The effect of three hemostatic agents on early bone healing in an animal model

Abstract

Background: Resorbable bone hemostasis materials, oxidized regenerated cellulose (ORC) and microfibrillar collagen (MFC), remain at the site of application for up to 8 weeks and may impair osteogenesis. Our experimental study compared the effect of a water-soluble alkylene oxide copolymer (AOC) to ORC and MFC versus no hemostatic material on early bone healing.

Methods: Two circular 2.7 mm non-critical defects were made in each tibia of 12 rabbits. Sufficient AOC, ORC or MFC was applied to achieve hemostasis, and effectiveness recorded. An autologous blood clot was applied to control defects. Rabbits were sacrificed at 17 days, tibiae excised and fixed. Bone healing was quantitatively measured by micro-computed tomography (micro-CT) expressed as fractional bone volume, and qualitatively assessed by histological examination of decalcified sections.

Results: Hemostasis was immediate after application of MFC and AOC, after 1-2 minutes with ORC, and >5 minutes for control. At 17 days post-surgery, micro-CT analysis showed near-complete healing in control and AOC groups, partial healing in the ORC group and minimal healing in the MFC group. Fractional bone volume was 8 fold greater in the control and AOC groups than in the MFC group (0.42 ± 0.06, 0.40 ± 0.03 vs 0.05 ± 0.01, P < 0.001) and over 1.5-fold greater than in the ORC group (0.25 ± 0.03, P < 0.05). By histology, MFC remained at the application site with minimal healing at the defect margins and early fibrotic tissue within the defect. ORC-treated defects showed partial healing but with early fibrotic tissue in the marrow space. Conversely, control and AOC-treated defects demonstrated newly formed woven bone rich in cellular activity with no evidence of AOC remaining at the application site.

Conclusions: Early healing appeared to be impaired by the presence of MFC and impeded by the presence of ORC. In contrast, AOC did not inhibit bone healing and suggest that AOC may be a better bone hemostatic material for procedures where bony fusion is critical and immediate hemostasis required.

Figures

Figure 1

Representative micro-CT images of excised rabbit tibiae at 17 days post-surgery. Left column: Cross-section views through the center of the defect showing newly formed bone as slightly opaque in untreated (control), alkylene oxide copolymer- and oxidized regenerated cellulose-treated defects, and an absence of opacity in microfibrillar collagen-treated defects (bar = 5.0 mm). Center and right columns: micro-CT generated binarized images of mineralized tissue of the defect area (center) and area analyzed within the defect (right). AOC-treated and untreated (control) defects show substantial, well developed mineralized tissue, ORC-treated defect shows a more diffuse structure of mineralized tissue, and minimal mineralized tissue is observed within the MFC-treated defect.

Figure 2

Bone mineralization at 17 days post-surgery. Mean values for micro-CT derived fractional bone volume (BV/TV) showing an 8 fold greater BV/TV value for untreated (control) and alkylene oxide copolymer-versus microfibrillar collagen-treated defects (0.42 ± 0.06, 0.40 ± 0.03 vs 0.05 ± 0.01 respectively, P < 0.001) and over 1.5 fold greater BV/TV value versus oxidized regenerated cellulose-treated defects (0.25 ± 0.03, P < 0.05). Number of defects per group = 12, P values determined from Tukey's Multiple Comparison Test.

Figure 3

Microscopy of control defects at 17 days post-surgery. Untreated (Control) adjacent 5 μm demineralized bone sections stained with H&E (upper) and Trichrome-EVG at ×2 (left) and ×10 magnification. The edge of the defect is identified with black/green arrows, and the area of magnification shown with a dotted box. Defects show good healing with newly formed woven bone (light blue arrows) rich in cellular activity (osteoid shown with dark blue arrows). Blue stars indicate native collagen meshwork throughout the tissue.

Figure 4

Microscopy of alkylene oxide-treated defects at 17 days post-surgery. Alkylene oxide copolymer-treated adjacent 5 μm demineralized bone sections stained with H&E (upper) and Trichrome-EVG at ×2 (left) and ×10 magnification. The edge of the defect is identified with black/green arrows, and the area of magnification shown with a dotted box. Defects show good healing with newly formed woven bone (light blue arrows) rich in cellular activity (osteoid shown with dark blue arrows). Blue stars indicate native collagen meshwork throughout the tissue.

Figure 5

Microscopy of oxidized regenerated cellulose-treated defects at 17 days post-surgery. Oxidized regenerated cellulose-treated adjacent 5 μm demineralized bone sections stained with H&E (upper) and Trichrome-EVG at ×2 (left) and ×10 magnification. The edge of the defect is identified with black/green arrows, and the area of magnification shown with a dotted box. Defects show partial healing with newly formed woven bone (light blue arrows) rich in cellular activity (osteoid shown with dark blue arrows). Early fibrotic tissue is observed within the defect (white stars). Blue stars indicate native collagen meshwork within the marrow and defect.

Figure 6

Microscopy of microfibrillar collagen-treated defects at 17 days post-surgery. Microfibrillar collagen-treated adjacent 5 μm demineralized bone sections stained with H&E (upper) and Trichrome-EVG at ×2 (left) and ×10 magnification. The edge of the defect is identified with black/green arrows, and the area of magnification shown with a dotted box. Defects show minimal healing with no evidence of newly formed woven bone. Dense bundles of residual collagen are observed (yellow arrows) and early fibrotic tissue within the marrow space (white stars). Blue stars indicate native collagen meshwork within the marrow space.