The Quantitative Assessment of Osteoinductivity of Human Demineralized Bone Matrix and cDNA Array Analysis of Osteogenic Differentiation in Human Periosteal Cells

<p>Demineralized bone matrix (DBM) has been extensively utilized in orthopeadic, periodontal, and maxillofacial applications. DBM comprises bone morphogenetic proteins (BMPs) that are essential regulators for endochondral bone formation. The current study was undertaken to establish the quantitative <em>in vitro</em> assays for determining the osteoinductive potential of human DBM and the use of cDNA gene expression array technology for examining gene expression profiles during osteoblast differentiation in human periosteal cells.</p> <p>BMP-4 is one of the most osteoinductive factors and has been investigated for clinical applications. For this reason, BMP-4 was designated as the osteoinductive protein for use in the quantitative <em>in vitro</em> assay. At the same time, the osteoinductivity of DBM was measured by histomorphometric analysis of new bone formation in an <em>in vivo</em> athymic mouse bioassay. DBM exhibiting high osteoinductivity in the nude mouse bioassay contained higher amounts of extractable BMP-4 than did DBM samples possessing low osteoinductivity.</p> <p>In the study of the effect of residual calcium on extractability of BMP-4, the extractable BMP-4 content was undetectable in nondemineralized bone matrix. As the residual calcium level decreased, the extractability of BMP-4 in DBM increased. DBM particles less than 250 microns yielded the lowest amounts of extractable BMP-4 among all size groups tested. Bone particles in the 500–710 micron size range provided for DBM with the highest extractability of BMP-4. In the donor age study, the BMP-4 content in the protein extracts of DBM appears to be age-dependent, with DBM from younger donors being most likely to have greater BMP-4 quantity.</p> <p>In the <em>in vitro</em> dose-response studies, DBM-conditioned media (DBM-CM) enhanced alkaline phosphatase activities of human periosteal cells in a dose-dependent fashion. Alkaline phosphatase activities in both biochemical and histochemical analysis increased in the DBM-CM treated flasks compared to non-treated flasks after five days of incubation. In gene expression analysis of osteogenic differentiation using cDNA array analysis, a few differentially expressed genes, including biglycan, TGF-β1, and TGF-βR1 were up-regulated, whereas collagen14A1 was down-regulated in response to DBM-CM treatment. In the present study, gene expression array technology provides insight to the biological process of osteogenic differentiation.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Year dc:date.available

2003

Author dc:creator

• Honsawek, Sittisak

Contributors dc:contributor

• Lloyd Wolfinbarger
• Christopher J. Osgood
• Patricia A. Pleban
• Ralph M. Powers, Jr.

• BMP-4
• Demineralized bone matrix
• Osteoinductivity
• Cell Biology
• Molecular Biology

Identifier

9780496385331

OAI identifier oai:identifier

oai:digitalcommons.odu.edu:biomedicalsciences_etds-1041