Fabrication and characterization of bioprints with Lactobacillus crispatus for vaginal application

Anthony J Kyser; Mohammadali Masigol; Mohamed Y Mahmoud; Mark Ryan; Warren G Lewis; Amanda L Lewis; Hermann B Frieboes; Jill M Steinbach-Rankins

doi:10.1016/j.jconrel.2023.04.023

Fabrication and characterization of bioprints with Lactobacillus crispatus for vaginal application

J Control Release. 2023 May:357:545-560. doi: 10.1016/j.jconrel.2023.04.023. Epub 2023 Apr 21.

Authors

Anthony J Kyser¹, Mohammadali Masigol², Mohamed Y Mahmoud³, Mark Ryan⁴, Warren G Lewis⁵, Amanda L Lewis⁶, Hermann B Frieboes⁷, Jill M Steinbach-Rankins⁸

Affiliations

¹ Department of Bioengineering, University of Louisville Speed School of Engineering, Louisville, KY 40202, USA. Electronic address: ajkyse01@louisville.edu.
² Center for Predictive Medicine, University of Louisville, Louisville, KY 40202, USA. Electronic address: m.masigol@louisville.edu.
³ Center for Predictive Medicine, University of Louisville, Louisville, KY 40202, USA; Department of Toxicology and Forensic Medicine, Faculty of Veterinary Medicine, Cairo University, Egypt. Electronic address: myabde01@louisville.edu.
⁴ Department of Bioengineering, University of Louisville Speed School of Engineering, Louisville, KY 40202, USA. Electronic address: mark.ryan@louisville.edu.
⁵ Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Diego, La Jolla, CA, USA; Glycobiology Research and Training Center, University of California San Diego, La Jolla, CA, USA. Electronic address: wglewis@ucsd.edu.
⁶ Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Diego, La Jolla, CA, USA; Glycobiology Research and Training Center, University of California San Diego, La Jolla, CA, USA. Electronic address: a1lewis@ucsd.edu.
⁷ Department of Bioengineering, University of Louisville Speed School of Engineering, Louisville, KY 40202, USA; Center for Predictive Medicine, University of Louisville, Louisville, KY 40202, USA; Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40202, USA; UofL Health - Brown Cancer Center, University of Louisville, KY 40202, USA. Electronic address: hbfrie01@louisville.edu.
⁸ Department of Bioengineering, University of Louisville Speed School of Engineering, Louisville, KY 40202, USA; Center for Predictive Medicine, University of Louisville, Louisville, KY 40202, USA; Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40202, USA; Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY, USA. Electronic address: jmstei01@louisville.edu.

Abstract

Bacterial vaginosis (BV) is characterized by low levels of lactobacilli and overgrowth of potential pathogens in the female genital tract. Current antibiotic treatments often fail to treat BV in a sustained manner, and > 50% of women experience recurrence within 6 months post-treatment. Recently, lactobacilli have shown promise for acting as probiotics by offering health benefits in BV. However, as with other active agents, probiotics often require intensive administration schedules incurring difficult user adherence. Three-dimensional (3D)-bioprinting enables fabrication of well-defined architectures with tunable release of active agents, including live mammalian cells, offering the potential for long-acting probiotic delivery. One promising bioink, gelatin alginate has been previously shown to provide structural stability, host compatibility, viable probiotic incorporation, and cellular nutrient diffusion. This study formulates and characterizes 3D-bioprinted Lactobacillus crispatus-containing gelatin alginate scaffolds for gynecologic applications. Different weight to volume (w/v) ratios of gelatin alginate were bioprinted to determine formulations with highest printing resolution, and different crosslinking reagents were evaluated for effect on scaffold integrity via mass loss and swelling measurements. 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 greatest structural stability with dual genipin and calcium crosslinking, showing minimal mass loss and swelling over 28 days. 3D-bioprinted L. crispatus-containing scaffolds demonstrated sustained release and proliferation of live bacteria over 28 days, without impacting viability of vaginal epithelial cells. This study provides in vitro evidence for 3D-bioprinted scaffolds as a novel strategy to sustain probiotic delivery with the ultimate goal of restoring vaginal lactobacilli following microbiological disturbances.

Keywords: 3D bioprinting; Bacterial vaginosis; Female reproductive tract; Lactobacillus crispatus; Probiotics; women's health.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Alginates
Female
Gelatin
Humans
Lactobacillus / metabolism
Lactobacillus crispatus*
Probiotics*
Vagina
Vaginosis, Bacterial* / drug therapy
Vaginosis, Bacterial* / microbiology

Substances

Gelatin
Alginates

Grants and funding

R01 AI168475/AI/NIAID NIH HHS/United States