BMP2/BMP4 Knock Out in Murine Osteoblasts Affects Cellular Differentiation, Cell Cycling, and Extracellular Matrix Remodeling.

摘要

Bone morphogenetic proteins (BMPs) are currently utilized in periodontics for ridge preservation and sinus augmentation; however, clinical results with these materials can be unpredictable. This unpredictability may be explained by gaps in our knowledge of BMP's complete role in osteoblast cellular processes. This study was undertaken to create a novel immortalized murine BMP-2/4 knock out (iBMP-2/4 KO) osteoblast model to determine the role of BMP-2 and 4 (BMP-2/4) in osteoblast differentiation, cell proliferation, and extracellular matrix (ECM) remodeling.;Immortalized murine floxed BMP-2/4 (ifBMP-2/4) osteoblast cells were doubly knocked out by a Cre recombinase positive adenovirus to generate a murine iBMP-2/4 KO osteoblast cell line. Successful KO was confirmed through immunofluorescence and PCR. Expression of bone-specific genes of both ifBMP-2/4 and iBMP-2/4 KO cells were compared by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell differentiation and mineralization of the ifBMP-2/4 and iBMP-2/4 KO cells were tested by alkaline phosphatase and alizarin red S staining. Rescue of BMP-2/4 KO mineralization was attempted by addition of either 100 ng/ml BMP-2, 20 ng/ml BMP-4, or both 100 ng/ml BMP-2 and 20 ng/ml BMP-4. Cell cycling was observed by flow cytometry. ECM remodeling was determined by in-situ and gelatin zymography assays.;ifBMP-2/4 murine osteoblast cells were successfully infected, knocking out the genes for BMP-2 and BMP-4. The morphology of iBMP-2/4 KO osteoblasts was smaller and more spindle shaped than their floxed counterparts as observed under light microscopy. Expression of bone-specific genes such as ATF4, Bsp, Coll1, Dlx 3, Oc, Osn, and Osx were significantly decreased in the iBMP-2/4 KO cells compared to that of the ifBMP-2/4 osteoblasts. Cell differentiation and mineralization were also reduced in KO cells, as shown by decreased alkaline phosphatase and alizarin red S staining. Mineralization of iBMP-2/4 KO cells was rescued by addition of recombinant BMP-2 and BMP-4 to the media. The cell cycle was delayed in G2 phase, as detected by flow cytometry, in iBMP-2/4 KO cells. Furthermore, iBMP-2/4 KO cells had defects in ECM remodeling as reflected by a reduction in collagen type I, IV, and gelatin processing resulting from a decrease in matrix metalloproteinase (MMP)-2 and MMP-9 expression.;In this study, data showed that BMP-2 and BMP-4 are necessary for osteoblast differentiation and mineralization. It was also demonstrated that BMP-2 and BMP-4 play important substantive roles in regulating cell cycle, proliferation, and ECM remodeling. Furthermore, BMP-2 and 4 are involved in ECM remodeling by regulating MMP-2 and MMP-9 activity. In clinical application, excessive BMP concentrations could lead to over expression of MMPs resulting in unpredictable osseous gains.