The influence of hyperbaric oxygen treatment on the healing of experimental defects filled with different bone graft substitutes

Abstract

To assess potential effects of hyperbaric oxygen (HBOT) on artificial bone grafts, β - Tricalcium phosphate (β-TCP) and calcium phosphate coated bovine bone (CPCBB) substitutes were applied to standard bone defects in rat tibiae. The control defects were left empty. Half of the animals received 60 minutes of 2.4 atmosphere absolute (ATA) of HBOT. Rats were sacrificed at one, two and four weeks. Bone healing was assessed histologically and histomorphometrically using light microscopy. The periosteum over the bone defects was examined ultrastructurally. Cardiac blood was collected to determine the serum osteocalcin levels. The HBOT increased new bone formation in the unfilled controls and β-TCP groups and significantly decreased cartilage matrix and fibrous tissue formations in all groups. Active osteoblasts and highly organized collagen fibrils were prominent in the periosteum of β-TCP and control groups. Serum osteocalcin levels also increased with HBOT. The healing of defects filled with CPCBB was similar to the controls and it did not respond to HBOT. These findings suggested that the HBOT had beneficial effects on the healing of unfilled bone defects and those filled with β-TCP bone substitute but not with CPCBB, indicating a material-specific influence pattern of HBOT.

Keywords: Hyperbaric oxygen; beta tricalcium phosphate; calcium phosphate coated bovine bone; light microscopy; rat; ultrastuctural.

Figure 1

(a) Endochondral bone formation characterized by abundant cartilage matrix in the histological slide of the control group without HBOT at one week time point (H&E×100). (b) The histological slide of the control group which received HBOT at one week time point, the new bone growth and blood vessel formations (arrows) are clearly visible (H&E×250). (c) Histological appearance of the CPCBB group without HBOT at one week time point, note the arrows showing new bone trabeculae around the bone graft (H&E×40) (Cm; Cartilage matrix, Nbf; New bone formation, Gm; Graft material). (d) Light micrography of the β-TCP group without HBOT taken at two week time point (H&E ×100).

Figure 2

New bone formation and residual graft particles were observed in the histological section taken from the β-TCP group with adjunctive HBOT at four week time point (H&E×200) (Nbf; New bone formation, Gm; Graft material, Ct; Connective tissue).

Figure 3

CPCBB graft material which is gradually resorbing and resembles the necrotic bone can be seen in the middle of this slide. This section has been taken from the CPCBB + HBOT group which was sacrified at four week time point (H&E×200) (Nbf; New bone formation, Gm; Graft material, Ct; Connective tissue).

Figure 4

The histological view of the defect which is nearly filled with newly formed bone in the control groups which had received four week of HBOT (H&E×200) (Nbf; New bone formation, Bm; Bone marrow).

Figure 5

The ultrastructural appearance of two periosteal osteoblast cells with well developed intracytoplasmic organelles in the control group without HBOT at one week time point ( TEM ×6000) (N; Nucleus, Ger; Granular endoplasmic reticulum, Mi; Mitochondrion; Cf; Collagen Fibrils).

Figure 6

Residual β-TCP bone graft material is seen between two osteoblast cells of the periosteum, note the cytoplasmic processes (arrows) extending from the cell membrane to the bone graft substitute. This electron micrograph was taken from the β-TCP + HBOT group which were sacrificed at one week time point (TEM×7500) (N; Nucleus, R; Ribosome, Ger; Granular Endoplasmic Reticulum).

Figure 7

(a) The ultrastructural view of the periosteum in β-TCP group following four week of HBOT. Note well developed membranes of the highly active intracytoplasmic organelles (TEM×6000) (N;Nucleus, Mi; Mitochondrion, Li; Lisosome, Ger; Granular endoplasmic reticulum, Cf; Collagen fibrils) (b) Four week of HBOT could not prevent the damages in the periosteum of the CPCBB group. This electron micrograph shows the extent of degeneration in two periosteal cells. The swollen membranes, the pyknotic nuclei and the dispersed collagen fibrils can be seen as the evidences of deterioration in the cellular structure of this group (TEM×7500) (N; Nucleus, Mi; Mitochondrion, Ger; Granular endoplasmic reticulum, Pli; Phagocytic lisosome, Cf; Collagen Fibrils).

Figure 8

Ultrastructural findings of extensive degeneration in the periosteum of CPCBB + HBOT group at one week time point. It is clear that the the integrity of the cell membrane could not be maintained and there was also evidence of vacuolar degeneration (stars) in the intracytoplasmic compartment (TEM×7500) (Gm; graft material, Cf; Collagen fibrils).