Fabrication and characterization of Lactobacillus Crispatus containing bioprints for bacterial vaginosis application.

Bacterial vaginosis (BV) is a condition in which healthy lactobacilli are replaced by an overabundance of pathogenic bacteria in the female reproductive tract. Current antibiotic treatments often fail to “cure” infection, resulting in recurrence in more than 50% of women, 6 months post-treatment. Recently, probiotics have demonstrated promise to restore vaginal health; however, as with other active agents, delivery requires once-to-twice daily administration. Recently, three-dimensional (3D)-bioprinting has enabled the fabrication of well-defined cell-laden architectures with tunable agent release, thereby presenting a novel approach with which to deliver probiotics. One promising bioink, gelatin alginate, was selected for study, due to its ability in other work to provide structural stability, host compatibility, viable probiotic incorporation, and nutrient diffusion. The focus of this study was to formulate and characterize 3D-bioprinted Lactobacillus crispatus (L.cr.)-containing gelatin alginate scaffolds for reproductive health applications. Different weight to volume (w/v) ratios of gelatin alginate were bioprinted to determine the formulation with the highest printing resolution, and different crosslinking reagents were evaluated for effect on scaffold integrity, via mass loss and swelling measurements. Additionally, post-print viability, sustained-release, and vaginal keratinocyte cytotoxicity assays were conducted. A 10:2 (w/v) gelatin alginate formulation was selected based on line continuity and resolution, while degradation and swelling experiments demonstrated the greatest structural stability with dual-crosslinking, showing minimal mass loss and swelling over 28 days. Last, 3D-bioprinted L.cr.-containing scaffolds demonstrated sustained-release of therapeutically-relevant levels of probiotics over 28 days, while maintaining the viability of vaginal epithelial cells. For the first time, this study shows that 3D-bioprinted scaffolds may provide a new alternative to sustain probiotic delivery with future goals to help maintain or restore female reproductive health after BV infection