infection drug resistance model, the formation of a biomembrane is one of important mechanisms of drug resistance characteristics generated by bacteria fixedly planted on the surface of a prosthesis, in-vitro experiment results show that an epidermal staphylococcus (RP62A) strain used by the invention has strong capability of generating the biomembrane, and animal experiments detect a titanium alloy steel plate polluted by bacteria, and the bacterial biomembrane exists on the surface of the titanium alloy steel plate, which shows that the internal fixation infection model manufactured by the invention generates the effect of resisting the antibiotics drug resistance through the bacterial biomembrane.

The invention provides an animal model for fracture internal fixation and low-toxicity infection, which is not reported in domestic patents at present, the experimental method is designed rigorously, the fracture internal fixation and low-toxicity infection is simulated to a great extent, the method is feasible and can provide a treatment template and a detection index for the fracture internal fixation and low-toxicity infection, a rat after molding is suitable for long-term feeding, and the economic and time losses caused by the midway death of animals are reduced.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.