Healing Large Segmental Defects in Dogs and Sheep with Recombinant Human Bone Morphogenetic Protein (rhBMP-2)
Authors: Wang Elizabeth A. , Toriumi D. M. , Gerhart T. N. 
Published: March 2011

Human recombinant BMP-2 is known to induce cartilage and bone in a rat subcutaneous site. Here we demonstrate that BMP-2 can induce therapeutic bony union in segmental bone defects in three different animal models: internally fixed defects in sheep femur (2.5 cm) and dog mandible (3.0 cm). In each model, inactive bone matrix from the same species as the test animal was prepared by acid demineralization and extraction in guanidinium chloride. Human recombinant BMP-2 was reconstituted with the bone matrix and inserted in the defect site. Inactive matrix served as the negative control. Healing was monitored weekly by x-ray, At the final time points, specimens were evaluated for union by biomechanical testing and for bone formation by histology. In the first model, a 3 cm mandibular defect in dogs was made and stabilized with a stainless steel reconstruction plate. Animals were terminated at 3 and 6 months. In the second model, an osteoperiosteal defect was created by excising a 2.5 cm midshaft segment from the femur of a skeletally mature sheep. The gap was stabilized by an anterolateral fixation plate. Animals were sacrificed at 12 weeks postoperation. In both studies, BMP-2 mixed with inactive carrier induced bony union judged by radiography, biomechanical testing, and histology; by the same criteria, the defect implanted with the carrier matrix alone did not unite.

The ability of demineralized bone to induce cartilage and bone formation in ectopic sites was described by Urist [1]; this activity was named bone morphogenetic protein (BMP). The in vivo progression of demineralized bone to bone has been well-studied [1,2]. After initial proliferation of mesenchymal cells in the first five days after implantation, a cartilage model forms at days 5–10. The cartilage mineralizes and hypertrophies, and is gradually replaced by bone. The newly formed bone then continues to remodel, so that in rats a thin ossicle of bone surrounding bone marrow has formed by day 21. The therapeutic potential of a material which could induce bone formation and replace bone graft is very high, and demineralized bone or partially purified protein prepared from demineralized bone has been used to repair large bony defects in animal models and in human therapy.

More recently, some of the individual components of the bone morphogenetic activity have been isolated and characterized from bovine bone [3]. Extensive biochemical purification and molecular cloning have demonstrated that there are at least 7 distinct proteins present in bone. Molecular cloning of the bone morphogenetic proteins (BMPs) 2 through 7 demonstrated that they are related and all are members of the transforming growth factor/inhibin family of genes [4,5,6]. There may also be additional factors involved in cartilage and bone induction, as other investigators have described components different from these [7,8]. Expression of the recombinant human BMP-2, BMP-3, BMP-4, BMP-5, and BMP-7 in mammalian cells, followed by purification, allowed the demonstration that an individual BMP is sufficient to induce cartilage and bone in an ectopic site. A detailed study in rats of BMP-2-induced bone formation showed that the progression seen with the recombinant protein is the same as observed with demineralized bone or highly purified bone-derived, bone-inductive proteins [9].

With this background of rhBMP-2-induced ectopic bone formation, this study demonstrates the efficacy of BMP-2 in healing a bony defect. Using allogeneic bone as a matrix, rhBMP-2 shows efficacy in healing large defects in dog mandible and sheep femur.