Ultrastructural Observation of Calcification Preceding New Bone Formation Induced by Demineralized Bone Matrix Gelatin

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Abstract
Demineralized bone matrix gelatin (BMG) was implanted into the skeletal muscle of Sprague-Dawley rats, and the resulting ultrastructural changes of the BMG were examined 3, 5, 7, 10, 15 and 20 days later. Most of the implanted BMG became calcified 7–20 days after implantation. Calcification (‘acellular mineral deposition’) was first observed as needle-shaped crystalline deposits in the BMG matrix on day 7 after implantation, which gradually increased in size and fused with one another in some deposits. They appeared to be divided into small partitions consisting of denatured collagen fibers unlike those of noncalcified BMG. Some deposits had electron-lucent areas in their center adjacent to well-calcified peripheral areas, and the central area contained many collagenlike fibers and spherical vacuoles. Osteoblast-like cells were not present around these calcified deposits 7 days after implantation. After that, new bone formation was often seen near the area of acellular mineral deposition, and the fused calcified deposits remained until day 15 after implantation. The noncalcified BMG was mainly absorbed by macrophages, and the sites of acellular mineral deposition were absorbed by multinucleated cells resembling osteoclasts which were considered to be activated by the implants. Acellular mineral deposition probably started as calcium and phosphate deposits on some materials in the BMG matrix, and by heterogenic nucleation without osteoblasts or matrix vesicles, inducing bone formation. Thus the BMG may be not only a carrier of bone morphogenetic protein, gradually supplying it to the surrounding tissue, but also a storage site for minerals that are indispensable for bone formation. Furthermore, these osteoblasts may be activated by osteoclast-like cells that absorb minerals from sites of acellular mineral deposition.

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