Marjorie M. Tucker

Complete Regeneration of Bone in the Baboon by Recombinant Human Osteogenic Protein-1 (hOP-1, Bone Morphogenetic Protein-7)

We examined the efficacy of a single application of recombinant human osteogenic protein-1 (hOP-1, bone morphogenetic protein-7) for its ability to regenerate large calvarial defects in adult male baboons (Papio ursinus). Recombinant hOP-1, in conjunction with baboon or bovine guanidinium-extracted insoluble collagenous bone matrix (0.1, 0.5 and 2.5 mg per g of collagenous matrix as carrier), was implanted in 46 calvarial defects surgically prepared in 14 baboons, whilst 18 defects were implanted with the carrier matrix without hOP-1. Specimens were harvested on d 15, 30, 90 and 365 and subjected to histomorphometry on serial undecalcified sections cut at 7 microm to study the temporal sequence of tissue morphogenesis after the single application of hOP-1. Histological analysis indicated that the induction of new bone formation proceeded from the periphery to the central core of hOP-1 treated specimens after rapid angiogenesis and mesenchymal cell migration in apposition to the collagenous matrix. Whilst chondrogenesis was limited, newly formed bone has already filled with fully differentiated bone marrow elements as early as d 15, even with the 0.1 mg dose of hOP-1. On d 30 and 90, doses of 0.1 and 0.5 mg of hOP-1 showed greater amounts of bone than controls, and on d 90, they induced complete regeneration of the defects. Doses of 2.5 mg hOP-1 per g of matrix induced extensive osteogenesis initially with heterotopic ossification and displacement of the temporalis muscle above the defects. One year after implantation of hOP-1 there was restoration of the internal and external cortices of the calvaria. These results show that hOP-1 induces complete regeneration of calvarial bone in the adult primate, and suggest that the optimal activity of hOP-1 to achieve regeneration is between 100 and 500 microg of hOP-1 per g of matrix. These results in the primate may form the scientific basis for future clinical applications of hOP-1.

Long-Term Evaluation of Bone Formation by Osteogenic Protein 1 in the Baboon and Relative Efficacy of Bone- Derived Bone Morphogenetic Proteins Delivered by Irradiated Xenogeneic Collagenous Matrices

To investigate the long‐term efficacy of irradiated recombinant human osteogenic protein 1 (hOP‐1) in bone regeneration and morphogenesis, hOP‐1 was combined with a bovine collagenous matrix carrier (0, 0.1, 0.5, and 2.5 mg hOP‐1/g of matrix), sterilized with 2.5 Mrads of γ‐irradiation, and implanted in 80 calvarial defects in 20 adult baboons (Papio ursinus). The relative efficacy of partially purified bone‐derived baboon bone morphogenetic proteins (BMPs), known to contain several osteogenic proteins, was compared with the recombinant hOP‐1 device in an additional four baboons. Histology and histomorphometry on serial undecalcified sections prepared from the specimens harvested on day 90 and day 365 showed that γ‐irradiated hOP‐1 devices induced regeneration of the calvarial defects by day 90, although with reduced bone area compared with a previous published series of calvarial defects treated with nonirradiated hOP‐1 devices. One year after application of the irradiated hOP‐1 devices, bone and osteoid volumes and generated bone tissue areas were comparable with nonirradiated hOP‐1 specimens. Moreover, 365 days after healing regenerates induced by 0.5 mg and 2.5 mg of irradiated hOP‐1 devices showed greater amounts of bone and osteoid volumes when compared with those induced by nonirradiated hOP‐1 devices. On day 90, defects treated with 0.1 mg and 0.5 mg of bone‐derived baboon BMPs, combined with irradiated matrix, showed significantly less bone compared with defects receiving irradiated devices containing 0.1 mg and 0.5 mg hOP‐1; 2.5 mg of partially purified BMPs induced bone and osteoid volumes comparable with the 0.1‐mg and 0.5‐mg hOP‐1 devices. Control specimens of γ‐irradiated collagenous matrix without hOP‐1 displayed a nearly 2‐fold reduction in osteoconductive bone repair when compared with nonirradiated controls. These findings suggest that the reduction in bone volume and bone tissue area on day 90 may be caused by a reduced performance of the irradiated collagenous matrix substratum rather than to a reduction in the biological activity of the irradiated recombinant osteogenic protein. This is supported by the results of in vitro and in vivo studies performed to determine the structural integrity of the recovered γ‐irradiated hOP‐1 before application in the baboon. Recoveries by high‐performance liquid chromatography (HPLC) and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE)/immunoblot analyses indicated that doses of 2.5‐3 Mrads of γ‐irradiation did not significantly affect the structural integrity of the recovered hOP‐1. Biological activity of the recovered hOP‐1 was confirmed in vitro by showing induction of alkaline phosphatase activity in rat osteosarcoma cells (ROS) and in vivo by de novo endochondral bone formation in the subcutaneous space of the rat. These findings in the adult primate indicate that a single application of γ‐irradiated hOP‐1 combined with the irradiated xenogeneic bovine collagenous matrix carrier is effective in regenerating and maintaining the architecture of the induced bone at doses of 0.5 mg/g and 2.5 mg/g of carrier matrix.

Recombinant human osteogenic protein-1 induces dentin formation: An experimental study in miniature swine

It was the aim of the present study to investigate the induction of dentin formation by recombinant human osteogenic protein-1 (hOP-1). In 4 adult miniature pigs a total of 16 teeth with artificially exposed dental pulps were capped with 3 mg of a complex of recombinant hOP-1 in collagen matrix (2.5 micrograms/mg), collagen matrix alone, or calcium hydroxide paste. Teeth were removed in block section after a healing period of 5 weeks. Decalcified sections were processed for light microscopy and histomorphometric analysis. In hOP-1 treated teeth substantial amounts of hard tissue formation (osteodentin and tubular dentin) had consistently led to a complete bridging of the defects. Less dentin formation was seen after calcium hydroxide application. In control defects collagen matrix alone failed to form complete dentin bridges. Recombinant human osteogenic protein-1 in a collagen carrier matrix appeared to be suitable as a bio-active capping agent for surgically exposed dental pulps.