Effects of hydroxyapatite implants on primary mineralization during rat tibial healing: biochemical and morphometric analyses.

The effect of 40- to 60-mesh hydroxyapatite (HA) granules (Calcitek, Inc., Carlsbad, CA) on the process of primary mineralization during bone healing was examined following insertion of the HA granules into rat tibial bone after marrow ablation. Response to HA was assessed by monitoring morphometric and biochemical changes in matrix vesicles, which are extracellular organelles associated with initial calcification. Following insertion of HA, matrix vesicle-enriched membranes (MVEMs) were isolated from the tissue adjacent to the implant and from the endosteum of the contralateral limb at 3, 6, 14, and 21 days and from a nonimplanted control group (t = 0). MVEM alkaline phosphatase- and phospholipase A2-specific activities were increased on days 6 (peak) and 14; phosphatidylserine content was also elevated on days 6 and 14 (peak). Comparable changes were seen in the contralateral limb but at lesser magnitudes. Morphological changes were observed as well. The number of matrix vesicles/micron2 matrix increased on days 6 (peak) and 14. The mean diameter of the matrix vesicles was elevated on days 6 (peak), 14, and 21. Mean distance from the calcifying front increased on day 6 but was decreased on days 14 and 21. These results indicated that HA behaves like bone-bonding implants in that there is a stimulation of matrix vesicle enzymes, increased phosphatidylserine content, and increase numbers of matrix vesicles. However, the increases occur only after 6 days postimplantation, indicating a delay in response when compared to normal healing. This delay is confirmed by the morphometric measurements. HA causes a reduction in the response associated with marrow ablation. In addition, the effects of HA are comparable locally and systemically but with different intensity. These observations suggest that osteogenic cells are able to compensate for the inhibitory effects of HA and primary calcification involves normal matrix vesicle production and maturation, if somewhat delayed and reduced in magnitude. The ability to support primary mineral formation may contribute to the successful bonding of HA with surrounding osseous tissue.