(W1030-10-64) 3D Bioprinted Artificial Buccal Mucosal Membranes: A Single Platform for Conducting Permeation, Efficacy and Safety Testing of Treatment Modalities for Oral Cavity Disorders

Purpose: Oral cavity disorders affect a large percentage of the population worldwide irrespective of their age and demographics. The diseases are usually seen in the mucosal surfaces such as the buccal mucosa, lips, and tongue. Not only these diseases affect the individual restricting them from speech, eating and swallowing, they are extremely painful. In addition to these, majority of the treatment options are provide symptomatic relief and are steroidal in nature, which are contraindicated in co-morbid conditions, and intolerance. The pursuit for new and effective medicines has not seen much light due to the absence of viable preclinical models that resemble the human pathophysiology to justify the condition. Therefore, in the study the diseased human buccal mucosal cells were fabricated as 3D bioprinted scaffolds, which served the purpose of a viable model to test the permeation, efficacy and safety of the formulations.

Methods: Artificial membranes were fabricated from isolated buccal tissues procured from the commercial vendors of the patients suffering from oral cavity disorders such as oral lichen planus. The cells were cultured in growth media with fetal bovine serum, and observed for their morphology, growth patterns, and viability. Once the cells reached confluence ( > 85%), they were used to prepare the scaffolds. The 3D bioprinting technique was employed in the study to provide robustness to the membranes, and the possibility of tuning the structures. The 3D bioprinter had three pneumatic heads (3 mL volume), and a UV curing bed to crosslink the polymer. Prior to the bioprinting, the bioink was optimized using various hydrophilic polysaccharides that served as a medium for growing of the cells. The membranes were printed in various patterns and had an average size of 20 (L) x 20 (W) x 10 (H) mm, and were nearly transparent to clearly see the growth of the cells. The fabricated membranes were placed in a petri-dish of appropriate size, and simulated saliva to replicate the oral cavity conditions. The assembly was then applied with the model anti-inflammatory drug formulations in the form of gel (0.1 to 5%), and evaluated for permeation (24 h), efficacy (48 h) and safety (72 h). To compare the utility of the fabricated system, the experiments were run in parallel to the conventional cell culture technique using MTT assay, the biomarker estimation, and the in vitro permeation tests using porcine mucosa

Results: The scaffolds were fabricated successfully using the bioink (polysaccharide content of 5 to 10%), which were able to hold the buccal mucosal cells, and maintained the healthy growth of the cells (up to 4 days). The scaffolds were also printable in different mesh sizes (0.25 to 0.25 mm, hexagonal to circular patterns), and structures allowing the flexibility of printing larger structures if needed for the testing of mucoadhesion properties. The cell viability was ~98% during the start of the experiment, and the optimized scaffold was easy to handle and sufficiently rigid to be placed on the petri-dish. The formulations were applied under sterile condition, and subjected to the analysis. The drug showed a permeation of ~20%, ~55% and ~89% at the end of 3, 12 and 24 h; while the cell viability reduced from initial ~98% to 90% at the end of 24 h, and 84% at 72 h. The efficacy study showed approximately 65% reduction in the inflammatory mediators such as TNF-α, and IL that were released into the medium. In comparison experiments, the drug permeation through the porcine mucosa about 10% and 27% at the end of 3 and 13 h respectively, after which the tissues were not appropriate for the testing. In the MTT assay, the cell viability reduced to 34% at the end of 24 h, which may be attributed to the mono-layer of the cells showing limited growth as compared to the scaffold like structure. In addition, the cell culture technique required separate setups for the estimation of inflammatory mediators which reduced to 76%. Due to the lack of cells at the end of the treatment, the mono-layer cell study had to be stopped, and redone using the fresh setups.

Conclusion: Oral buccal mucosal cells were able to be fabricated as membranes using polysaccharide bioinks, and used for the testing of treatments. Upon comparison of the results, the 3D bioprinted scaffolds of buccal mucosa served as a single platform to carry out the permeation testing, efficacy and safety of the formulations, while the conventional testing needed multiple platforms and porcine tissues. The developed scaffolds can be a cost-effective and human relevant model for the discovery of newer treatments for oral cavity disorders.

References: 1. Koopaie M., Nassar DHMA, Shokrolahi M. Three-dimensional bioprinting of mucoadhesive scaffolds for the treatment of oral mucosal lesions; an in vitro study. 3D Printing in Medicine. 2022;8:30.
2. Nesic D., Schaefer BM., Sun Y., Saulacic N., Sailer I. 3D Printing approach in dentistry: The future for personalized oral soft tissue regeneration. Journal of Clinical Medicine. 2020;9(7):2238.

Acknowledgements: The authors acknowledge the support from Yenepoya Technology Incubator and Yenepoya (Deemed to be University), Mangalore, India for the formulations, analytical and characterization facility.

Representative Image of 3D Printed Scaffold