Effects of enamel matrix derivative and transforming growth factor-β1 on human osteoblastic cells

Human alveolar bone fragments (explants) were obtained from adult healthy donors (ranging from 15 to 25 years old), using palatal/lingual and/or interradicular alveolar bone associated with either premolars or third molars extracted for orthodontic reasons, with clinically healthy periodontium. Osteoblastic cells were obtained from these explants by enzymatic digestion using collagenase type II (Gibco - Life Technologies, Grand Island, NY) as described by Mailhot and Borke[28]. Importantly, to avoid contamination with periosteal, periodontal ligament, and gingival cells, bone fragments were scrapped and the first 2 digestions were discarded. Primary cells were cultured in α-minimum essential medium (α-MEM - Gibco), supplemented with 10% fetal bovine serum (FBS - Gibco), 50 μg/mL gentamicin (Gibco), 0.3 μg/mL fungizone (Gibco), 10^-7 M dexamethasone (Sigma, St. Louis, MO), 5 μg/mL ascorbic acid (Gibco), and 7 mM β-glycerophosphate (Sigma). Such osteogenic culture condition supports the development of the osteoblastic phenotype[29, 30].

Subconfluent cells in primary culture were harvested after treatment with 1 mM ethylenediamine tetraacetic acid (EDTA - Gibco) and 0.25% trypsin (Gibco) and subcultured cells under osteogenic culture condition were used in all experiments. The progression of the subcultured cells and the acquisition of the osteoblastic phenotype have been well characterized by the work of de Oliveira et al. [31]. During the culture period, cells were incubated at 37°C in a humidified atmosphere of 5% CO₂ and 95% air; the medium was changed every three or four days. All experiments were performed using three different sets of subcultures, and each experiment conducted in quadruplicate. All patients were informed about the study's purpose before they consented to participate. The local Research Ethics Committee approved the protocol.

Emdogain gel (EMD - Biora, Malmo, Sweden) was dissolved in acidic water, pH 5.9, whereas TGF-β1 (Sigma Chemical Co., St. Louis, MO, USA) was dissolved in acetonitrile plus trifluoracetic acid (Sigma). Both solutions were aliquoted and stored at -70°C. Two concentrations had to be chosen because the osteoblastic cell subculture would not allow a more extensive experimental design than the one proposed herein. Thus, based on previous studies[10, 32], treatment with EMD and TGF-β1 was performed at concentrations of 100 μg/mL and 5 ng/mL, respectively. Four experimental conditions were established: 1) medium supplemented with 10% FBS (control); 2) 100 μg/mL EMD in medium supplemented with 10% FBS (EMD group); 3) 5 ng/mL TGF-β1 in medium supplemented with 10% FBS (TGF-β1); 4) combination of 100 μg/mL EMD and 5 ng/mL TGF-β1 in medium supplemented with 10% FBS (EMD+TGF-β1 group). The final pH for all groups was in the 7.2-7.4 range. A negative control was not possible because culture medium with either no FBS or a minimum concentration of FBS did not support the progression of the osteoblastic cell cultures (data not shown).

The cell growth assay was performed using a modified method of Coletta et al. (1998)[33]. Osteoblastic cells were plated in a 24-well culture plate (Corning Inc., NY, USA) at a density of 20,000 cells/well in 1 mL of α-MEM supplemented with 10% FBS (Gibco), 50 μg/mL gentamicin (Gibco), 0.3 μg/mL fungizone (Gibco), 10^-7 M dexamethasone (Sigma), 5 μg/mL ascorbic acid (Gibco), and 7 mM β-glycerophosphate (Sigma). The cells were allowed to attach and spread for 24 h, and then washed with PBS and cultured in serum-free α-MEM for an additional 24 h. After treatments with the four experimental conditions for four and seven days, cells were enzymatically (1 mM EDTA, 1.3 mg/mL collagenase type II, and 0.25% trypsin - Gibco) detached. Aliquots of these solutions were incubated for 5 min with the same volume of trypan blue and directly counted in a hemocytometer (Fisher Scientific, Pittsburgh, PA, USA). For each time point, total cell number (×10⁴/well) was determined, which included trypan blue-stained cells.

Effect of EMD, TGF-β1 and the combination of both on osteoblastic cells proliferation was assessed by direct counting of cell number and BrdU incorporation into DNA. The BrdU is detecting in the tissue through primary antibodies. These primary antibodies are then labeled with a secondary antibody tagged with a substrate for diaminobenzidine (DAB, Nunc International, Naperville, IL, USA)[34]. The substitution of an endogenous DNA base, thymidine, with the BrdU analogue ensures specific labeling of only the dividing cells during S-phase (DNA synthesis). Osteoblastic cells were plated on 8-well glass culture chamber slides (Nunc International, Naperville, IL, USA) at a density of 20,000 cells/well in 500 μl of α-MEM supplemented with 10% FBS (Gibco), 50 μg/mL gentamicin (Gibco), 0.3 μg/mL fungizone (Gibco), 10^-7 M dexamethasone (Sigma), 5 μg/mL ascorbic acid (Gibco), and 7 mM β-glycerophosphate (Sigma), and were incubated at 37°C and 5% CO₂. Following 24 h of serum starvation, cells were exposed to the four experimental culture conditions for 24 h. After treatment, cells were incubated with BrdU (diluted 1:1,000) for 1 h under the same conditions, washed in PBS and fixed in 70% ethanol for 15 min. BrdU incorporation in proliferating cells was revealed using immunohistochemistry (Amershan Pharmacia Biotech Inc., Piscataway, NJ). Briefly, the anti-5-bromo-2'-deoxyuridine monoclonal antibody, diluted 1:100 in nuclease with deionized water, were added to the wells and incubated for 1 h. The wells were then washed three times with 500 μL of PBS and the peroxidase anti-mouse IgG2a (15:1,000) were added to the wells and incubated for 1 h. After another washing step, the reaction was developed with 0.6 mg/mL of 3,3'-diaminobenzidine tetrahydrochloride (Sigma) containing 1% H₂O₂ and 1% DMSO for 5 min at 37°C. The cells were then stained with Crazzi hematoxylin and examined under transmitted light microscopy. The BrdU labeling index, expressed as the percentage of cells labeled with BrdU, was determined by counting 1,500 cells using an image analysis system (Kontron 400, Zeiss, Eching bei Munich, Germany).

For immunofluorescence labeling of noncollagenous matrix proteins, cells were treated with the four experimental culture conditions for five days. At day 5, cells were fixed for 10 min at room temperature (RT) using 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.2. After washing in PB, they were processed for immunofluorescence labeling[31]. In addition, cell adhesion and spreading were morphologically evaluated by direct fluorescence with fluorophore-conjugated probes. Briefly, cells were permeabilized with 0.5% Triton X-100 in PB for 10 min followed by blocking with 5% skimmed milk in PB for 30 min. Primary monoclonal antibodies to bone sialoprotein (anti-BSP, 1:200, WVID1-9C5, Developmental Studies Hybridoma Bank, Iowa City, IA, USA), alkaline phosphatase (anti-ALP, 1:100, B4-78, Developmental Studies Hybridoma Bank), and osteopontin (anti-OPN, 1:800, MPIIIB10-1, Developmental Studies Hybridoma Bank) were used, followed by a mixture of Alexa Fluor 594 (red fluorescence)-conjugated goat anti-mouse secondary antibody (1:200, Molecular Probes) and Alexa Fluor 488 (green fluorescence)-conjugated phalloidin (1:200, Molecular Probes), which labels actin cytoskeleton. Replacement of the primary monoclonal antibody with PB was used as control. All antibody incubations were performed in a humidified environment for 60 min at RT. Between each incubation step, the samples were washed three times (5 min each) in PB. Before mounting for microscope observation, samples were briefly washed with dH₂O and cell nuclei stained with 300 nM 4', 6-diamidino-2-phenylindole, dihydrochloride (DAPI, Molecular Probes) for 5 min. After mounting with an antifade kit (Prolong, Molecular Probes), the samples were examined under epifluorescence using a Leica DMLB light microscope (Leica, Bensheim, Germany), with N Plan (X2.5/0.07, X10/0.25, X20/0.40) and HCX PL Fluotar (X40/0.75, X100/1.3) objectives, outfitted with a Leica DC 300F digital camera. The acquired digital images were processed with Adobe Photoshop software (version 7.0.1, Adobe Systems).

Osteoblastic cells were plated in 24-well culture plates at a density of 20,000 cells/well in 2 mL of α-MEM supplemented with 10% FBS (Gibco), 50 μg/mL gentamicin (Gibco), 0.3 μg/mL fungizone (Gibco), 10^-7 M dexamethasone (Sigma), 5 μg/mL ascorbic acid (Gibco), and 7 mM β-glycerophosphate (Sigma) at 37°C in a humidified atmosphere with 5% CO₂. Following serum starvation, cells were exposed to the four experimental culture conditions described previously for seven and fourteen days. Media was changed and supplemented every three or four days. Total protein content was determined using a modification of the Lowry method. Briefly, proteins were extracted from each well with 0.1% sodium lauryl sulphate (Sigma) for 30 min, resulting in a lysates of the cells, and mixed 1:1 with Lowry solution (Sigma) for 20 min at RT. The resulting solution was diluted in Folin and Ciocalteau's phenol reagent (Sigma) for 30 min at RT. Absorbance was measured at 680 nm using a spectrophotometer (Cecil CE3021, Cambridge, UK). The total protein content was calculated from a standard curve and expressed as μg/mL.

Osteoblastic cells were plated in 24-well culture plates at a density of 20,000 cells/well in 2 mL of α-MEM supplemented with 10% FBS (Gibco), 50 μg/mL gentamicin (Gibco), 0.3 μg/mL fungizone (Gibco), 10-7 M dexamethasone (Sigma), 5 μg/mL ascorbic acid (Gibco), and 7 mM β-glycerophosphate (Sigma) at 37°C in a humidified atmosphere with 5% CO₂. Following serum starvation, cells were exposed to the four experimental culture conditions described previously for seven and fourteen days. Media was changed and supplemented every three or four days. Alkaline phosphatase (ALP) was extracted from each well with 0.1% sodium lauryl sulphate (Sigma) for 30 min, resulting in a lysates of the cells ALP activity was measured as the release of thymolphthalein from thymolphthalein monophosphate using a commercial kit (Labtest Diagnostica, MG, Brazil). Briefly, 50 μl thymolphthalein monophosphate was mixed with 0.5 ml 0.3 M diethanolamine buffer, pH 10.1, and left for 2 min at 37°C. The solution was then added to 50 μl of the lysates obtained from each well for 10 min at 37°C. For color development, 2 ml 0.09 M Na₂CO₃ and 0.25 M NaOH were added. After 30 min, absorbance was measured at 590 nm and ALP activity was calculated from a standard curve using thymolphthalein to give a range from 0.012 to 0.4 μmol thymolphthalein/h/ml. Data were expressed as ALP activity normalized for total protein content at 7 and 14 days.

Osteoblastic cells were plated in 24-well culture plates at a density of 20,000 cells/well in 2 mL of α-MEM supplemented with 10% FBS (Gibco), 50 μg/mL gentamicin (Gibco), 0.3 μg/mL fungizone (Gibco), 10^-7 M dexamethasone (Sigma), 5 μg/mL ascorbic acid (Gibco), and 7 mM β-glycerophosphate (Sigma) at 37°C in a humidified atmosphere with 5% CO₂. Following serum starvation, cells were exposed to the four experimental culture conditions described previously with differentiation medium for 21 days. Media was changed and supplemented every three or four days. At day 21, cultures were washed in PBS and fixed with 10% formaldehyde in PBS, pH 7.2, for 16 h at 4°C. The samples were then dehydrated in a graded series of ethanol and stained with 2% Alizarin red S (Sigma), pH 4.2, for 8 min at RT. Using an inverted light microscope (X10 objective; Carl Zeiss, Jena, Germany), equipped with a digital camera (Canon EOS Digital Rebel Camera, 6.3 Megapixel CMOS sensor, Canon USA Inc., Lake Success, NY, USA), the formation of mineralized areas was analyzed. Ten microscopic fields in each sample were randomly selected and the mineralized area was measured as a percentage area of the well using an image analyzer (Image Tool; University of Texas Health Science Center, San Antonio, TX, USA).

Data represent the pooled results of three independent experiments. Each experiment was conducted using cells of a single donor. All experiments were performed in quadruplicate for each set of subculture. All results are presented as mean ± standard deviation, and the non-parametric Kruskal-Wallis test for independent samples was used for statistical analyses. If the result of the Kruskal-Wallis test was significant (P < 0.05), the Fischer's test for multiple comparisons, computed on ranks rather than data, was performed[35].

Nuclear immunoreactivity for BrdU was clearly noticed in osteoblastic cells under all treatments. Both treatments and their combination affected the proliferation at the first 24 hours of experiments compared to the control (EMD, P < 0.001; TGF-β1, P < 0.001; EMD + TGF-β1, P < 0.05) (Figure 1). In addition, treatment with EMD significantly increased total cell number compared to TGF-β1 (P < 0.05) and the combination of the factors (P < 0.001). Treatments with EMD, TGF-β1 and EMD+TGF-β1 significantly increased total cell number at day 4 compared to the control (P < 0.001, P < 0.01, and P < 0.001, respectively); the treatment with only EMD resulted in higher values compared to the TGF-β1 treatment (P < 0.001) and the combination of the factors (P < 0.01), whereas total cell number for EMD+TGF-β1 was significantly higher compared to TGF-β1 (P < 0.01). On day 7, no statistical differences among TGF-β1, EMD+TGF-β1 and control groups were detected. However, all these groups showed a significantly lower number of cells compared to the EMD group (control, P < 0.01; TGF-β1, P < 0.05; EMD + TGF-β1, P < 0.01) (Figure 2).

Epifluorescence of actin cytoskeleton labeling revealed that cells were adherent and spread, showing a polygonal elongated morphology, with focal areas of multilayer formations (Figure 3A-D). Indirect immunofluorescence using a primary antibody anti-OPN showed that such protein was expressed in the majority of cells, mostly in the perinuclear area suggestive of Golgi apparatus, and in a dot pattern throughout the cytoplasm. No differences in terms of OPN labeling pattern and fluorescence intensities among control and EMD, TGF-β1 e EMD+TGF-β1 groups were noticed; for all groups, no extracellular OPN labeling was detected (Figure 3A-D). Immunolabeling for ALP was more intense for control than for the treated groups, with a labeling pattern characterized by punctate deposits throughout the cell surface and cytoplasm (Figure 3E-H). At day 5, no bone sialoprotein labeling was detected for all groups (data not shown).

Total protein synthesis was not significantly affected by the treatments (P > 0.05) (Figure 4); however, a tendency for greater values of total protein was clearly seen at day 7 for all treated groups and for the EMD group at day 14. ALP activity was negatively affected by EMD, TGF-β1 and EMD+TGF-β1 treatments compared to the control both at days 7 and 14. On day 14, the treatments with EMD and EMD+TGF-β1 exhibited lower ALP activity than TGF-β1 group (P < 0.01 and P < 0.001, respectively) (Figure 5). At day 21, matrix mineralization was significantly higher for the control group compared to EMD (P < 0.05), TGF-β1 (P < 0.001) and EMD+TGF-β1 groups (P < 0.01) (Figures 6 and 7).