Oral Biofilm Cryotherapy as a Novel Ecological Modulation Approach

Abstract

Oral cryotherapy is used in dentistry as a safe, simple, and low-cost treatment for a variety of oral lesions. It is well known for its ability to aid in the healing process. However, its effect on oral biofilms is unknown. As a result, the purpose of this study was to assess the effects of cryotherapy on in vitro oral biofilms. In vitro multispecies oral biofilms were grown on the surface of hydroxyapatite discs in symbiotic or dysbiotic states. CryoPen X+ was used to treat the biofilms, whereas untreated biofilms served as control. One set of biofilms was collected for study immediately after cryotherapy, whereas another group was reincubated for 24 h to permit biofilm recovery. Changes in biofilm structure were analyzed with a confocal laser scanning microscope (CLSM) and a scanning electron microscope (SEM), while biofilm ecology and community compositional changes were analyzed with viability DNA extraction and quantitative polymerase chain reaction (v-qPCR) analysis. One cryo-cycle immediately reduced biofilm load by 0.2 to 0.4 log10 Geq/mL, which increased with additional treatment cycles. Although the bacterial load of the treated biofilms recovered to the same level as the control biofilms within 24 h, the CLSM detected structural alterations. Compositional alterations were also detected by SEM, corroborating the v-qPCR findings that showed ≈≤10% incidence of pathogenic species compared to nontreated biofilms that encompassed ≈45% and 13% pathogenic species in dysbiotic and symbiotic biofilms, respectively. Spray cryotherapy showed promising results in a novel conceptual approach to the control of oral biofilms. Acting selectively by targeting oral pathobionts and retaining commensals, spray cryotherapy could modify the ecology of in vitro oral biofilms to become more symbiotic and prevent the evolution of dysbiosis without the use of antiseptics/antimicrobials.

Electrolyzed Saline Targets Biofilm Periodontal Pathogens In Vitro

“…Biofilms were grown on standardized (12 mm diameter, 0.05 mm thickness) hydroxyapatite discs (HADs) (HiMed Inc.) microaerophilicaly for 48 h using bioreactor-derived communities. Two biofilm models were investigated: symbiotic and dysbiotic, simulating homeostatic healthy and periodontitis-associated biofilms, respectively (Zayed et al 2023). Dysbiosis was induced using BHI-2 medium with higher concentrations of L-cysteine, which neutralize the H 2 O 2 oxidative stress of the oral streptococci in the multispecies biofilms, leading to imbalances simulating neutralizing factors that initiate dysbiosis in the oral cavity.…”

“…Scanning electron micrographs of the rinsed biofilms (hydrated) were taken using a scanning electron microscope (FEI XL30-FEG; FEI) at high vacuum with a 10-keV acceleration voltage. Biofilms were fixed for visualization as described earlier (Zayed et al 2023). Afterward, biofilms were dried at room temperature and then coated with Pt/Pd 80/20 using a sputtering device (Quorum Q150T S; Quorum Technologies).…”

“…Fourteen key oral bacterial species were included in this study (Appendix Table 1) (Zayed et al 2022(Zayed et al , 2023. All the strains were maintained as described in the table.…”