Supplementary Materials and Methods Section

S 1. Preparation of Saliva-containing Cell Culture Media

Saliva samples of the individual donors were incorporated to cell culture medium (saliva group) following lyophilisation in MEMalpha medium supplemented with Glutamax (Intvitrogen, Karlsruhe, Germany), 10 % fetal bovine serum and 1 % antibiotics (both PAA, Pasching, Austria). The volume of the culture medium used for solubilisation equated the volume of the respective non-lyophilized saliva sample (150 ml). For sterilisation, the saliva-containing medium was sterile filtrated (0.22 µm strainer, Millipore, Schwalbach, Germany) twice and subsequently UV-irratdiated for 2 h. Saliva-containing culture media were aliquoted and stored at -20 °C until usage. The small portion of saliva remnants which did not go into solution was effectively removed by the filtration process.

S 2. Metabolic Activity (EZ4U) Assay

In the EZ4U assay, the quantity of soluble, coloured formazan derivates – which are generated via the reduction of non-toxic tetrazolium salts by enzymes of the mitochondrial respiratory chain – corresponds with the number of viable cells. To monitor the cell number, MC3T3 osteoblasts were washed with PBS and incubated with fresh cell culture medium containing dye substrate (1:8) at 37 °C for 3 hours. The absorption was measured in triplicate within each cell culture replicate.

S 3. Quantitative RT-PCR

For assessing the RNA integrity and quantity with the Experion RNA StdSens Chip technology (Bio-Rad, München, Germany), heat-denatured RNA samples and RNA ladder were loaded onto chips primed with filtered gel stain, vortexed and measured immediately in the Experion electrophoresis station. Electropherograms were checked using the Experion software version 3.0 (Bio-Rad, München, Germany) for RNA integrity and quantity. The Quant-iT PicoGreen dsDNA, which served for quantifying the cDNA, specifically avoids interferences by single-stranded nucleic acids. Herein, fluorescence was measured in duplicate in a 200 µl reaction mixture containing 10 mM Tris-HCl and 1 mM EDTA at pH 7.5, PicoGreen (1:200) and 2 µl of cDNA sample in a black microplate with transparent bottom at 480 nm excitation and 520 nm emission wavelength and compared to a standard curve ranging from 1 ng/ml to 1 µg/ml nucleic acids after subtracting blank fluorescence values. The iQ-SYBR Green Super Mix used in qPCR reactions contained 100 mM KCl, 40 mM Tris-HCI, pH 8.4, 0.4 mM of each dNTP, iTaq DNA polymerase, 50 units/ml, 6 mM MgCl2, SYBR Green I and 20 nM fluorescein. Pre-validated gene-specific primer assays for the following molecules were purchased from SABiosciences, Frederick, MD, USA: ubiquitin (Ubc; RefSeq Accession Number NM_019639.4, band size 73 bp, reference position relative to the start of the relevant RefSeq sequence 2296), RUNX2 (NM_009820.4, 181 bp, 1733), osteopontin (NM_009263.2, 133 bp, 1147), alpha-1 collagen (Col1a1; NM_007742.3, 66 bp, 3370), osteocalcin (OCN; NM_007541.2, 63 bp, 46) and alkaline phosphatase (AP; NM_007431.2, 150 bp, 558). The primer conditions were as follows: 95 °C 15 sec, TA 55 °C 40 sec, 72 °C 30 sec. The products’ specificity of each amplicon was checked by examining the melting temperatures (heating at 0.05 °C/s to 95 °C). The melting temperatures (heating at 0.05 °C/s to 95 °C) of the amplicons were as follows: TM RUNX2 84 °C, TM osteopontin,Ubc 78 °C, TM Col1a1 81 °C, TM ALP 86 °C. Negative reverse transcription and negative template controls were included in all PCR runs. Gene expression was quantified by calculating the difference between the Ct values (deltaCt) for each gene of interest and the corresponding Ct value of the housekeeping gene ubiquitin (Ubc).

S 4. BCA Protein Assay and Cellular Alkaline Phosphatase Assay

For assessing the content of active cellular alkaline phosphatase, cells were lysed in 25 mM Tris-HCl pH 8.5, 0.5 % TritonX 100 by repeated freeze-thaw-cycles and stored at -80 °C until usage. The BCA protein assay kit was used to examine the total protein quantity. Herein, Cu2+ is chelated by proteins and the resulting cuprous cations are detected by bicinchoninic acid, which was quantified for each sample at 562 nm after 30 min incubation at 37 °C. The absorbance of serially diluted bovine serum albumin samples was simultaneously measured in duplicate as a standard curve and protein concentrations were calculated after subtracting blank OD values. The alkaline phosphatase substrate for evaluating the quantity of active cellular alkaline phosphatise was disodium 3-(4-methoxyspiro{l,2-dioxetane-3,2’-(5’-Chloro) Tricyclo [3.3.1.13,7] Decan}-4-yl) phenylphosphat (CSPD), which emits a chemoluminescent signal upon enzymatic conversion. The chemoluminescent signal was detected in triplicate within each cell culture replicate in a white flat-bottomed microplate, which was immediately read at 37 °C. Averaged blank chemoluminescence values were subtracted from raw data and alkaline phosphatase activity was normalized to the corresponding total protein amount.

S 5. Calcium Assay

For examining the extracellular matrix mineral content, cells were scraped off using 0.5 N acetic acid, incubated at 4 °C over night and stored at -80 °C until usage. After being vortexed thoroughly, the lysate was spun down (1000 rpm, 3 min) at room temperature (RT). For the Quantichrom Calcium Assay, all samples were diluted 1:40 in a reaction mixture containing a phenolsulphonephthalein dye which forms a blue coloured complex with free calcium. The colour intensity was measured in triplicate in each cell culture replicate after 3 min incubation (RT) at 612 nm. The absorbance of serially diluted calcium standards was measured in duplicate simultaneously in each run. After subtracting blank OD values, the calcium concentrations were calculated and normalized to the total protein amount.

S 6. Enzyme-linked Immunosorbent Assays (ELISA)

S 6.1 Lactoferrin

Standard series ranged from 390 pg/ml to 100 ng/ml lactoferrin. Standards and samples derived from each saliva-containing cell culture medium (n=6) were loaded in triplicate onto wells pre-coated with anti-lactoferrin antibodies (1 h, RT). After rinsing, wells were incubated with 0.5 % peroxide (5 min, RT) in order to inactivate adhered salivary peroxidase. A biotinylated anti-human lactoferrin detection antibody was added (1 h, RT) to bind to the captured lactoferrin. After four wash steps, a streptavidin-conjugated HRP was added (30 min, RT) which catalyzed a colorimetric reaction with the chromogenic substrate TMB (3,3’,5,5’-tetramethylbenzidine, 30 min, RT, in the dark). The absorbance of TMB at 450 nm displays the amount within the sample after addition of dilute sulfuric acid. The microtiter plate was rinsed with 0.5 % peroxide (5 min) at room temperature (RT) before adding the corresponding detection antibody in order to inactivate adhered peroxidase comprised in saliva medium. Both a test kit solution standard without lactoferrin as well as cell culture medium without saliva served as blank measurements. The standard curve was plotted and extrapolated with linear OD values (y) vs. logarithmic lactoferrin concentrations (x).

S 6.2 IL-1beta and TNFalpha

The cell culture supernatant was collected from all cell culture replicates of the respective saliva or control subject and pooled to yield 4 ml each. Amicon Ultra-4 Centrifugal Filter Devices 3 kD (Millipore, Schwalbach, Germany) were used to concentrate cell culture supernatants up to a final volume of 500 µl each. Standard series of IL-1beta ELISA ranged from 2.74 pg/ml to 2 ng/ml, standard series of TNFalpha ELISA detected as less as 123.5 pg/ml. Samples and standards were examined in duplicate each with the following incubation times: sample/standard incubation 2.5 h at RT, biotinylated anti-mouse IL-1beta or TNFalpha detection antibody 1 h at RT, HRP solution 45 min at RT, TMB solution 30 min at RT in the dark. All incubation periods were alternated with intense plate washing. The microtiter-plate was rinsed with 0.5 % peroxide (5 min, RT) before the corresponding detection antibody was incubated in order to inactivate adhered peroxidase comprised in saliva medium. Standard curves were plotted using logarithmic x- and y-scales.

S 7. Enzyme Activity Assays

All measurements were carried out at RT.

Alpha-amylase: In brief, 10 µl aliquots of each sample were incubated in alpha-amylase test solution (1:30, 10 min) consisting of 5 mM 2-chloro-4-nitrophenyl-4-O-b-D-galactopyranosylmaltotrioside (GalG2CNP), 5 mM CaCl2, 50 mM NaSCN, 0.03 % serum albumin and 0.03 % NaN3 in 50 mM MES buffer (2-N-(Morpholino)-ethansulfonacid, pH 6.0). The alpha-amylase within the salivary medium samples hydrolyzed GalG2CNP yielding the free aglycone 2-chloro-4-nitrophenolate (CNP) with a maximum of adsorption at 405 nm for 5 min. One unit of alpha-amylase was defined as hydrolytic production of 1 µmol CNP/min*ml.

Unspecific protease: The unspecific protease activity was measured using an EnzCheck protease assay kit (Molecular Probes, Leiden, Netherlands). 100 µl of saliva medium samples were incubated with 100 µl of BODIPY FL-labelled casein (10 µg/ml), yielding a release of fluorescent peptides. The product formation was measured fluorimetrically (excitation: 485 nm, emission 530 nm) during 60 min.

Salivary alkaline phosphatase: For assessing the alkaline phosphatase activity in salivary cell culture medium preparations, 6 µl samples were incubated in 294 µl of diethanolamin (1 M, pH 9.8, 10 min), mixed with 60 mM 4-nitrophenylphosphate-Na after 30 min, and the absorption was recorded immediately at 405 nm for 5 min.

Lysozyme: Lysozyme activity within the saliva medium samples was detected using the EnzCheck fluorimetric lysozyme assay kit measuring the hydrolysis of fluorescein-labelled M. lysodeicticus (Molecular Probes, Leiden, Netherlands). A volume of 5 µl of salivary medium samples or hen egg white lysozyme controls, respectively, were added to 95 µl of a reaction mixture containing substrate solution (0.05 mg/ml), 0.1 M sodium phosphate, 0.1 M NaCl and 2 mM sodium azide at pH 7.5 and fluorescence was recorded continuously during 10 min (excitation 494 nm, emission 518 nm).

Peroxidase: Peroxidase activity in 4 µl of salivary medium samples was determined using 240 µl freshly hydrolysed 2’,7’-dichlorofluorescin (LDCF) added to phosphate buffer (0.15 M, 1 mM KSCN, pH 6 at 37.8 °C) and hydrogen peroxide (2.2 mM). According to the amount of peroxidase, LCDF was oxidised to the fluorescing dichlorofluorescin (DCF), which was recorded continuously over 10 min (excitation 488 nm, emission 530 nm). One unit of peroxidase was defined as one µmol DCF released per min*ml.

Collagenase: Collagenase activity within the saliva-containing culture media was analyzed using the EnzCheck Gelatinase/Collagenase Assay kit (Invitrogen, Karlsruhe, Germany) measuring the cleavage of fluorescein-labelled gelatine. 100 µl of the samples or collagenase controls were added to 100 µl reaction mix containing 25 µg/ml gelatine, 50 mM Tris-HCl, 0.15 M NaCl, 5mM CaCl2 and 0.2 mM sodium azide at pH 7.6 and the product formation was measured fluorimetrically during 10 min (excitation: 495 nm, emission 515 nm).

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