The broad, long-term objective of our research is to develop a new technology that addresses two of the primary limitations of existing bone replacement therapeutics by: (1) improving the low osteoinductive capacity of SBGS (synthetic bone graft substitutes);and (2) developing an improved, compression-resistant biomimetic synthetic bone graft substitute that contains the osteogenic growth factor BMP-2 (bone morphogenetic protein-2) plus cBBP (cyclic BMP binding peptide: a novel cyclic, synthetic peptide that acts as a high-affinity pro-osteogenic BMP-binding carrier) for use in applications such as posterolateral spine fusion, where mechanical stability and resistance to compression by the surrounding muscular tissue are essential. BMP-2/-7 are osteogenic cytokines that stimulate pluripotent mesenchymal cells to proliferate and differentiate into the chondrocytes and osteoblasts that elaborate new cartilage and bone. Recombinant human BMP-2 (rhBMP-2) and rhBMP-7 with col I (type I collagen) carrier are FDA-approved for use in orthopedic surgery, but BMP-2 and -7 are expensive, water-soluble, and exhibit short retention times at the sites where they are implanted. cBBP binds rhBMP-2 and -7 with high affinity, reduces [125I]-rhBMP-2 loss from 59% to 17% at 24 hr, and reduces the total amounts of rhBMP-2/-7 required to stimulate significant ectopic bone formation or achieve successful spinal fusion or femoral bone defect healing in vivo. cBBP reduces the amount of rhBMP-2 required for rat spinal fusion by 90% and to heal critical-sized femoral defects in the rat by >70%. We now propose to identify the mechanisms by which cBBP enhances BMP retention and to extend our successful rodent studies to the dog, an essential step in preclinical testing. In addition to binding rhBMP-2 and -7 with high affinity and specificity, cBBP is incompletely soluble in aqueous solutions at physiological pH. We hypothesize that cBBP enhances BMP retention, in part, by binding to BMPs with high affinity and forming insoluble aggregates or complexes that retard BMP diffusion out of the tissue.
In Specific Aim 1, we will test this hypothesis by elucidating the biochemical and biophysical mechanisms that account for cBBP-enhanced retention of BMP-2. The collagen binding, solubility, and aggregation properties of cBBP and rhBMP-2, alone and in combination, will be assessed analytically by solid phase binding assay, filtration/centrifugation/BMP ELISA, nephelometry, analytical ultracentrifugation, FTIR spectroscopy, and light scattering. We also hypothesize that the kinetics of cBBP binding to BMP-2 is the single most important determinant of the effects of cBBP on BMP-2-stimulated osteogenesis.
In Specific Aim 2, we will test this hypothesis by modifying the amino acid sequence of cBBP to maximize rhBMP-2 binding and assess the effects of these modifications on retention of [125I]-rhBMP-2 and BMP-2-stimulated osteoinduction in vivo. Binding of cBBP analogs will be assessed by surface plasmon resonance. Retention will be assessed pharmacokinetically. cBBP analog-enhanced BMP-2- stimulated osteoinduction in the murine ectopic bone forming bioassay will be assessed by densitometry, histomorphometry, and mCT scanning. The mechanistic insights gained in Specific Aims 1 and 2 will be used (if possible) to develop a cBBP analog that enhances more BMP-2-stimulated osteogenesis than native cBBP. This analog, rather than native cBBP, will be used in Specific Aims 3 and 4 (if available).
In Specific Aim 3, we will optimize the cBBP (or cBBP analog): BMP-2 ratios required for canine lumbar spinal fusion using the FDA-approved ACS (absorbable collagen sponge) carrier and low-dose BMP-2. Single level L4-L5 lumbar fusion will be assessed by palpation, radiography, mCT scanning, biomechanics, and histology in dogs treated with vehicle or graded doses of cBBP analog alone, rhBMP-2 alone, or cBBP + rhBMP-2.
In Specific Aim 4, we will test a resorbable, compression-resistant scaffold (sintered bone + col I) with cBBP and low-dose rhBMP-2 in the canine lumbar spinal fusion model, with testing as outlined above.
Impaired bone healing affects many patients with spinal fusions, compound fractures, non-unions, and large osseous defects. Autogenous bone grafts and banked bone can restore form and function, but the failure and complication rates can be high. BMPs (bone morphogenetic proteins) are used to enhance repair, but their use is limited by high cost, poor retention at the site of implantation, and side effects, such as swelling and difficulty swallowing if implanted in the cervical spine. We have developed an inexpensive material (cBBP or cyclic BMP binding peptide) that binds BMPs, increases their retention in tissue, and reduces the amount of BMP required to fuse the spine and heal critical-sized defects in long bones by 90%. The proposed studies could lead to the development of cBBP as a new BMP carrier for synthetic bone graft substitutes that reduces health care costs and improves treatment outcomes for orthopedic surgery patients, particularly recent combat veterans with skull and extremity wounds who are treated at VA facilities.