Melt electro written three-dimensional scaffolds engineered as oral microcosm models-an in vitro study.

Abstract

Introduction: Biofilms are 3-dimensional (3D) aggregates of microorganisms that are associated with a wide range of diseases. Although there have been several studies investigating biofilm formation on two-dimensional substrates, the use of 3D substrates may result in more representative and clinically relevant models. Accordingly, the aim of this study was to compare the growth of biofilms in the 3D substrates against biofilms grown in 2D substrates.
Material and Methods: Two grams of medical grade polycaprolactone (PCL) were loaded into a plastic Luer-lock 3 ml syringe and a 23G needle was used as a spinneret. The syringe was placed in a melt electro-writing (MEW) device to obtain fine fibers under controlled parameters. The 3-dimensional MEW PCL scaffolds were manufactured and characterised with an overall thickness of ~ 0.8 mm, with ~ 15 μm diameter fibers and ordered pore sizes of either 100 or 250 µm. PCL films employed as 2D substrates were manufactured by dissolving 10 gms of PCL in 100 ml chloroform and stirred for 3 h to obtain a transparent solution. Then, the solution was cast in glass petri dishes and dried to remove all organic solvents. In addition, commercial hydroxyapatite discs were also used as 2D controls. Unstimulated saliva from six healthy donors (gingival health) were used to grow biofilms. The formed biofilms were assessed at day 4, day 7 and day 10 using crystal violet assay, confocal microscopy, scanning electron microscopy and next-generation 16s sequencing.
Results: The results demonstrates that 3D PCL scaffolds dramatically enhanced biofilm biomass and thickness growth compared to that of the 2D controls. Confocal microscopy of biofilms at day 4 stained with SYTO 9 and propidium iodide showed thickness of biofilms in 2D substrates were 39 µm and 81µm for hydroxyapatite discs and PCL films, respectively. Biofilms in 3D substrates were 250 µm and 338 µm for MEW PCL 100µm pore size and MEW PCL 250 µm pore size, respectively. Similar results were noticed at day 7 and day 10. Scanning electron microscopy showed biofilm bridges formed over the fibers of the MEW scaffolds. Pilot trials of next generation sequencing detected similar taxa in biofilms formed in 3D scaffolds compared to that of 2D substrates.
Discussion: We have successfully investigated a 3D biofilm growth model using 3D medical grade PCL scaffolds. Thicker biofilms can be conveniently grown using this inexpensive static model. This will facilitate 3D microbial community studies that are more clinically relevant and improve our understanding of biofilm-associated disease processes.