Experimental Study Of The Intervention Effects Of Tuoguan San On The Internal Fixation Infected Animal Model Of Staphylococcus Aureus Biofilm

OBJECTIVE:1. To interfere with the internal fixation infected animal model of Staphylococcus aureus(S. aureus) biofilm by Tuoguan sanand observe the suppression effect of Tuoguan san on internal fixation infected animal model of S. aureus biofilm under laboratory environment.2. To interfere with the mature bacterial biofilm in animal model with Tuoguan san;and observe the suppression effect of continuous medication on internal fixation infected animal model of S. aureus biofilm with SEM(scanning electron microscopy).METHODS:1. Isolated single colonies of S. aureus strain were inoculated to L-Broth culture medium, and then inoculated in 24-well microtest plates. The internal fixations were utilized as carriersfor bacterial biofilm growth, and the bacterial growth and variations were observed with SEM. Colonies in the sample bacterial biofilms of culture plates were counted using plate-dilution method.2. Tuoguan san was prepared into suspension of 0.1g/ml.3. A total of 60 healthy chinchilla rabbits were randomly divided into 3 groups(20 rabbits in each group). The first group was the control group, the second group was the experimental group and the third group was the blank group. Ten rabbits from each group were sacrificed respectively on Day 4 and Day 10, and then the internal fixations were collected and tissue samples were cut off.4. Internal fixations from each group underwent the following sequential processes: rinsed with PBS, fixed with glutaraldehyde solution, dehydrated with ethanol of different gradients, displaced with isoamyl acetate, critical point-dried in sample CO2, solid surface coating with ion sputtering, and the samples were finally prepared into SEMdetection samples for observation of the morphological structure variations of S. aureus biofilm.5. The surrounding tissues of internal fixations from each groupwere collected andprepared into paraffin sections.The preparation process was completed after the sections have received HE stainingand then the variations of inflammatory cells(neutrophils, phagocytes, macrophages) and fibroblasts of the sectionsat two periods of time were observed througha microscope.RESULTS:1. During the cultivation of S. aureus, obvious turbid LB broth was observed in culture plates, and mature S. aureus biofilmswere formed on the surfaces of internal fixations 7 days later. According to the statistics of bacterial colonies counting: the concentration of S. aureus in LB broth presented an increasing tendency with the extension of culture time, while that ofthe S. aureus in the LB broth group administrated with Tuoguan san presented a decreasing tendency with the extension of culture time.2. The Chinchilla rabbits from different groups were sacrificed graduallyfor sample preparation and examination. The S. aureus biofilm of the control group increased gradually on the surfaces of internal fixations;Tuoguan san administrated in the experimental group presented increasing suppression effects on the S. aureus biofilms on the surface of internal fixations;no bacteria or biofilm was observed in the blank groupwhile fibroblasts were observed, indicatingthat Tuoguan san has strong suppression effects on S. aureus biofilm.3. 3. The results of histopathologicalexaminations of different groupssuggested that: theinflammatory cells increased gradually and a few fibroblasts were formed in the control group; the inflammatory cells decreased gradually and fibroblasts increased gradually in the Tuoguan san intervention group; very few inflammatory cells were formed and fibroblasts increased gradually in the blank group; the Tuoguan san intervention group has promoting effects on the healing of S. aureus biofilm infected wounds.CONCLUSION:1. Tuoguan san has a suppression effect on the infectionof in vitro model of internal fixation infected S. aureus biofilm.2. Tuoguan san has a suppression effect on the morphological structure of internal fixation infected S. aureus biofilm.3. Tuoguan san has a promoting effect on the woundhealing of internal fixation infected S. aureus biofilm.