The cells were harvested and prepared from 5 voluntary patients with written informed consent from the Department of Orthopedics, Koenig-Ludwig-Haus. The study was approved by the Ethics Committee of the Medical Faculty of the University of Wuerzburg (12/06). Bone marrow was collected under aseptic conditions and cells were isolated according to the protocol described by Lee et al (23). Ficoll density-gradient centrifugation was used in order to isolate hMSC (30 min, 1,300 rpm, density=1,077 g/ml, Biochrom AG, Berlin, Germany). After collection of the cells from the interphase, a washing step with phosphate buffered saline (PBS) (Roche Diagnostics GmbH, Mannheim, Germany) containing 2% FCS followed. After centrifugation the cell pellet was resuspended in expansion medium (DMEM-EM), which was made of Dulbecco’s modified Eagle’s medium (DMEM) (Gibco Invitrogen, Karlsruhe, Germany) containing 10% FCS (Linaris, Wertheim-Bettingen, Germany), 1% penicillin and streptomycin (Sigma-Aldrich, Schnelldorf, Germany). Cells were incubated for 24 h at 37°C and 5% CO₂. The medium was changed every other day. The morphology of hMSC was analyzed by inverted microscopy (Leica DMI 4000B Inverted Microscope, Leica Microsystems, Wetzlar, Germany).

Osteogenic differentiation was carried out in a 24-well plate (BD Falcon, Heidelberg, Germany) with 1×10⁴ cells/well until 70% confluence was reached. The expansion medium DMEM-EM additionally contained 10⁻⁷ M dexamethasone, 10⁻³ M β-glycerophosphate and 2⁻⁴ M ascorbate-2-phosphate (all Sigma-Aldrich). The von Kossa method was used to show the presence of calcium mineral components. Adipogenic differentiation medium was made of DMEM-EM containing 10⁻⁷ M dexamethasone and 10⁻⁹ g/ml recombinant human insulin (both Sigma-Aldrich). Staining with Oil Red O confirmed the presence of intra-cellular lipid droplets.

For chondrogenic differentiation the pellet culture system was used. The cell pellets were cultured in a defined chondrogenic differentiation medium (Lonza, Basel, Switzerland) supplemented with 10⁻⁹ g/ml transforming growth factor-β3 (Sigma-Aldrich). Cells were cultivated for 3 weeks. Thereafter, the pellets were embedded in Optimal Cutting Temperature Paraffin (Tissue-Tek^® O.C.T^TM; Sakura Finetek, Zoeterwoude, The Netherlands). Cryosections were stained with Alcian blue to show the presence of glycosamineglycane.

Flow cytometry was used in order to confirm surface antigen markers. hMSC (1×10⁶) were incubated with anti-CD105, anti-CD90, anti-CD44 and anti-CD34 (all antibodies were purchased from BD Bioscience, Heidelberg, Germany). Cell surface analyses were performed by flow cytometry (FACSCanto™; BD Bioscience).

The head and neck squamous carcinoma cell lines FaDu and HLaC78 were used (24,25). Cells were grown in DMEM-EM and cultured at 37°C with 5% CO₂ in culture flasks. Every other day the medium was replaced. After reaching 70–80% confluence cells were trypsinized with 0.25% trypsin (Gibco Invitrogen), washed with PBS and seeded in new flasks or treatment wells. Experiments were performed using cells in the exponential growth phase. FaDu and HLaC78 were incubated with the supernatants of hMSC (hMSC-sup). The following experiments were carried out in order to evaluate the effects of cytokines released by hMSC on FaDu and HLaC78.

The dot blot assay (RayBiotech Inc., Norcross, GA, USA) was used as a semi-quantitative method for determining hMSC cytokine secretion. After an incubation period of 48 h in DMEM without supplements, the supernatants were collected and investigated for the presence of cytokines. The assay was performed according to the manufacturer’s protocol. The labeled proteins were observed by enhanced chemiluminescence using detection buffer and exposure to X-ray film. The cytokines were represented as dots with different intensity and growth size.

Supernatants of hMSC were collected. IL-6 concentration was measured using the ELISA kit human IL-6 (Disclose SAS, Besancon Codex, France). All experiments were investigated in duplicate. The plate was read at 450 nm (Titertek Multiskan PLUS; Labsystems, Helsinki, Finland). IL-6 concentration (pg/ml) was ascertained by creating a standard curve using recombinant IL-6. DMEM without supplements served as the control.

Cells (2×10⁴) (FaDu and HLaC78) were incubated with hMSC-sup at 37°C with 5% CO₂ for 4 days, while electronically counting the cell number each day (Casy^® Technologies, Innovatis AG, Reutlingen, Germany). DMEM-EM served as the control (n=5).

Simultaneously, the effect of IL-6 on cell proliferation was evaluated by using the MTT assay. Cells were seeded at a density of 1×10⁴ in a 96-well rounded bottom plate. After an incubation period of 24 h in DMEM-EM, hMSC-sup +/− anti-IL-6 500 ng/ml (R&D Systems, Wiesbaden-Nordenstadt, Germany) cells were washed with PBS. Then all plates were incubated with 100 μl of MTT solution (1 mg/ml) followed by 5 h of incubation at 37°C with 5% CO₂. After removal of MTT, 100 μl of isopropanol was added for 1 h at 37°C with 5% CO₂. The plate was read at a wavelength of 570 nm (Titertek Multiskan) (n=5).

The expression of EGFR in FaDu and HLaC78 were investigated as follows: According to the manufacturer’s protocol, the TRIzol method was used in order to extract total RNA from cells. Next, the extracted total RNA was reverse transcribed to cDNA. For the quantification of gene expression, the SYBR Green PCR master mix kit (Applied Biosystems, Foster City, CA, USA) was used. The EGFR primer was purchased from Applied Biosystems (forward primer 5′-GCGTTCGGCACGGTGTATAA-3′ and reverse primer 5′-GGCTTTCGGAGATGTTGCTTC-3′). The conditions for gene amplification were as follows: 50°C for 2 min; 95°C for 10 min, and 40 cycles at 95°C for 15 sec and 60°C for 1 min. As an endogenous control, the GAPDH gene was used.

Cells (FaDu and HLaC78) were harvested by trypsinization and dissolved in RIPA buffer (PBS, containing 1% NP40, 0.5% sodium deoxycholate and 0.1% SDS), supplemented with 10 μg/ml phenylmethanesulfonyl fluoride (PMSF). Protein concentration was then determined. Equal amounts of total protein lysates were loaded on a 10% SDS-polyacrylamide gel and transferred by electroblotting to a polyvinylidene difluoride membrane. Blots were blocked for 1 h at room temperature with TBST (10 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 8.0), containing 5% nonfat dry milk. Afterwards, the membrane was incubated with primary antibody ERK1/2 (Cell Signaling Technology, Beverly, MA, USA) overnight at 4°C. Subsequently, the membrane was washed and incubated with a species-specific IgG secondary antibody for 1 h to visualize the specific bindings. The protein expression was detected with a chemiluminescence system (ECL, Amersham Biosciences, Freiburg, Germany), according to the manufacturer’s protocol.

All data were transferred to standard spreadsheets. Differences between groups were examined for significance by the Kruskal-Wallis test using GraphPad Prism 6.0 statistics software (GraphPad Software, Inc., San Diego, CA, USA). Differences were considered statistically significant when the P-value was <0.05.

hMSC exhibited a fibroblast-shaped morphology. They were able to differentiate into chondrocytes, adipocytes and osteocytes, which were confirmed by Alcian blue, Oil Red O and van Kossa staining. The flow cytometric analysis revealed the presence of typical surface markers. hMSC were positive for CD105, CD90 and CD44, and negative for CD34 (Figs. 1 and 2).

Morphology of FaDu and HLaC78 was evaluated before and after cultivation with hMSC-sup for 48 h. Microscopy revealed no differences in cell morphology between each group for both cell lines.

The dot blot assay was used to evaluate different cytokines in the hMSC supernatants. hMSC supernatants contained a variety of cytokines responsible for pro- and anti-inflammation, chemotaxis, angiogenesis and growth factors. The dots of the following cytokines had a strong intensity: interleukin (IL)-3; IL-6, IL-8, IL-10, Growth regulated oncogene (GRO), GRO-α, Monocyte chemotactic protein (MCP)-1, Macrophage colony-stimulating factor, Stem cell factor, Tumor necrosis factor (TNF)-α and OncostatinM (Fig. 3 and Table I).

The secretion of IL-6 was quantified with the ELISA method. hMSC in monolayer culture released 260 pg/ml IL-6. DMEM without supplements as control did not contain IL-6 (Fig. 4).

The cultivation of FaDu and HLaC78 with hMSC-sup induced a significant enhancement of cell proliferation compared to cultivation in DMEM-EM. The MTT assay confirmed the results of the cell counting. FaDu and HLaC78 showed a significant enhancement of cell proliferation. The impact of IL-6 on cell proliferation was investigated by the addition of anti-IL-6 into hMSC-sup. Anti-IL-6 induced the inhibition of cell proliferation. FaDu and HLaC78 showed a reduction in cell proliferation compared to cultivation with hMSC-sup without anti-IL-6 (Figs. 5 and 6).

fGFR gene expression was assessed by real-time PCR in FaDu and HLaC78. Values were expressed using the ΔΔCt method to derive relative fold change. Both cell lines were positive for EGFR expression. The EGFR expression was almost 2-fold more in HLaC78 compared to FaDu (Fig. 7).

The expression of ERK1/2 and the corresponding phosphorylated protein in FaDu and HLaC78 was investigated with the western blotting. Western blot analysis revealed expression of ERK1/2 as well as its phosphorylated protein after cultivation in hMSC-sup. The cultivation in DMEM-EM revealed total absence of the phosphorylated protein (Fig. 8).