| Objective:(1)Explore new methods for making osteomyelitis models.(2)Explore the immunomodulatory effect of MDSCs in Staphylococcus aureus infected osteomyelitis.Methods:(1)Osteomyelitis model production:Inoculate Staphylococcus aureus(ATCC25923)on blood agar plate,incubate it in a constant temperature incubator at37°C for 24 hours,take it out,pick a single colony aseptically from the inoculation loop to 15ml of sodium chloride meat The broth was placed in the constant temperature shaking bacteria box at 37°C for 18 hours,and the concentration of the bacterial solution was adjusted to 1×10~7 CFU/m L.A sterile K-wire with a diameter of 1 mm and a length of 5 mm was placed in the bacterial suspension.Continue to incubate in an incubator for 18 hours,take out the K-wires and stain with crystal violet to confirm the presence of bacterial biofilm before use.Rats were anesthetized by intraperitoneal injection of 3%sodium pentobarbital(30 mg/kg)before the operation.After the anesthesia took effect,the limbs of the rats were immobilized,and the right hind limb was shaved to prepare the skin.Make an incision of about 1 cm in length along the medial side of the anterior tibial crest under the knee joint,enter the periosteum from the medial side of the tibialis anterior muscle,expose the anterolateral tibial crest at the upper end of the tibia,scrape the periosteum,and drill here with a hand drill with a diameter of 1.5mm Bone holes were probed with a small curette to remove the cancellous bone.The K-wires with bacterial biofilms were inserted into the tibia medullary cavity,and the drilled holes were closed with bone wax.After repeated washing with normal saline,the incisions were sutured layer by layer,and the incisions were kept in a single cage.4 weeks postoperatively.Postoperative detection:(1)Body temperature detection:The body temperature of the rats was detected before the operation,and the changes of the body temperature of the rats were continuously detected 1 week after the operation.Depth,read after 20seconds,and compare the changes in rat body temperature before and after surgery.(2)Observation of incision healing:observe the healing of the incision site after operation.(3)Imaging examination:After the 4th week after operation,all rats underwent X-ray detection of the modeling site,and frontal and lateral radiographs were used to observe the healing of bone tissue and the formation of osteomyelitis.(4)Gross specimen observation:The rats were killed by excessive anesthesia in the fourth week after operation,and the tibia was removed to remove the soft tissue.(5)Bone H&E pathological staining:put the removed tibia into EDTA decalcification solution for decalcification,change the decalcification solution every day,test the degree of decalcification with a fine needle,and send the specimen to the pathology department of the hospital for paraffin when the fine needle can penetrate the bone cortex Embedded pathological sections were stained with H&E.(6)Bacterial culture and identification:After the rats were killed,the secretions from the drilling site were aseptically taken and streaked on a blood agar plate to observe the growth of bacterial colonies.The cultured bacteria were analyzed by mass spectrometer to identify whether they were Bacteria inoculated on K-wires cause infection.(7)Detection of inflammatory factors:Pentobarbital was used to anesthetize rats in the fourth week,blood was drawn by cardiac blood collection,collected in sterile tubes,the blood coagulated naturally at room temperature for 10-20 minutes,and centrifuged at 2-8°C for about 20 minutes(2000-3000 rev/min),collect the supernatant,and use the ELISA kits of rat TNF-a,PCT,IL-4,IL-10,IL-17,TGF-βand IFN-γfor detection,using a multifunctional microplate reader Measure the absorbance and calculate the concentration.(8)The changes of MDSCs and Treg,Th1,Th2 and Th17 were detected by flow cytometry analysis.(9)The expression and proportion of MDSCs were detected by immunofluorescence.(2)Model intervention,18 SD rats were randomly divided into 3 groups,6 rats in each group,which were respectively the Test group:osteomyelitis model+gemcitabine drug injection(10 mg/kg,intraperitoneal injection,1 time/week);Model group:osteomyelitis model+injection and experimental group with the same volume of normal saline;Control group:healthy rats without treatment.The development of osteomyelitis in rats was detected by imaging analysis,gross specimen observation,histopathology and ELISA,the proportion of MDSCs was detected by flow cytometry and immunofluorescence,and whether the changes of Treg,Th1,Th2 and Th17 in rat inflammatory factor secreting cells were detected.It was directly inhibited by gemcitabine inhibitor to evaluate the healing of osteomyelitis in rats after inhibiting MDSCs.Results:(1)Detection of changes in rat body temperature:The body temperature was detected from 1 day before surgery to 1 week after surgery.After taking the average value,statistical analysis showed that the body temperature of the rats detected before surgery was below 37°C,and the body temperature began to rise on the second day after surgery.,the temperature rose to a maximum of 38.8°C on the4th-5th day after the operation,and the temperature gradually returned to the preoperative body temperature range on the 7th-8th day after the operation.(2)Incision healing,fistula formation,and general tibia specimens:in the second week after operation,all rats had different degrees of redness,swelling and pus,and purulent secretions were seen in the incision;fistula formation could be seen after subcutaneous incision of the skin;the tibia was removed.After removing the soft tissue,purulent secretions can be seen at the drilling site,the drilling is not healed,and new bone is formed inside.(3)X-ray examination of the modeling site:X-ray examination of the right tibia was performed at the fourth week after modeling.It was observed that the tibial medullary cavity where the Kirschner wire was located was enlarged,sequestered bone was formed,and new bone was formed to surround it.Kirschner wire,the thinned part of the cortex is discontinuous,and the bone at the drilled hole is not healed well.(4)H&E pathological examination of bone tissue:the right tibia was removed,the soft tissue was removed,decalcified with EDTA decalcification solution for about 4weeks,the Kirschner wires were removed,and the sections were stained with H&E.A large number of inflammatory cells were seen infiltrating the bone marrow cavity under the light microscope.,the cortical bone at the drilling site was not healed,and new bone formation could be seen in the bone marrow cavity.(5)Bacterial secretion culture identification:After disinfection,the skin of the right tibia modeling site was incised,the soft tissue was separated,and the secretions from the drilled part of the rat were aseptically dipped on a blood agar plate for streak culture.After culturing for 24 hours A single Staphylococcus aureus colony was seen,with a haemolytic ring.(6)Bacterial mass spectrometry identification:The bacterial secretion culture colonies were sent to the laboratory for bacterial mass spectrometry identification.The bacteria formed after the identification of rat secretion culture were all Staphylococcus aureus ATCC25923.(7)MDSCs flow cytometry analysis showed that there were MDSCs cells in the bone marrow and spleen of normal rats.The proportion of MDSCs increased during osteomyelitis,and gemcitabine could significantly inhibit the MDSCs cells;in addition,it could be observed that the proportion of MDSCs in spleen cells was higher than that in bone marrow.slightly lower.Treg flow cytometry showed that Treg cells increased in rats with osteomyelitis,but there was no significant difference in Th1,Th2,and Th17(P<0.05).(8)Detection of inflammatory factors by ELISA:The detection of TNF-a,PCT,IL-4,IL-10,IL-17 and IFN-γin rat serum by ELISA showed that it was significantly increased during osteomyelitis infection.The MDSCs cell inhibitor gemcitabine was used.After the infection of TGF-β,there was no significant change.(9)Immunofluorescence MDSCs:Immunofluorescence method was used to detect the number and morphology of MDSCs in the bone marrow of rats in each group.The results of immunofluorescence showed that MDSCs were expressed in normal rats and rats with osteomyelitis.The expression of MDSCs was increased and significantly decreased after the use of gemcitabine inhibitors.Conclusions:(1)A rat model of osteomyelitis infected with Staphylococcus aureus was successfully made.(2)MDSCs play a negative immunoregulatory role in Staphylococcus aureus-infected osteomyelitis,and inhibiting MDSCs may reduce the inflammatory effect of Staphylococcus aureus-infected osteomyelitis. |
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