Analyses Using Micro-CT Scans and Tissue Staining on New Bone Formation and Bone Fusion According to the Timing of Cranioplasty via Frozen Autologous Bone Flaps in Rabbits : A Preliminary Report

Abstract

Objective: The timing of cranioplasty and method of bone flap storage are known risk factors of non-union and resorption of bone flaps. In this animal experimental study, we evaluated the efficacy of cranioplasty using frozen autologous bone flap, and examined whether the timing of cranioplasty after craniectomy affects bone fusion and new bone formation.

Methods: Total 8 rabbits (male, older than 16 weeks) were divided into two groups of early cranioplasty group (EG, 4 rabbits) and delayed cranioplasty group (DG, 4 rabbits). The rabbits of each group were performed cranioplasty via frozen autologous bone flaps 4 weeks (EG) and 8 weeks (DG) after craniectomy. In order to obtain control data, the cranioplasty immediate after craniectomy were made on the contralateral cranial bone of the rabbits (control group, CG).The bone fusion and new bone formation were evaluated by micro-CT scan and histological examination 8 weeks after cranioplasty on both groups.

Results: In the micro-CT scans, the mean values of the volume and the surface of new bone were 50.13±7.18 mm(3) and 706.23±77.26 mm(2) in EG, 53.78±10.86 mm(3) and 726.60±170.99 mm(2) in DG, and 31.51±12.84 mm(3) and 436.65±132.24 mm(2) in CG. In the statistical results, significant differences were shown between EG and CG and between DG and CG (volume : p=0.028 and surface : p=0.008). The histological results confirmed new bone formation in all rabbits.

Conclusion: We observed new bone formation on all the frozen autologous bone flaps that was stored within 8 weeks. The timing of cranioplasty may showed no difference of degree of new bone formation. Not only the healing period after cranioplasty but the time interval from craniectomy to cranioplasty could affect the new bone formation.

Keywords: Autologous bone; Cranioplasty; Frozen stored; Rabbits; Timing.

Fig. 1. Diagram and time-line of experimental schedule. Eight rabbits were divided into two groups of the delayed cranioplasty group (DG, 4 rabbits) and the early cranioplasty group (EG, 4 rabbits). The craniectomy was performed on the right side cranial bone of the DG rabbit at day 0, and EG rabbit at day 28. The obtained bone flap was stored in a freezer of -80℃. At day 56, cranioplasty was performed on both groups via the frozen bone flap. In order to obtain control data, the craniectomy was made on the left cranial bone of the rabbit, and the bone flap was immediately fixed (control group). At day 112, the EG and DG rabbits were sacrificed.

Fig. 2. Preparation of the experimental model of formation of bone defect and bone flap. After incision was made along the midline of the cranial skin, the muscles and periosteum were incised layer by layer to expose the cranial bone (A). The bone flap margin was formed 3-mm lateral to the sagittal suture, using a trephine drill with a 10-mm outer diameter and 9-mm inner diameter. To avoid damaging the dura mater, the inner table of the cortical bone was left (B and C). Using a 1-mm round burr, the space between the bone-flap margin and the cranial bone was expanded out of the bone flap in order to create a 12-mm bone defect diameter (D), and the dura mater was carefully exposed (E). The 9-mm bone flap was lifted up and the bone defect was formed (F).

Fig. 3. The fibrous tissues were removed in order to expose the bone defect area (A). Afterwards, the autologous bone flap (B), which has been kept in the freezer, was applied to each labeled rabbit in order to perform cranioplasty, and the bone flap was fixed to cranial bone using a titanium-alloy miniplate and screws (C). Using the same method, the control group was formed on the left cranium of the rabbit (D).

Fig. 4. The axial sectional image (A), and coronal and sagittal sectional images (B) of computed tomography scans. The 3D reconstructed images showed outer surface (C) and inner surface (D) of cranium. The left side of cranium is experimental craniectomy site, and the right side is the control group.

Fig. 5. We defined the region of interest (ROI) as the region within a 14-mm diameter from center point of bone flap. We measured the bone volume (VOL_ROI) and the bone surface (SUR_ROI) of ROI (A). From VOL_ROI and SUR_ROI, the bone volume (VOL_{bone flap}) and the bone surface (SUR_{bone flap}) of the bone flap, and the volume (VOL_metal) and the surface (SUR_metal) of metal fixture were removed. Afterwards, the computed tomography scan images (B) and the 3D reconstruction images (C) of the new bone volume (VOL_new) and the new bone surface (SUR_new) were quantified and measured.

Fig. 6. The histological patterns of new bone formation. The new bone formation was observed in the microscopic examination of the hematoxylin and eosin stain and Goldner's stain in all of the rabbit tissues. The new bone formation was observed as a form of 1) a bony islet (arrow) between the bone flap and the edge of the cranial bone (A), 2) a bony islet (arrow) at the lower part of the bone flap (B), 3) a new bone formation (arrow) at the edge of the cranial bone (C), and 4) a bone flap incorporation (arrow) between the bone flap and the edge of the cranial bone (D).