Transforming Growth Factor-β Supports the Rapid Morphogenesis of Heterotopic Endochondral Bone Initiated by Human Osteogenic Protein-1 via the Synergistic Upregulation of Molecular Markers

Members of the transforming growth factor-β (TGF-β) superfamily of proteins, the bone morphogenetic proteins (BMPs) and the TGF-β isoforms, are involved in the coordination of cartilage and bone differentiation both in embryonic development and in postnatal life. Both osteogenic protein-1 (OP-1) and TGF-β1 have been shown to be potent regulators and inducers of heterotopic endochondral bone induction in non-human primates. In marked contrast, TGF-β1 does not induce heterotopic endochondral bone in rodents. In the primate, the osteogenic properties of OP-1 are synergistically enhanced by the combined administration of TGF-β1. The binary application of OP-1 (0.1, 0.3, 1.0 and 3.0μg) and TGF-β1 (0.01, 0.03 and 0.1 μg) to 25 mg of guanidinium-inactivated insoluble collagenous bone matrix as carrier in the rodent heterotopic bioassay for 7, 12 and 21 days resulted in a classical synergistic, dose-dependent and temporal up-regulation of OP-1-induced endochondral bone formation. There were significant increases in alkaline phosphatase activity (day 12) and calcium content (days 12 and 21). mRNA expression of OP-1, TGF-β1, BMP-3 and collagens type II and IV, markers of bone formation, showed an up-regulation of the genes (days 12 and 21) by the binary applications of the morphogens. Histologically, single applications of OP-1 elicited a dose dependent induction of endochondral bone formation while the binary applications resulted in a temporal acceleration of the morphogenetic cascade. The optimal ratio of OP-1/TGF-β1 was 30:1 by weight for endochondral bone formation and expression of molecular markers. The present data provides insights to the mechanisms of synergistic molecular therapeutics for endochondral bone formation in clinical contexts.

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