Purification and characterisation of a growth factor from porcine bone

Abstract

A growth factor was extracted from porcine bone matrix by demineralisation and purified by heat and acid treatment, hydroxyapatite chromatography and gel filtration under dissociative conditions and reverse-phase HPLC. Using the mitogenic response of osteoblast-progenitor cells from embryonic chicken, a mitogenic activity was purified 3000-fold. The mitogenic protein thus purified shows an apparent molecular mass of 13.5 kDa in both the nonreduced and reduced form on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The mitogenic activity is sensitive to proteinase K, dithiothreitol, and resistant to DNAse, RNase, heat (70°C) and pH (3 -10). The factor stimulates the proliferation of osteoblast-progenitor cells from embryonic chick at a concentration of 1 ng/ml. It is active on cells from skin, periosteum and sternum and has no or little activity on cells of the calvaria, intestine or kidney of embryonic chick or on mouse AKR-2B/Balb c/3T3 cell line.Bone growth is characterised by a localized remodeling in which periods of bone resorption are followed by subsequent new bone formation. It is likely that the coupling of bone resorption to bone formation is mediated by growth factors in the micro-environment, which may modulate their biological action through complex modes of release and presentation to responding cells [l]. A basic assumption is that bone regeneration occurs by a combination of induced proliferation of predifferentiated osteoprogenitor cells and induced differentiation of mesenchymal cells. The osteoprogenitor cell proliferation process is well known and seen in the reaction of periosteum and endosteum to injury, diet, vitamins and hormones. The process of induced cell differentiation of niesenchymal cells has been observed from quantitative measurement of bone formed in response to implants of either bone matrix or purified proteins in extraskeletal and intraskeletal sites [2-41.Endogeneous bone growth factors of possible relevance are produced from organ culture or extracted from bone matrix and include: bone-derived growth factor, a 11.6-kDa protein produced by cultured fetal rat calvaria [l, 51; transforming growth factor-p, a 25-kDa protein recently demonstrated by the same organ culture system [5, 61; cartilagederived factor, a substance like somatomedin C and insulin growth factor I [7]; cationic cartilage-derived growth factor, a 19-kDa protein like fibroblast growth factor [S]; human skeletal growth factor, a 9 -12-kDa form now recognized as being like insulin growth factor I1 [9, lo]; bone morphogenetic protein, an 18-kDa protein isolated from bovine bone matrix [ll]; osteogenin, an 18.5-kDa bovine protein with bone-inducing activity [3]; a 22-kDa mitogenic factor from rat bone [12]; and a 26-kDa cartilage induction factor A from bovine bone 1131, now recognized to be identical to transforming growth factor [14], and cartilage induction factor B and [l, 5, 6, 121, ox [3, 11, 19, 211 and human [9, 101. The present report describes a growth factor, isolated from porcine bone m...