Ex-vivo Modelling of Antimicrobial Control Measures in Pulpal Disease

Streptococcus anginosus group (SAG) bacteria are opportunistic pathogens which are a major cause of pulpal infection resulting in abscesses. Treatment is generally broad-spectrum antibiotics which can promote an increased incidence of bacterial resistance. Antimicrobial peptides (AMPs) are peptides expressed in the pulp that may have therapeutic actions. To investigate the novel use of such AMPs in controlling pulpal infection a suitable model is required. Objectives: To develop an ex-vivo culture system of pulpal disease. Methods: Pulps were extirpated from upper and lower incisors of freshly culled 28-day old male Wistar rats and freeze-thawed 3 times to devitalise the matrix. Clinical isolates of SAG bacteria obtained from Cardiff Dental School were cultured in DMEM+10% brain-heart infusion (BHI) broth and seeded at approximately 10⁹ cfu/ml on devitalised pulps and incubated at 37ºC, 5% CO₂, 60rev/min for 4, 8 or 12 h prior to staining with BacLight Live/Dead stain or were fixed with 3% glutaraldehyde for examination by SEM. Pulps incubated in DMEM+BHI without SAG were used as controls to identify pulpal contaminants from the extraction process. Following culture, pulps were mascerated, bacteria cultured on fastidious anaerobe agar plates and identified by 16s rDNA sequencing. Results: Bacterial suspensions in the presence of devitalised pulpal matrix for 4, 8 and 12 h demonstrated adherence of bacteria to the matrix as shown by SEM and Live/Dead staining confirmed viability. 16s rDNA analysis revealed that initial bacterial inoculates persisted within the matrix during the three culture periods. Conclusions: Clinical isolates have been shown to adhere to devitalised matrix following 12 h in culture and viability of these isolates has been maintained. This model may be further developed to determine the bactericidal effect of AMPs and biocides in the presence of pulpal matrix (JLR acknowledges receipt of a Richard Whipp Studentship).