Date of Award

Spring 1996

Document Type


Degree Name

Doctor of Philosophy (PhD)


Biomedical Sciences

Committee Director

Lloyd Wolfinbarger, Jr.

Committee Member

James Yuan

Committee Member

Mickey Castle

Committee Member

R. James Swanson

Committee Member

Ralph M. Powers, Jr.


The osteoinductive potential (osteoinductivity) is a very important aspect of demineralized bone matrix (DBM) as a biomaterial. This study is directed at establishing qualitative and quantitative assays of the osteoinductivity of human DBM. Further aspects of this study include an assessment of the effects of the production process, matrix characterization, and donor related factors on osteoinductivity of the DBM.

Athymic mice were chosen as the animal model for use in the in vivo bioassay. Implantation of DBM in muscle pouches provided for much higher osteoinductivity (remineralization to 10% calcium) than subcutaneous site implantation (remineralization to 1.6% calcium). Histological observations revealed that new bone and bone marrow formed after four weeks of intramuscular implantation. In the in vivo dose-response studies, 20 mg of DBM provided for the highest levels of osteoinductivity.

Human periosteal cells (HPO/CB-MZ01) were chosen for the in vitro bioassays of the osteoinductivity of DBM. Both biochemical and histochemical studies revealed that the DBM can induce significantly higher alkaline phosphatase (ALP) activities when compared to DBM-free control flasks after four days of incubation. In the in vitro dose-response studies 5 and 10 mg of DBM produced the highest osteoinductivity.

The study of the effect(s) of residual calcium on osteoinductivity revealed that nondemineralized bone matrix lacked significant osteoinductivity. As the residual calcium content decreased, the degree of remineralization produced by the bone matrix increased, reaching maximum remineralization when the residual calcium content of bone matrix approached 2%. At residual calcium levels below 2%, osteoinductivity of the DBM decreased. The study of the effect of particle size revealed that DBM particles less than 250 micron produced the lowest osteoinductivity among all size groups studied. Bone particles in the 500-710 micron size range provided for DBM with the highest osteoinductivity. The effect of donor age on osteoinductivity of DBM revealed that demineralized bone matrix from 16 to 60 year old donors provided for nearly equivalent levels of remineralization, all in excess of 7% calcium in DBM explants.

In summary, this study established and validated the in vivo and in vitro bioassays for the assessment of osteoinductivity of DBM. Further, the study investigated the relationships between osteoinductivity and the residual calcium content in the DBM, the relationship between the osteoinductivity and particle size, and the relationship between the osteoinductivity and donor age and gender. The study validated the biochemical assays used for the assessment of the osteoinductivity of DBM. The results provided scientific evidence for the standards to be applied to the bone demineralization process and donor.


A Dissertation Submitted to the Faculty of Old Dominion University and Eastern Virginia Medical School in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.