Isolation of a native osteoblast matrix with a specific affinity for BMP2.
During their commitment and differentiation toward the osteoblast lineage, mesenchymal stem cells secrete a unique extracellular matrix (ECM) that contains large quantities of glycosaminoglycans (GAGs). Proteoglycans (PGs) are major structural and functional components of the ECM and are composed of a core protein to which one or more glycosaminoglycan sugar chains (GAGs) attach. The association of BMP2, a member of the TGF-beta super-family of growth factors, and a known heparin-binding protein, with GAGs has been implicated as playing a significant role in modulating the growth factor's in vitro bioactivity. Here we have characterised an osteoblast-derived matrix (MX) obtained from decellularised MC3T3-E1 cell monolayers for its structural attributes, using SEM and histology, and for its functional ability to maintain cell growth and viability. Using a combination of histology and anion exchange chromatography, we first confirmed the retention of GAGs within MX following the decellularisation process. Then the binding specificity of the retained GAG species within the MX for BMP2 was examined using a BMP2-HBP/EGFP (BMP2 Heparin-Binding Peptide/Enhanced Green Fluorescent Protein) fusion protein. The results of this study provide further evidence for a central role of the ECM in the regulation of BMP2 bioactivity, hence on mesenchymal stem cell commitment to the osteoblast lineage.
Stem Cells and Tissue Repair Group, Institute of Molecular and Cell Biology, Proteos, Singapore, Singapore. martin.grunert@imb.a-star.edu.sg
Martin
M
Grünert
Christian
C
Dombrowski
Murali
M
Sadasivam
Kerry
K
Manton
Simon M
SM
Cool
Victor
V
Nurcombe
D016475:3T3 Cells; D000704:Analysis of Variance; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D002810:Chondroitinases and Chondroitin Lyases / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D005109:Extracellular Matrix / Q000378:metabolism* / Q000648:ultrastructure; D006025:Glycosaminoglycans / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D019763:Heparin Lyase / Q000378:metabolism; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding; D011509:Proteoglycans / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*
C525718:BMP2 protein, human; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D006025:Glycosaminoglycans; D011509:Proteoglycans; D016212:Transforming Growth Factor beta; D049452:Green Fluorescent Proteins; D002810:Chondroitinases and Chondroitin Lyases; D019763:Heparin Lyase