Animal model
A total of 92 male Fischer 344 rats (Japan SLC, Hamamatsu, Japan) were used, with approval from the institutional ethical committee of our institute. Surgeries were performed on all animals at age 12 weeks under general anesthesia with isoflurane and pentobarbital. After exposing the femoral shaft, two pairs of tip-threaded 1.4-mm-diameter K-wires were placed in the proximal and distal femur. A bone defect of 1 mm was created between the proximal and distal pins by an oscillating saw, and a custom-made external fixator was attached to connect the pairs of pins (Fig. 1). Euthanasia was performed by intraperitoneal administration of a pentobarbital overdose before assessment.
Topical cutaneous application of CO2
Rats were assigned into two groups: CO2 group (n = 46) and control group (n = 46). After sedation using a minimum dose of isoflurane, the hair of the affected limb was shaved and CO2 absorption enhancing hydrogel (NeoChemir Inc., Kobe, Japan) was applied. The hydrogel had a pH of 5.5 and consisted of carbomer, glycerin, sodium hydroxide, sodium alginate, sodium dihydrogen phosphate, methylparaben, and deionized water. In the CO2 group, the affected limb was sealed in a polyethylene bag filled with 100% CO2 for 20 min a day (Fig. 2). This treatment was performed 5 days a week. In the control group, the affected limb, coated in the same way with CO2 absorption enhancing hydrogel, was sealed in a polyethylene bag filled with air.
Radiographic assessment of bone healing
At 1, 2, 3, and 4 weeks after surgery, 58 animals (29 in the CO2 group and 29 in the control group) were anesthetized and fixed in the supine position with the hip joints fully abducted, and radiographs of the defect site were acquired. Radiographically, each callus on the two cortices on the lateral images were evaluated by three orthopedic surgeons blinded to the groups. Bone healing was defined as the absence of a bony gap or the presence of a bridging callus at both the anterior and posterior cortices.
Histological assessment for bone healing
At 2 and 4 weeks after surgery, the femur was harvested from 6 animals in each group, and the tissues were fixed in 4% paraformaldehyde, decalcified with ethylenediaminetetraacetic acid (EDTA), and embedded in paraffin wax. Sagittal sections were cut to a thickness of 5 μm and stained with Safranin-O/Fast Green for histological assessment of the bone defect area. The degree of bone healing was assessed with Allen’s grading system (grades 0 through 4), which was originally designed for histological evaluation of fracture healing [18]. Specimens were examined by three blinded examiners.
Biomechanical assessment of bone healing
Five femurs at 4 weeks after surgery were used for biomechanical evaluation. After euthanasia, the femur was dissected, and the muscle surrounding the defect site was removed. A standardized three-point bending test was performed with a load torsion and bending tester (MZ-500D, MZ-500S, Maruto Instrument Co., Ltd., Tokyo, Japan) for rats in both groups. The ultimate stress (N), extrinsic stiffness (N/mm), and failure energy (N.mm) were assessed by outsourcing to Kureha Special Laboratory, Co., Ltd. (Tokyo, Japan).
Micro-computed tomography (μ-CT) measurement of bone healing
At 4 weeks after surgery, the femur with bone defect was harvested, and the external fixator and pins were removed from the bone. For quantification of bone regeneration, μ-CT imaging analysis was performed. The femurs were scanned and evaluated using a μ-CT scanner (R_mCT2, RIGAKU, Tokyo, Japan). The region of interest (ROI) was set as 3 mm proximal and distal from the midline of the defect site on sagittal view. Tissue mineral density (TMD), total callus volume (TV), bone mineral content (BMC), and volumetric bone mineral density (vBMD; BMC/TV) of the callus were evaluated by TRI/3-D-BON (Ratoc System Engineering, Tokyo, Japan).
Assessment of gene expression
At 1, 2, and 3 weeks after surgery, gene expression was measured in 6 animals in each group by real-time polymerase chain reaction (PCR). Newly generated callus tissue was harvested. Total RNA was extracted from the tissue with a RNeasy Mini Kit (Qiagen, Valencia, California) and reverse-transcribed into single-stranded DNA with a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, California). Real-time PCR was performed in duplicate on the cDNA with an ABI PRISM 7700 Sequence Detection System and SYBR Green reagent (Applied Biosystems). We examined the expression of genes for VEGF to evaluate angiogenesis. The expression level of each gene was first normalized with respect to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which served as an internal control, and the results are presented as the fold change relative to the control group (ΔΔCt method).
Statistical analysis
The Fisher’s exact test was used for radiographic assessment and the Mann-Whitney U-test was used for histological assessment, μ-CT assessment, biomechanical assessment, and genetic assessment with real-time PCR. A p-value less than 0.05 was considered statistically significant.