Athanasios E. Mavrakos

The mineralized matrix of osseous tissue harbors abundant mitogenic activity which is extractable by demineralizing solvents. In bovine bone powder free of blood and cartilage contamination, the volume concentration of mitogens is up to 20 times greater than in serum. Growth factor activity in bone extracts was quantitated on quiescent mouse BALB/c/3T3 fibroblasts, where [3H]thymidine incorporation for 48 h was stimulated up to 200-fold in a linear, dose-dependent manner. Six distinct bone-derived growth factors (BDGFs) have been resolved and partially purified (up to 44,000-fold) on heparin-Sepharose using NaCl gradient elution. Provisionally named by the NaCl molarity at which they elute, these BDGFs include BDGF-0.45 (25% of total activity). This factor is heat-stable and sensitive to dithiothreitol, and displaces 125I-labelled bovine platelet-derived growth factor in a radioreceptor assay. BDGF-0.45 (approximately 50 ng/g of bone) is closely related or identical to bovine platelet-derived growth factor. BDGF-1.1 (10%) has a pI of 5.2 and shows a 16,600-dalton doublet on sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western blots stained with antiserum to bovine anionic fibroblast growth factor. Two activities with high heparin affinity resemble cationic forms of fibroblast growth factor. BDGF-1.5 is the dominant factor in fetal membranous bone (50%), but is less abundant in adult bone (20%). BDGF-1.7, a 17,500-18,400-dalton triplet, is virtually absent in fetal bone (7%) but abundant (30%) in adult bone and may be related to cartilage derived growth factor. Two minor activities, BDGF-0.1 (10%) and BDGF-2.0 (7%) have not been characterized. Proliferation of bovine capillary endothelial cells was strongly supported by BDGFs 1.1, 1.5, and 1.7, but not by 0.45. These four purified BDGFs and the crude bone extract were also strongly mitogenic for rat osteoblasts while depressing alkaline phosphatase specific activity by 2-3-fold. Bone exhibits the most complex spectrum of growth factor activities of any tissue yet described. Bone cells and other indigenous cell types must be considered as possible sources of the BDGFs, in addition to sequestration from blood. Mechanisms for unmasking or release of BDGFs from the mineralized matrix resulting in local action on target cells are undoubtedly important for the development and maintenance of bone tissue.

The presence of many types of polypeptide growth factors in the mineralized extracellular matrix of bone is now well established. These factors are generally referred to as bone-derived growth factors (BDGFs), and are similar, or possibly identical, to the following species; platelet-derived growth factor (PDGF); acidic and basic forms of fibroblast growth factor (aFGF, bFGF); transforming growth factor beta (TGF-beta); and insulin-like growth factor 1 (IGF-1). Several osteoinductive factors, such as bone morphogenetic protein (BMP) and osteogenin, a skeletal growth factor (SGF), and osteoblast-derived BDGFs, have also been identified. Complete description of the biological functions of these BDGFs which are relevant to bone will ultimately require specific bioassays involving specific cell types in vitro, as well as in vivo animal implant models. Studies with primary rat osteoblast-like cells exposed either to mixed BDGFs, pure TGF-beta, or heparin-purified PDGF, aFGF, or bFGF from bovine bone have shown a general dose-dependent mitogenic effect. Phenotypic changes which accompany the BDGF-induced wave of proliferation include: decreased osteocalcin secretion and a reduction in 1,25-(OH)2 vitamin D3-stimulated osteocalcin synthesis; reduced alkaline phosphatase specific activity; decreased cyclic AMP responsiveness to parathyroid hormone (PTH); and increased collagen synthesis. Bone exhibits the most complex spectrum of growth factor activities of any tissue yet described. In bovine bone powder free of blood and cartilage contamination, the volume concentration of mitogens is up to 20 times greater than that in serum. Bone cells and other indigenous cell types must be considered as possible sources of the BDGFs, in addition to sequestration from blood. Mechanisms for the unmasking or release of BDGFs from the mineralized matrix that result in local action on osteoblasts, endothelial cells, and other target cells are undoubtedly important for the development and maintenance of bone tissue.