Demineralized bone matrix (DBM) is a well-established osteoinductive bone material derived from pulverized cortical bone specimens. To develop an implantable product, acellular particles are added to a substrate such as glycerol or a biodegradable polymer, resulting in an osteoinductive bone graft material. DBM has been used successfully as bone-graft substitute material in the treatment of many periodontal procedures, in craniofacial defect repairs, and also in orthopedic and other types of surgery. In this clinical study, DBM was used as the graft material to help complete the alveolar bone healing before placement of a dental implant. To further analyze the amount of new bone growth versus the residual DBM in the site, histological serial sections were prepared of each bone core removed from patients, and histomorphometry was performed on the sections stained using Methylene Blue/Basic Fuchsin (hematoxylin and eosin-like stain) and von Kossa stains. We found that von Kossa staining offered opportunities for the identification of specimen components when compared with the hematoxylin and eosin-like stain for identifying the DBM versus new bone. The von Kossa stains the DBM biomaterial light brown, whereas it stains the phosphate ion of mineralized bone black. This staining creates an excellent contrast to a transparent background, facilitating quantification with the use of image analysis software on the basis of color. Furthermore, the combined use of von Kossa staining and polarized light created much more consistent results for the histomorphometric analysis through considerably lower intraobserver variability.
