ANALYSIS OF OSTEOBLAST ACTIVITY AT BIOMATERIAL-BONE INTERFACES BY IN-SITU HYBRIDIZATION

To investigate the effects of bioactive materials on bone formation in vivo, a new experimental model using in situ hybridization has been d eveloped. A hole was drilled bilaterally in the distal epiphysis of ra bbit femurs with subsequent implantations of beta-tricalcium phosphate (beta-TCP) cylinders in a press-fit manner. Specimens were collected at 3, 7, 14, and 28 days after operation. Femurs with empty drilling h oles, and normal distal femurs without operation were used as controls . All specimens were decalcified and hybridized with a procollagen alp ha 1(I) complementary RNA probe labeled with digoxygenin. In normal-bo ne sections, procollagen alpha 1(I) RNA was clearly demonstrated in pe riosteal osteoblasts, in osteoblasts in the mineralizing zone adjacent to growth plates, and in osteoblasts lining remodeling canals. As for beta-TCP, labeled osteoblasts around the material were not found at d ay 3, whereas they were most intensively observed at day 7 and a littl e less at day 14, in accordance with new-bone formation around the mat erial. Weaker signals were also detected in fibroblasts at day 7. At d ay 28, osteoblasts lining the surface of newly formed bone were mainly negative, whereas those adjacent to the resorption sites of the beta- TCP showed positive signals, demonstrating an active remodeling at the material surface. The temporal expression of procollagen alpha 1(I) R NA in the beta-TCP specimens was fundamentally the same as that in the empty-hole specimens, suggesting no remarkable acceleration or suppre ssion of bone-forming activity of osteoblasts by beta-TCP, which is co nsistent with osteoconductive bone formation. This in situ hybridizati on method was suggested to be a powerful tool in analyzing the biologi cal effects of bioactive materials. (C) 1996 John Wiley & Sons, Inc.