Ossification Of Cells Research Articles

Osteogenic Potential of the Transcription Factor c-MYB.

The transcription factor c-MYB is a well-known marker of undifferentiated cells such as haematopoietic cell precursors, but recently it has also been observed in differentiated cells that produce hard tissues. Our previous findings showed the presence of c-MYB in intramembranous bones and its involvement in the chondrogenic steps of endochondral ossification, where the up-regulation of early chondrogenic markers after c-myb overexpression was observed. Since we previously detected c-MYB in osteoblasts, we aimed to analyse the localisation of c-MYB during later stages of endochondral bone formation and address its function during bone matrix production. c-MYB-positive cells were found in the chondro-osseous junction zone in osteoblasts of trabecular bone as well as deeper in the zone of ossification in cells of spongy bone. To experimentally evaluate the osteogenic potential of c-MYB during endochondral bone formation, micromasses derived from embryonic mouse limb buds were established. Nuclear c-MYB protein expression was observed in long-term micromasses, especially in the areas around nodules. c-myb overexpression induced the expression of osteogenic-related genes such as Bmp2, Comp, Csf2 and Itgb1. Moreover, alizarin red staining and osteocalcin labelling promoted mineralised matrix production in c-myb-overexpressing cultures, whereas downregulation of c-myb by siRNA reduced mineralised matrix production. In conclusion, c-Myb plays a role in the osteogenesis of long bones by inducing osteogenic genes and causing the enhancement of mineral matrix production. This action of the transcription factor c-Myb might be of interest in the future for the establishment of novel approaches to tissue regeneration.

Effect of recombinant adeno-associated BMP-4/7 fusion gene on the biology of BMSCs

The aim of the study was to investigate the effect of recombinant adeno-associated virus (AAV) with morphogenetic protein 4/7 fusion gene on the ossification of rabbit bone marrow stromal cells (BMSCs). The genes BMP-4 and BMP-7 were obtained through one-step reverse transcriptase polymerase chain reaction from human placental cells. The BMP-4/7 fusion gene was generated through recombination. The rabbit BMSCs were transfected with the recombinant AAV vectors carrying AAV-BMP-4/7 with various multiplicity of infection (MOI) values. Expression of fusion gene BMP-4/7 protein was determined by the ELISA method. The ossification of cells was evaluated by observing alkaline phosphatase (ALP) and osteocalcin (OC) activity after transfection for 7 and 14 days. We successfully constructed the recombinant AAV with the BMP-4/7 fusion gene. A 29-20 kDa protein was shown by SDS-PAGE electrophoresis following transfection with AAV-BMP-4/7. The fusion protein BMP-4/7 in BMSCs transferred by AAV showed a positive correlation with various MOI values. There was significantly higher ALP and OC activity in the AAV-BMP-4/7 transfection groups than in the AAV-EGFP groups (t(ALP)=896.88, P<0.001, t(OC)=543.24, P<0.01). The fusion gene BMP-4/7 is capable of enhancing the expression of BMPs and possesses significant ossification activity through AAV.

Recombinant adeno-associated virus BMP-4/7 fusion gene confers ossification activity in rabbit bone marrow stromal cells

The biological effects of transfection of an adeno-associated virus (AAV) vector with bone morphogenetic proteins 4 and 7 (BMP-4/7) fusion gene (AAV-BMP-4/7) were determined in rabbit bone marrow stromal cells (BMSCs). BMP-4 and BMP-7 genes were obtained through one-step reverse transcriptase polymerase chain reaction from human placental cells. The BMP-4/7 fusion gene was then generated through recombination. Rabbit BMSCs were transfected with the recombinant AAV vectors carrying AAV-BMP-4/7 with multiplicity of infection values. Cell growth curves were drawn to evaluate the biological effects of AAV-BMP-4/7 on cell activity. The transfection efficiency was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The ossification of cells was evaluated by observing alkaline phosphatase (ALP) and osteocalcin (OC) activity after transfection for 7 and 14 days. The cells were then transfected with AAV-BMP-4/7 and AAV-enhanced green fluorescent protein. We successfully constructed the recombinant adeno-associated virus with the BMP-4/7 fusion gene. The transfection efficiency of AAV-BMP-4/7 was approximately 72% without significant biological effects on cell activity. Cell ossification was significant after transfection with AAV-BMP-4/7. The 1 x 10(5) vg/cell multiplicity of infection value of transfection efficiency was more than 5 x 10(4) vg/cell (59.38%). Significantly higher ALP and OC activity occurred in the AAV-BMP-4/7 transfection groups than in the AAV-enhanced green fluorescent protein groups (t(ALP) = 896.88, P < 0.001; t(OC) = 543.24, P < 0.01). The AAV-BMP-4/7 fusion gene can highly efficient transfect rabbit BMSCs cultured in vitro and it has significant ossification activity.