A quantitative assessment of osteoinductivity of human demineralized bone matrix

Abstract

Demineralized bone matrix (DBM) is widely used in the repair of pathologies associated with skeletal defects and periodontal diseases. The present study was directed at establishing in vivo and in vitro models for a quantitative assessment of the osteoinductivity of DBM before clinical use. Athymic mice were used in an in vivo assay to overcome the species limitations (for human DBM) found in xenogeneic animal models. Calcium contents of explants, as an indicator of new bone formation, were assayed and expressed as a change in the weight percent calcium in the explant as compared to the weight percent of calcium in the implanted material. A total of 82 mice (2 implants per mouse) were used in this study. Significant amounts of new bone were induced in this animal model in response to implantation of DBM. Muscular implantation was found to be more osteoinductive (increases of 10.0 +/- 0.4 calcium weight percent of explant) than subcutaneous implantation (increases of 1.62 +/- 0.27 calcium weight percent of explant) and new bone formation in muscular implantation sites of athymic mice mimics endochondral bone formation. Between weeks 1 to 4, the weight of explanted materials did not significantly differ from the weight of the implanted material; however, by week 5 the explant weight began to increase. Calcium deposition over the 5 weeks of implantation increased in a nearly linear fashion. Consequently week 4 was chosen as the optimum time for explantation in the in vivo assay in that sufficient calcium levels had been achieved without a significant increase in explant dry weight. Aliquots of 10, 20, 30, and 40 mg per implantation site were used in dose response studies in the in vivo bioassay. Dose response curves with DBM exhibited maximal activity at the 20 mg DBM implant dose in the in vivo bioassay. An in vitro bioassay was also developed where human periosteal (HPO) cells were chosen because osteoprogenitor cells found in bone repair typically come from periosteal tissue. Alkaline phosphatase (ALP) activity in confluent cell cultures of HPO cells exposed to DBM, as an indicator of osteoblast induction, reached its highest level on day 5 of DBM treatment. Aliquots of 2, 5, 10, 20, 30, and 40 mg DBM per flask were chosen in dose response studies using the in vitro bioassay. These dose response studies with DBM revealed that quantities approximating 5 to 10 mg DBM in the in vitro model provided for maximal levels of ALP in cell extracts. A linear correlation (R2 = 0.7397) was demonstrated between the in vivo calcium remineralization assay and the in vitro ALP assay of osteoinductivity of DBM, suggesting that the in vitro assay can be used to quantitatively assess the osteoinductive potential of DBM where production and distribution of clinically usable DBM dictates rapid analysis.