Establishment of a mandible defect model in rabbits infected with multiple bacteria and bioinformatics analysis

Abstract

Objective: A model of chronic infectious mandibular defect (IMD) caused by mixed infection with Staphylococcus aureus and Pseudomonas aeruginosa was established to explore the occurrence and development of IMD and identify key genes by transcriptome sequencing and bioinformatics analysis. Methods: S. aureus and P. aeruginosa were diluted to 3 × 10⁸ CFU/mL, and 6 × 3 × 3 mm defects lateral to the Mandibular Symphysis were induced in 28 New Zealand rabbits. Sodium Morrhuate (0.5%) and 50 μL bacterial solution were injected in turn. The modeling was completed after the bone wax closed; the effects were evaluated through postoperative observations, imaging and histological analyses. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and protein‒protein interaction (PPI) network analyses were performed to investigate the function of the differentially expressed genes (DEGs). Results: All rabbits showed characteristics of infection. The bacterial cultures were positive, and polymerase chain reaction (PCR) was used to identify S. aureus and P. aeruginosa. Cone beam CT and histological analyses showed inflammatory cell infiltration, pus formation in the medullary cavity, increased osteoclast activity in the defect area, and blurring at the edge of the bone defect. Bioinformatics analysis showed 1,804 DEGs, 743 were upregulated and 1,061 were downregulated. GO and KEGG analyses showed that the DEGs were enriched in immunity and osteogenesis inhibition, and the core genes identified by the PPI network were enriched in the Hedgehog pathway, which plays a role in inflammation and tissue repair; the MEF2 transcription factor family was predicted by IRegulon. Conclusion: By direct injection of bacterial solution into the rabbit mandible defect area, the rabbit chronic IMD model was successfully established. Based on the bioinformatics analysis, we speculate that the Hedgehog pathway and the MEF2 transcription factor family may be potential intervention targets for repairing IMD.

Keywords: animal model; bioinformatics analysis; infectious mandible defects; mixed bacterial solution; the hedgehog pathway.

FIGURE 1

Schematic diagram of the research process Legend: In this study, the mandibular bone defect of rabbits was first established, and then the prepared bacterial solution was injected to successfully establish the IMD model, and bioinformatics analysis was conducted by transcriptomic sequencing.

FIGURE 2

Model of the surgical procedure Legend: (A) Skin preparation and iodophor disinfection of the operative area. (B) The surgical area was dissected and separated the lateral surface of the mandible. (C) A 6 × 3 × 4 mm rectangular bone defect was prepared; and (D) 0.1 mL 5% sodium morate was injected and maintained for 5 min. (E) After rinsing with normal saline, 0.1 mL of 3 × 10⁸ CFU/mL mixed bacterial solution was injected; and (F) the bone wax was used for closure and the area was sutured layer by layer.

FIGURE 3

Post-modeling monitoring results and imaging results Legend: (A). The difference in body temperature before and after modeling. (B). The difference in food intake before and after modeling. (C). The temperature changes before and after modeling. *** means p < 0.001 (n = 28). (D). The result of wound healing at 14 days after surgery; (E). The result of wound healing at 28 days after surgery. (F). Gross specimens at 14 days after modeling. (G). Gross specimens at 28 days after modeling. (H). The results of imaging changes at 14 days. (I). The results of imaging changes at 28 days.

FIGURE 4

Bacterial culture and identification Legend: (A). The bacterial culture of the secretions of defects at 14 days. (B). The bacterial culture of the secretions of defects at 28 days. (C). PCR results of the suspected SA sample and the SA standard strain, where M is the standard band, SA is the standard Staphylococcus aureus band, and the rest are the suspected Staphylococcus aureus samples. (D). qPCR results of suspected PA samples and PA standard strains. Green is the dissolution curve of PA standard bacteria, and red is the dissolution curve of suspected PA. The curve trend and peak value were consistent.

FIGURE 5

The histopathological results Legend: (A). HE 14 days after modeling and (B). 28 days after modeling, and the red circles in a and bindicate acute inflammation. The red box indicates chronic inflammation, the red arrows indicate macrophages, the red triangle indicates the loss of bone nucleus, and the yellow arrow shows free dead bone. (C). Goldner staining at 14 days after modeling; (D). Goldner staining at 28 days after modeling. (E). TRAP staining results at 14 days and (F). TRAP staining results at 28 days. (G). Differences in the Smeltzer score were determined according to the Smeltzer scoring rules; the pathological changes between the two groups have no significant difference (p > 0.05, n = 14). (H). Analysis of osteoclast count per unit area in the two groups. The blue arrows are osteoclast-positive areas (* indicates p < 0.05 and n = 14).

FIGURE 6

Differentially expressed gene volcano plot and heatmap Legend: Red represents upregulated genes, blue represents downregulated genes, and gray represents non-differentially expressed genes. (A) The differentially expressed gene volcano plot. (B) The differentially expressed gene heatmap.

FIGURE 7

GO/KEGG cluster histogram and enrichment analysis bubble diagram Legend: (A). The DEGs GO cluster histogram. (B). The DEGs GO enrichment bubble diagram. (C). The KEGG cluster histogram of DEGs. (D). The KEGG enrichment bubble diagram of DEGs; the darker the color, the more significant the enrichment, and the larger the bubble, the larger the GeneRatio.

FIGURE 8

Protein interaction network diagram Legend: Each node represents the proteins produced by a single, protein-coding gene locus. The lines between nodes represent the interactions between proteins.

FIGURE 9

Core genes and modules and iRegulon predictions from PPI Legend: (A). Core module 1. (B). Core genes from cytoHubba; the darker the color, the higher the score and ranking; circles represent genes in the core module and lines represent the mutual regulation of genes. (C). Transcription factors and their regulatory networks; Yellow indicates the predicted transcription factor, blue indicates DEGs, and the lines show the regulatory relationship between transcription factors and DEGs.