The Effect of Residual Calcium in Decalcified Freeze-dried Bone Allograft in a Critical-sized Defect in the Rattus norvegicus Calvarium
Demineralized freeze-dried bone allograft (DFDBA), a widely used graft material in periodontal regenerative procedures, is processed with hydrochloric acid in the attempt to expose proteins located within the bone matrixes that are capable of inducing new bone formation. However, the degree of DFDBA demineralization varies between tissue banks, which may have an effect on clinical regeneration. This study uses the critical-sized defect (CSD) model to evaluate the wound-healing response to the residual calcium of donor bone. If the percentage of residual calcium in a graft were demonstrated to significantly enhance wound healing, then periodontal patients may benefit from further standardization of human-allograft processing. Sixty adult, male, Harlan Sprague-Dawley rats (Rattus norvegicus) were randomly and equally divided into 4 test groups (ie, DFDBA at 1%, 2%, and 3% to 6% residual calcium levels and FDBA at 23% residual calcium) and a control group (no allograft). An 8-mm-diameter craniotomy was made in the rat calvarium, and polytetrafluoroethylene membranes with pore sizes of 0.50 μm were placed intracranially and ectocranially. Treatment materials were carefully placed into the CSD with a new sterilized dental amalgam carrier. Tetracycline hydrochloride was injected intraperitoneally for labeling new bone growth, and animals were euthanized 12 weeks postsurgery. As a result, histomorphometric bone fill at 12 weeks showed a statistically significant increase in the 2% DFDBA group as compared to all other groups. The authors conclude that a 2% residual calcium level in human DFDBA appears to significantly (P ≤ .05) enhance osseous wound healing in the rat calvarium.Abstract
Decalcified hematoxylin and eosin histology of the critical-sized defect: control group. New vital bone (VB; original magnification ×125). High magnification of the central area of the defect showing a large bone island and bone on both sides of the membrane (M). Figure 2. Decalcified hematoxylin and eosin histology of the critical-sized defect: 1% demineralized freeze-dried bone allograft (DFDBA). Central area of the defect (original magnification ×31.25). The center of the defect showing decalcified bone particles (DBPs; black arrow) surrounded by connective tissue (yellow arrows), the edges consist of solid bone with an amalgamation of DBPs and new bone (green arrow). Bone linking is not as evident as with 2% DFDBA (Figure 3).Figure 3. Decalcified hematoxylin and eosin histology of the critical-sized defect (CSD): 2% demineralized freeze-dried bone allograft. Center of CSD (original magnification ×31.25). Center of defect showing an amalgamation of the decalcified bone particles and the new vital bone that is forming a linked bridge over the CSD. New haversian canals that indicate maturation (green arrow) and the linking of new vital bone and decalcified-bone particle can be appreciated (black arrows).Figure 4. Decalcified hematoxylin and eosin histology of the critical-sized defect: 3% to 6% demineralized freeze-dried bone allograft (DFDBA). Central area of the defect (original magnification ×31.25). The center of the defect shows an abundance of connective tissue compared to the 2% DFDBA group. Minimal haversian systems are seen indicating lack of complete maturation.Figure 5. Decalcified hematoxylin and eosin histology of the critical-sized defect (CSD): freeze-dried bone allograft. Central area of the CSD (original magnification ×31.25). Center of defect shows unresorbed freeze-dried bone particle (FDBP) surrounded by an abundance of connective tissue (CT). Minimal new bone formation is evident within the CSD.Figure 6. Undecalcified Modified Masson's trichrome histology of the critical-sized defect 2% demineralized freeze-dried bone allograft group (original magnification ×12.50). Arrows indicate amalgamation of decalcified bone particle and new bone.
Mean percent total area of bone. The area fraction (Aa) of regenerated bone within the defects was statistically significant compared to the various groups. The dashed line indicates no statistical difference (P ≥ .05) in the Aa between the 1% demineralized freeze-dried bone allograft (DFDBA), 3% to 6% DFDBA, and the control group, but the 2% DFDBA group showed statistically more vital bone compared to all other groups (P ≤ .05). The height of the bar graph indicates the mean; *, statistical significance.
Densitometric view of all 5 treatment groups (1024 × 1024 dpi). Representative sections from each of the 5 treatment groups are shown. The arrow indicates a more uniform mass of new bone with the 2% demineralized freeze-dried bone allograft.
Tetracycline hydrochloride labeling of new bone: control group. Representative photomicrograph from the control group (original magnification ×100). The arrow indicates new bone formation near edge of defect (only 4 rings visible). (VB), vital bone.Figure 10. Tetracycline hydrochloride labeling of new bone: 2% demineralized freeze-dried bone allograft (DFDBA) group. Two percent DFDBA particle at the center of defect (6 rings visible; original magnification ×100). The arrow shows the fourth administration of tetracycline hydrochloride at the 42-day time point. (DBP), decalcified bone particle.
Contributor Notes
James W. Turonis, DDS, MS, Michael F. Cuenin, DMD, and Mark E. Peacock, DDS, MS, are currently stationed with the US Army Dental Corps in Germany.
James C. McPherson III, PhD, is from the Department of Clinical Investigation at Eisenhower Army Medical Center in Fort Gordon, Ga.
Steven D. Hokett, DDS, is currently in the private practice of Periodontics in Vancouver, Wash. Address correspondence to Dr Steven D. Hokett, 18014 NE 85th Way, Vancouver, WA 89682 (e-mail: hoketts@ohsu.edu).
Mohamed Sharawy, BdS, PhD, is from the Medical College of Georgia in the Department of Oral Biology in Augusta, Ga.