Biochemical and histological sequences of membranous ossification in ectopic site

Porous hydroxyapatite ceramics alone (control) and ceramics combined with rat marrow cells were implanted subcutaneously in the back of syngeneic rats and harvested 1-8 weeks after implantation. The ceramics were examined biochemically and histologically. Alkaline phosphatase activity in the marrow cell/ceramic composites began to increase at 2 weeks and achieved a peak at 4 weeks, followed by a gradual decrease. Bone gla protein contents in the composites began to increase at 3 weeks and steadily increased as time passed. Histologically, osteoblastic cells were detected at 2 weeks and obvious de novo bone together with active osteoblasts began to appear at 3 weeks in the composites. The process was membranous ossification without cartilage formation and was observed in the pores of the composites. The pore areas occupied by bone increased as time passed. In contrast, ceramics alone did not show any bone formation and contained traces of these biochemical parameters. These results indicated that the biochemical sequences correlate with the histological sequences in the heterotopic membranous ossification.