The 3T3-L1 murine preadipocytes were incubated with growth medium containing 1-methyl-3-isobutyl-xanthine dexamethasone and insulin for 5 days in the presence of (A) propylene glycol alginate or (B) Emdogain® at dilutions equivalent to 100 µg/ml and subjected to a TaqMan® Array for Mouse Lipid Regulated Genes.

<p>The table indicates the genes at least 2-fold up-, or down regulated.</p

Emdogain® after heating at 96°C maintains the ability to activate TGF-β signaling.

<p>(A) The 3T3-L1 murine preadipocytes were exposed to Emdogain® (100 µg/ml) or Emdogain® previously heated up to 96°C within a basal adipogenesis-inducing medium containing rosiglitazone and indomethacin for 5 days and lipid staining was performed. (B) 3T3-L1 cells were also exposed to normal or heat-treated Emdogain® for 18 hours in serum-free medium before the expression of PPARγ and CTGF were determined.</p

SB-431542, an inhibitor of TGF-β RI kinase activity, reversed the inhibitory effects of Emdogain®.

<p>The 3T3-L1 murine preadipocytes were incubated with basal adipogenesis-inducing medium containing rosiglitazone and indomethacin for 5 days in the presence of Emdogain® (100 µg/ml) or TGF-β (10 ng/ml) and a selective inhibitor of the TGF-β type I receptor SB-431542 (10 nM). (A) Lipid staining; (B) SB-431542 overcomes the blocking effect of Emdogain® on PPARγ expression; (C) Emdogain® and TGF-β increase phosphorylation of Smad3 in 3T3-L1 cells. **P<0.01%.</p

3T3-L1 cells retain their adipogenic potential after transient exposure to Emdogain®.

<p>The 3T3-L1 murine preadipocytes were incubated for one day with growth medium containing Emdogain® (100 µg/ml) or TGF-β (10 ng/ml). Then the medium was replaced by basal adipogenesis-inducing medium containing rosiglitazone and indomethacin for 5 days and lipid staining was performed.</p

The Emdogain®-induced TGF-β signaling cascade increases CTGF expression.

<p>(A) The 3T3-L1 murine preadipocytes were exposed to Emdogain® (100 µg/ml) either with or without SB431542 or SB600125 for 18 hours in serum-free medium and the expression CTGF determined. (B) 3T3-L1 cells were transfected with siRNA CTGF and the respective MOCK siRNA before being stimulated with Emdogain® or TGF-β. (C) Inhibition of 38 kDa CTGF based on Western blot analysis.</p

Rosiglitazone and indomethacin failed to rescue the adipocyte differentiation of Emdogain®-treated cells.

<p>The 3T3-L1 murine preadipocytes were incubated with growth medium containing indomethacin and rosiglitazone together with the basal adipogenesis-inducing medium for 5 days in the presence of (A) propylene glycol alginate or (B) Emdogain® at dilutions equivalent to 100 mg/ml and stained for lipid droplets.</p

EMD promotes expression of connexin 43 and N-cadherin in cell clusters.

<p>At time point 5 days post seeding, primary human osteoblasts were stained for connexin 43 or N-cadherin (red), and nuclei (blue). Expression of connexin 43 and N-cadherin significantly increased on cell membranes of EMD coated samples. (bar = 50 µm).</p

EMD significantly increases mineral deposition as assessed through von Kossa staining.

<p>At time points 5, 7, 10 and 14 days, primary human osteoblasts were fixed and stained with silver nitrate to determine patterns of mineralization. A) EMD significantly increased mineralization in clustered regions 5 days post seeding. At 10 and 14 days post seeding, areas of mineralization for EMD-coated samples were enlarged when compared to control samples (A) (bar = 1000 µm). 10 fields of view per sample were captured and percentage area of staining was quantified (B). At all time points, EMD significantly increased von Kossa staining. Furthermore, significant increases in nodule size were also observed at all time points (C). Data represent means ± SE (results from 3 independent experiments).</p

EMD increases osteoblast mRNA and protein levels of connexin 43 and N-cadherin.

<p>After 2, 3, 5, 7 and 10 days post seeding, mRNA was extracted and realtime PCR was performed using specific primers for connexin 43 (A) and N-cadherin (B). When samples were pre-coated with EMD, up to 4 fold increases in gene expression were observed for connexin 43 at 2, 3 and 5 days post seeding (A). 3 fold increases in gene expression of N-cadherin were also observed (B). Additional samples were extracted for western blot analysis (C). Elevated levels of both connexin 43 and N-cadherin were observed at 2, 3, 5 and 7 days post seeding. * denotes significant difference between EMD treated sample and respective control sample (p<0.05). Data shown is the average value from 3 independent experiments (3 replicates per experiment) ± SE.</p

EMD increases extracellular matrix deposition of osteocalcin and osteoblast differentiation markers.

<p>At 5 and 14 days post seeding, human primary osteoblasts were labeled with specific antibodies to osteocalcin. Osteoblasts seeded on EMD-coated samples secreted higher levels of osteocalcin into the extracellular matrix when compared to control samples at 5 and 14 days (A) (bar = 200 µm). EMD-coated samples also increased mRNA levels of osteoblast differentiation markers (B–E). After 3, 5, 7, 10 and 14 days post seeding, mRNA was extracted and realtime PCR was performed using specific primers for Runx2, COL1A1, osteocalcin and bone sialoprotein. Levels of Runx2 were not significantly altered between EMD-coated and control samples (B). When samples were pre-coated with EMD, up to 3 fold increases in gene expression were observed for C) COL1A1, D) osteocalcin and E) bone sialoprotein (p<0.05). * denotes significant difference between EMD treated sample and respective control sample. Data shown is the average value from 3 independent experiments (3 replicates per experiment) ± SE.</p