Demineralization of allograft bone increases the bioavailability of matrix-associated bone morphogenetic proteins (BMPs), rendering these grafts osteoinductive. While osteoinductivity is related to BMP content, little is known about how the demineralization protocol, in particular, extended demineralization times, affects graft BMP levels. We characterized the BMP-7 content of <710 μm bovine bone powder demineralized under various conditions. Using 1 g of bone per 50 ml of 0.125 N, 0.25 N, or 0.5 N HCl, demineralization was performed at room temperature for 5 min to 24 h. Minimum residual calcium levels were obtained within 90 min and were <1 wt % using the 0.25 N and 0.5 N baths and 17 wt % using the 0.125 N bath. Measured peak BMP-7 levels were also obtained within 90 min and were 161-165 ng g(-1) using the 0.25 N and 0.5 N baths and 55.2 ng g(-1) using the 0.125 N bath. This compares to 5.1 ng g(-1) for undemineralized bone. Further acid bath exposure to 24 h resulted in BMP-7 decline to about 50% of the peak value, which was significant. The BMP-7 half-life was estimated to be 26 h. It is likely that the decline was due to diffusion of BMP-7 from the bone matrix into the acid. These results suggest the importance of not over demineralizing bone grafts and should stimulate further research that can be incorporated into the processing methodology followed by tissue banks.

Bone morphogenetic proteins (BMPs) are associated with bone extracellular matrix and impart osteoinductive properties to demineralized bone matrix (DBM) grafts. The first step of the osteoinductive process is BMP release from DBM in situ; however, this has not been characterized for human DBM. The authors investigated the release of BMPs 2, 4, and 7 from a clinical human DBM putty (Bonus II DBM, Biomet Inc, Warsaw, IN). The DBM was placed in Sorensen buffer and the BMP concentrations in the Sorensen buffer and guanidine extracts of the DBM were measured concurrently by enzymelinked immunosorbant assay for up to 7 days. The baseline DBM concentrations were BMP-2: 28.1 ± 1.3 ng/g DBM, BMP-4: 0.577 ± 0.056 ng/g DBM, and BMP-7: 92.9 ± 7.5 ng/g DBM. Relative to baseline, the proportions released by 7 days were 11.1%, 3.9%, and 29.3%, respectively. The early (0-8 hour) and late (8-168 hours) elution rates were BMP-2: 0.16 ± 0.24 and 0.0089 ± 0.012 ng/(g DBM hr), and BMP-7: 1.29 ± 2.1 and 0.086 ± 0.039 ng/(g DBM hr), respectively. Little BMP-4 elution occurred over the first 24 hours, with the rate for the remaining interval being 0.00014 ± 0.00021 ng/(g DBM hr). The apparent DBM BMP profiles were counterintuitive in that the concentrations increased from baseline for some, or all, of the 7 days instead of monotonically decreasing. Similar behavior has previously been reported in bovine studies. This provides further evidence that BMPs are associated with at least 2 compartments in DBM differing by their affinity for BMPs and that guanidine extraction of BMPs is not 100% efficient.

Bone morphogenetic proteins (BMPs), present in parts per billion in bone, endow demineralized bone matrix (DBM) with osteoinductive properties suitable for clinical use. Although BMPs are mainly associated with bone matrix, they also associate with other bone compartments as well, including the mineral phase. The purpose of this study was to gain a more complete understanding of the distribution of BMPs in undemineralized bone. Eleven discrete particle size ranges of bovine cortical bone were prepared, ranging between less than 25 μm and 600 to 710 μm for the smallest and largest sizes, respectively. The bone was extracted with 4-M guanidine-HCl/0.05-M Tris-HCl, and the amount of BMP-7 released was measured with enzyme-linked immunosorbant assay. In addition, 106- to 710-μm bone particles were demineralized and similarly extracted for comparison. The measured BMP-7 content of the DBM was 24.6 ± 1.56 ng/g. The values for bone increased nonlinearly with decreasing particle size, ranging from 1.13 ± 0.50 ng/g for the 600- to 710-μm particles to 4.18 ± 1.14 ng/g for the less than 25-μm particles (P < 0.001). However, modeling the bone particles as solid spheres to estimate total surface area showed that the extracted BMP-7 per unit area was greater for larger particle sizes. These seemingly opposing results suggest that BMPs may become proportionally damaged or altered in response to the increased forces required to generate smaller particles and, as such, may not be detectable with enzyme-linked immunosorbant assay. In addition, minimization of bone particle size is not an effective strategy to approach the BMP availability of DBM.