Figure 1.
EMD promotes clustering of primary human osteoblasts on EMD-coated samples.
On control and EMD coated samples, cells were evenly distributed as shown with DAPI staining at 4 hours post seeding (A–B). However after 24 hours, clustering of cells was apparent after 24 hours on EMD coated samples (D) when compared to control samples (C). (bar = 500 µm).
Figure 2.
EMD promotes expression of connexin 43 and N-cadherin in cell clusters.
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).
Figure 3.
EMD increases osteoblast mRNA and protein levels of connexin 43 and N-cadherin.
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.
Figure 4.
EMD increases alkaline phosphatase activity in osteoblast clusters.
At time points 1, 3, 5, 7 and 10 days, osteoblasts were fixed and stained for alkaline phosphatase. A) EMD significantly increased alkaline phosphatase activity in clustered regions 5 days post seeding. After 10 days, complete staining was observed on samples coated with EMD when compared to control samples. (bar = 1000 µm) B) 10 fields of view per sample were captured and percentage area of staining calculated. Data represent means ± SE (results from 3 independent experiments). Significant increases in alkaline phosphatase activity were observed on EMD treated samples at 5, 7 and 10 days post seeding. C) mRNA was extracted and realtime PCR was performed using specific primers for alkaline phosphatase. Samples pre-coated with EMD showed significant mRNA levels 3, 5 and 7 days post seeding when compared to control groups (p<0.05). Data represent means ± SE (results from 3 independent experiments).
Figure 5.
EMD increases extracellular matrix deposition of osteocalcin and osteoblast differentiation markers.
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.
Figure 6.
EMD significantly increases mineral deposition as assessed through von Kossa staining.
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).