Four-week histologic evaluation of grafted calvarial defects with adjunctive hyperbaric oxygen therapy in rats

Abstract

Purpose: The aim of this study was to characterize the healing in the grafted calvarial defects of rats after adjunctive hyperbaric oxygen therapy.

Methods: Twenty-eight male Sprague-Dawley rats (body weight, 250-300 g) were randomly divided into two treatment groups: with hyperbaric oxygen therapy (HBO; n=14) and without HBO (NHBO; n=14). Each group was further subdivided according to the bone substitute applied: biphasic calcium phosphate (BCP; n=7) and surface-modified BCP (mBCP; n=7). The mBCP comprised BCP coated with Escherichia-coli-derived recombinant human bone morphogenetic protein-2 (ErhBMP-2) and epigallocatechin-3-gallate (EGCG). Two symmetrical circular defects (6-mm diameter) were created in the right and left parietal bones of each animal. One defect was assigned as a control defect and received no bone substitute, while the other defect was filled with either BCP or mBCP. The animals were allowed to heal for 4 weeks, during which those in the HBO group underwent 5 sessions of HBO. At 4 weeks, the animals were sacrificed, and the defects were harvested for histologic and histomorphometric analysis.

Results: Well-maintained space was found in the grafted groups. Woven bone connected to and away from the defect margin was formed. More angiogenesis was found with HBO and EGCG/BMP-2 (P<0.05). None of the defects achieved complete defect closure. Increased new bone formation with HBO or EGCG/BMP-2 was evident in histologic evaluation, but it did not reach statistical significance in histometric analysis. A synergic effect between HBO and EGCG/BMP-2 was not found.

Conclusions: Within the limitations of this study, the present findings indicate that adjunctive HBO and EGCG/BMP-2 could be beneficial for new bone formation in rat calvarial defects.

Keywords: BMP-2; Biphasic calcium phosphate; Bone substitute; EGCG; Hyperbaric oxygen therapy.

Figure 1

(A, B) Histometric analysis (bar=2 mm). New bone, graft particles, soft tissue. Each slide was painted using Photoshop CS4 (Adobe, San Jose, CA, USA). Navy blue, new bone; Orange, graft particles; Green, soft tissue.

Figure 2

Histologic evaluation around the defect margin (bar=25 and 100 μm). It shows the osteogenic effect of HBO and BMP-2/EGCG. (A, D) NHBO-BCP subgroup, (B, E) NHBO-mBCP subgroup, (C, F) HBO-BCP subgroup. New bone formation was rarely present in (A) and (D). However, new bone in contact with the defect margin (C, F) and between particles (B, E) was increased. DM, defect margin; M, material; NB, new bone.

Figure 3

Histologic evaluation at the middle of defect (bar=25 and 100 μm). (A, D) NHBO-BCP subgroup, (B, E) NHBO-mBCP subgroup, (C, F) HBO-BCP subgroup. In the experimental groups, osteocytes (arrow) and bone marrow (arrow head) were observed. M, material; NB, new bone; BM, bone marrow.

Figure 4

Histological evaluation at the middle of the defect close to the defect base showing the osteogenic effect of HBO (H/E, bar=25 μm). (A) NHBO-BCP group, (B) HBO-BCP group. M, material; NB, new bone; BM (arrow head), bone marrow; Arrow, osteocyte.

Figure 5

Histologic evaluation (CD31 immunohistochemical staining, bar=50 μm) showing the angiogenic effect of HBO and BMP-2/EGCG. Blood vessels were observed in all groups. The CD31-positive endothelial cells were generally replaced (arrow head). (A) NHBO-control group, (B) HBO-control group, (C) NHBO-mBCP group. Arrow head, vessel; Arrow, red blood cell.

Figure 6

Immunohistochemical analysis showing the synergic effect of HBO with BMP-2/EGCG (CD31 staining, bar=50 μm). (A) NHBO-mBCP subgroup, (B) HBO-mBCP subgroup. M, material; Arrow head, vessel; Arrow, red blood cell.