Carnitine

The stored biofilms were thawed on ice, centrifuged and dispersed in Hanks' balanced salt solution (HBSS, Sigma-Aldrich) (Spano et al., 2016 (link)). A sample of biofilm was processed to determine CFU count. The remaining biofilm was applied topically onto RHG, with ~1 × 10⁷ CFU biofilm cells concentrated in a drop of 10 μl in HBSS as previously described (Buskermolen et al., 2017 (link)). The biofilm-RHGs were further cultured at the air liquid interface in medium containing DMEM/Ham's F12 (3/1) (Gibco, Grand Island, USA) supplemented with 1% Fetal Clone III (RHG, Logan, UT, USA), 0.1 μM insulin (Sigma-Aldrich, St. Louis, MO, USA), 2 μM hydrocortisone (Sigma-Aldrich), 1 μM isoproterenol (Sigma-Aldrich), 10 μM carnitine (Sigma-Aldrich), 10 mM L-serine (Sigma-Aldrich), 0.4 mM L-ascorbic acid (Sigma-Aldrich), and 2 ng/ml epidermal growth factor (Sigma-Aldrich), for 24 h at 37°C, 7.5% CO₂ and 95% humidity. After 24-h exposure RHG were harvested.

Adult CMs were isolated and seeded at a density of 6,000 cells per well in a 96-well plate for Seahorse measurements (Agilent Seahorse XFe96 Analyzer). Cells were washed with PBS and Seahorse base medium after attachment to the plate. The following substrates were added as energy substrates to the medium 1 h before measurements of the oxygen consumption rate: glucose 5 mM (Sigma), pyruvate 0.2 mM (Sigma), glutamine 4 mM (Sigma), palmitate–BSA 0.2 mM (Agilent), BSA control (Agilent), carnitine 0.2 mM (Sigma), valine 1 mM (Sigma), isoleucine 1 mM (Sigma), leucine 1 mM (Sigma), sodium propionate 0.05 mM (Sigma), sodium acetate 0.05 mM (Sigma), sodium octanoate 0.1 mM (Sigma), sodium decanoate 0.1 mM (Sigma). The oxygen consumption rate was measured using the Mito Stress Test kit (Agilent). The following inhibitors were injected: oligomycin (2 µM), FCCP (2 µM), rotenone and antimycin A (1 µM).

For [U-¹³C₁₆] palmitate tracer experiments, 2.5 mM [U-¹³C₁₆] sodium palmitate (Cambridge Isotopes, USA) was dissolved in 150 mM sodium chloride solution at 70 °C, and 40 mL of palmitate solution was added into 50 mL of 0.34 mM ultra-fatty acid free BSA (Sigma‐Aldrich, USA) solution at 37 °C to conjugate [U-¹³C₁₆] palmitate to BSA. 1 mM working BSA-conjugated [U-¹³C₁₆] palmitate solution was prepared via adjusting the pH to 7.4 and diluting to 100 mL with 150 mM sodium chloride. Finally, 50 µM BSA-conjugated [U-¹³C₁₆] palmitate and 1 mM carnitine (Sigma‐Aldrich, USA) were mixed with culture medium in 10% dialyzed FBS. For [U-¹³C₆] glucose tracer experiments, tracer media consisted of glucose free DMEM medium (Gibco, USA) with 10% FBS, supplemented with [U-¹³C₆] glucose (Cambridge Isotopes, USA). During tracer experiments, the medium was removed, cultured cells were rinsed with PBS, and tracer media added to the wells. Cells were cultured in tracer media for 24 - 48 h before metabolite extraction.

Telomerase reverse transcriptase–immortalized human gingiva keratinocyte and
fibroblast cell lines were cultured and used for the construction of human
gingiva equivalents exactly as previously described, except that no antibiotics
were used in the culture media (Buskermolen et al. 2016 (link)). The commensal,
gingivitis, and cariogenic microbiomes, grown as described from a pool of 10
saliva donors, were diluted in Hank’s Balanced Salt Solution (HBSS) with calcium
and magnesium (Gibco) to 10⁷, 10⁸, and 10⁹CFUs/mL. Each concentration (10 µL) was dripped onto the surface of the gingiva
equivalents, for a final exposure of 10⁵, 10⁶, or
10⁷ CFUs/equivalent. Controls were exposed to 10 µL of HBSS.
Exposed gingiva equivalents were cultured by air exposure for 24 h at 37 °C,
7.5% CO₂, and 95% humidity on 1.5 mL of DMEM/Ham’s F12 (3/1; Gibco),
supplemented with 1% Fetal Clone III (GE), 0.1μM insulin (Sigma-Aldrich), 1μM
isoproterenol (Sigma-Aldrich), 10μM carnitine (Sigma-Aldrich), and 10mM L-serine
(Sigma-Aldrich). Each experiment was performed with an intraexperiment
duplicate. Three experiments were performed, each with a different batch of
gingiva equivalents, which were exposed to different batches of the cultured
biofilms, grown independently from the same pool of 10 saliva donors, as
described earlier.