Non Clinical Model to Assess the Mechanism of Action of a Combined Hyaluronic Acid, Chondroitin Sulfate and Calcium Chloride: HA+CS+CaCl₂ Solution on a 3D Human Reconstructed Bladder Epithelium

Figures & data

Table 1 E. Coli Strains Characterization

Name	Strain	Growth Conditions
Escherichia coli strain CFT073 (pyelonephritis isolate)^Citation5,Citation19,Citation20	DSM 103538	TSYE broth or agar at 37 °C in aerobic conditions for 24–48h
Escherichia coli strain ATCC25922 (cystitis isolate)^Citation21	DSM 1103	Nutrient broth or agar at 37 °C in aerobic conditions for 24–48h

Table 2 Ialuril^® Dilutions Preparations for Microdilution Test

Test Mixture	Bacterial Suspension (10^8 CFU/mL in SU)	Culture Medium	Culture Medium 2X	IALURIL^®	IALURIL^® Diluted 1:1 in Sterile Water
Negative Control	1 mL	39 mL	–	–	–
IALURIL 80%	1 mL	–	7 mL	32 mL	–
IALURIL 50%	1 mL	19 mL	–	20 mL	–
IALURIL 25%	1 mL	19 mL	–	–	20 mL

Figure 1 Scheme of the anti-bacterial adhesion assay on HBE.

Table 3 Bacterial Viable Counts (Expressed as Log Values) at 4 h and 24 h

Series		Strain	Viable Counts 4h	Viable Counts 24h
Culture Media		E. coli DSM 103538	8.25	8.01
IALURIL	25%		7.93	8.16
	50%		7.94	8.05
	80%		7.73	8.19
Culture Media		E. coli DSM 1103	8.10	7.99
IALURIL	25%		8.10	7.82
	50%		7.89	7.71
	80%		7.48	8.04

Note: Bold values indicate E. coli control viable counts.

Figure 2 Caffeine quantification in the receptor compartment 1 h and 2 h after caffeine application, expressed as a percentage of applied caffeine. HA+CS+CaCl₂ significantly reduce the passage of caffeine compared to the untreated NC at both time points.

Figure 3 Statistic by “One-way ANOVA with post hoc Tukey HSD Test”: ***p<0.001. Caffeine permeation rate at 1 h (A) and at 2 h (B) after caffeine application, expressed as the percentage of caffeine permeated at 1 h and 2 h with respect to the amount of caffeine present in the apical compartment at each timepoint. Triplicate RHBE tissues for each treatment were used.

Figure 4 Statistic by “One-way ANOVA with post hoc Tukey HSD Test”: ***p<0.001 on viable counts (as per formula 1) on HBE tissues expressed as Log values after 4 h HA+CS+CaCl₂ pre-treatment followed by 4 h colonization. Triplicate HBE tissues for each treatment were used.

Figure 5 SEM analysis on colonized untreated 3D bladder epithelium (A): 2000x magnification and (B) 20,000x magnification, HA+CS+CaCl₂ pre-treated 3D bladder epithelium (4 h pre-treatment with HA+CS+CaCl₂ followed by 4 h colonization with E. coli DSM 1103 ((C): 2000x magnification and (D) 20,000x magnification, White arrows indicate bacterial pedestal structures and circles HA+CS+CaCl₂ product residues), not colonized untreated 3D bladder epithelium (negative control, (E) 2000x magnification) and Ialuril^® (F): 129,072 magnification).

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