The activity of alkaline phosphatase (ALP) of murine calvarial pre-osteoblasts (MC3T3-E1 cells) under the different treatments.

<p>1) no ATRA, no BMP-2/7; 2) 1 µM ATRA, no BMP-2/7; 3) 1 µM ATRA, 5 ng/ml BMP-2/7; 4) 1 µM ATRA, 50 ng/ml BMP-2/7; 5) no ATRA, 5 ng/ml BMP-2/7; 6) no ATRA, 50 ng/ml BMP-2/7 after for 4 day and 7 days. The ALP activity was normalized to total cellular protein content. All data are presented as mean values together with the standard deviation (SD). *: <i>p</i><0.05, **: <i>p</i><0.01, ***: <i>p</i><0.001.</p

Primer sequences for real-time quantitative polymerase chain reaction analysis of the expression of Runx2, collagen I, alkaline phosphatase (ALP) and osteocalcin (OCN) genes.

<p>Primer sequences for real-time quantitative polymerase chain reaction analysis of the expression of Runx2, collagen I, alkaline phosphatase (ALP) and osteocalcin (OCN) genes.</p

Heterodimeric BMP-2/7 Antagonizes the Inhibition of All-Trans Retinoic Acid and Promotes the Osteoblastogenesis

<div><p>Objectives</p><p>Hypervitaminosis A and alcoholism can result in a low mineral density and compromised regenerative capacity of bone, thus delaying implant osteointegration. The inhibitory effect of all-trans retinoic acid on osteoblastogenesis is considered to be one of the mechanisms. We hypothesized that heterodimeric bone morphogenetic protein-2/7 could antagonize all-trans retinoic acid and enhance osteoblastogenesis, with an aim to accelerate and enhance bone regeneration and implant osteointegration.</p><p>Materials and Methods</p><p>We applied 5 ng/ml or 50 ng/ml bone morphogenetic protein-2/7 to restore the osteoblastogenesis of pre-osteoblasts (MC3T3-E1 cell line) that was inhibited by 1 µM all-trans retinoic acid. We evaluated the efficacy by assessing cell numbers (proliferation), alkaline phosphatase activity (a marker for early differentiation), osteocalcin (a marker for late differentiation), calcium deposition (a marker for final mineralization) and the expression of osteoblastogenic genes (such as Runx2, Collagen Ia, alkaline phosphatase and osteocalcin) at different time points.</p><p>Results</p><p>All-trans retinoic acid significantly inhibited the expression of all the tested osteoblastogenic genes and proteins except alkaline phosphatase activity. In the presence of ATRA, 50 ng/ml bone morphogenetic protein-2/7 not only completely restored but also significantly enhanced all the osteoblastogenic genes and proteins. On the 28^th day, mineralization was completely inhibited by all-trans retinoic acid. In contrast, 50 ng/ml BMP-2/7 could antagonize ATRA and significantly enhance the mineralization about 2.5 folds in comparison with the control treatment (no ATRA, no BMP2/7).</p><p>Conclusions</p><p>Heterodimeric bone morphogenetic protein-2/7 bears a promising application potential to significantly promote bone regeneration and implant osteointegration for the patients with hypervitaminosis A and alcoholism.</p></div

The cell numbers of murine calvarial pre-osteoblasts (MC3T3-E1 cells) per well under the different treatments.

<p>1) no ATRA, no BMP-2/7; 2) 1 µM ATRA, no BMP-2/7; 3) 1 µM ATRA, 5 ng/ml BMP-2/7; 4) 1 µM ATRA, 50 ng/ml BMP-2/7; 5) no ATRA, 5 ng/ml BMP-2/7; 6) no ATRA, 50 ng/ml BMP-2/7 for 1 day and 4 days. All data are presented as mean values together with the standard deviation (SD). *: <i>p</i><0.05, **: <i>p</i><0.01, ***: <i>p</i><0.001.</p

The relative expression of four osteogenic genes under the different treatments.

<p>1) no ATRA, no BMP-2/7; 2) 1 µM ATRA, no BMP-2/7; 3) 1 µM ATRA, 5 ng/ml BMP-2/7; 4) 1 µM ATRA, 50 ng/ml BMP-2/7; 5) no ATRA, 5 ng/ml BMP-2/7; 6) no ATRA, 50 ng/ml BMP-2/7 for 1 day, 4 days and 7 days. (A) Runx2; (B) Collagen I; (C) Alkaline phosphatase; (D) Osteocalcin. The gene expression was first normalized to the corresponding β-actin gene expression for each sample. Then all the gene data were normalized to the gene data in control group on the 1^st day. All data are presented as mean values together with the standard deviation (SD). *: <i>p</i><0.05, **: <i>p</i><0.01, ***: <i>p</i><0.001.</p

The mineralization of murine calvarial pre-osteoblasts (MC3T3-E1 cells) under the different treatments.

<p>1) no ATRA, no BMP-2/7; 2) 1 µM ATRA, no BMP-2/7; 3) 1 µM ATRA, 5 ng/ml BMP-2/7; 4) 1 µM ATRA, 50 ng/ml BMP-2/7; 5) no ATRA, 5 ng/ml BMP-2/7; 6) no ATRA, 50 ng/ml BMP-2/7. (A) Light micrographs depicting the alizarin red staining on the 28^th day. (B) Graph depicting the calcification area on the 21^st day and the 28^th day. All data are presented as mean values together with the standard deviation (SD). *: <i>p</i><0.05, **: <i>p</i><0.01, ***: <i>p</i><0.001.</p

The expression of osteocalcin (OCN) of murine calvarial pre-osteoblasts (MC3T3-E1 cells) under the different treatments.

<p>1) no ATRA, no BMP-2/7; 2) 1 µM ATRA, no BMP-2/7; 3) 1 µM ATRA, 5 ng/ml BMP-2/7; 4) 1 µM ATRA, 50 ng/ml BMP-2/7; 5) no ATRA, 5 ng/ml BMP-2/7; 6) no ATRA, 50 ng/ml BMP-2/7 for 4 days and 7 days. All data are presented as mean values together with the standard deviation (SD). *: <i>p</i><0.05, **: <i>p</i><0.01, ***: <i>p</i><0.001.</p