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

Purpose: Medical Device Regulation (EU) 2017/745 requires the principal mode of action (MoA) to be demonstrated by experimental data. The MoA of Ialuril ® Prefill (combined as HA+CS+CaCl 2 : sodium hyaluronate 1.6%, sodium chondroitin sulphate 2% w/v and calcium chloride 0.87%) Class III medical device, indicated for intravesical instillation to reduce urinary tract infections, has been evaluated on a 3D reconstructed human bladder epithelium (HBE). Methods: Three experimental designs; i) E. coli strain selection (DSM 103538, DSM 1103) to investigate the HA+CS+CaCl 2 properties in modifying bacterial growth in liquid broth (CFU 4h and 24h) at 80%, 50% and 25% concentrations; ii) evaluation of film forming properties on HBE after 15 min exposure by quantifying caffeine permeation across the epithelium; iii) capacity to counteract E. coli adhesion and biofilm formation on colonized HBE by viable counts and ultrastructural analysis by scanning electron microscopy (SEM) using ciprofloxacin as the reference antimicrobial molecule. Results: No significant differences were observed in bacterial viability for both the E. coli strains. HA+CS+CaCl 2 reduced caffeine permeation of 51.7% and 38.1% at 1h and 2h, respectively and determined a significant decrease in caffeine permeation rate at both timepoints supporting HA+CS+CaCl 2 capacity to firmly adhere to the bladder epithelium creating a physical barrier on the surface. The viable counts in HBE treated tissues then infected with E. coli resulted not different from the negative control suggesting that the device did not inhibit E. coli growth. SEM images showed homogenous product distribution over the HBE surface and confirmed the capacity of HA+CS+CaCl 2 to adhere to the bladder epithelium, counteracting biofilm formation. Conclusion: The results support the capacity of HA+CS+CaCl 2 to counteract bacterial invasion by using a physico-mechanical mode of action: this medical device represents a valid alternative to antibiotics in the treatment of recurrent UTIs. Keywords: recurrent cystitis, urinary tract infections, E. coli UPEC, E. coli biofilm, hyaluronic acid, chondroitin sulphate, film forming properties, anti-bacterial adhesion, reconstructed 3D human bladder epithelium