PERIFERICOS Y MUSCULOS

The specimens containing the endoprosthesis were scanned using a GE eXplore Locus SP MicroCT scanner (GE Healthcare, Thermo Scientific, Waltham, MA, USA), with a focal spot of 8 µm and pixel size of 18 µm and scanning configuration isotropic voxels of 8 µm x 8 µm x 8 µm focal spot size and isotropic resolutions at 8 µm (Fig. 3).

Figure 3. Micro CT scan image; A) lateral view, B) occlusal view and cross sectional views, C) anterior stem, D) mid body and E) posterior stem.[B; Buccal and Li; Lingual]

The mini-plates and screws were removed before the scanning process to avoid scattering. The scan area was extended to the maximum diameter of scan view and covered beyond the region of interest (ROI) at the reconstruction site, including the entire anterior and posterior stems. The digitized signals were then transferred to a computer for reconstruction of the micro-CT slices. Standardized calibration was used compared to bone, air and water. All images were calibrated in Hounsfield units (HU) for quantitative analysis. New bone was analyzed using MicroView® 2.2 software (GE Healthcare, Waukesha, WI, USA).

Micro-CT slices were reconstructed perpendicular to the long axis of the mandibular reconstruction. The percent bone volume (BV %) around the stems of

the device was analyzed modified from the method described in a previous study.3

Briefly, three circular regions of interest (ROI) with standardized diameter of 0.52 mm were selected for each specimen. These were located directly next to stem surface in the buccal, lingual and inferior aspects. The selection of an ROI superior to the stem was not done as a tooth was frequently present in that area. In the case where a screw hole or tooth structure was seen to be within the ROI, the ROI was manually moved to the immediate adjacent area. Percent bone volume (BV %) was calculated using MicroViewTM 2.2 GE Healthcare computer software (Fig. 4).

Figure 4. The area of interest of bone growth around the Ti device’s stem. A standardized diameter of 0.52 mm in 3 areas was selected for each specimen. There was located directly nest to the stem surface at buccal, lingual and inferior region. Percent bone volume (BV %) in ROI was identified and analyzed using Microview computer software.

Mechanical Testing

The four specimens for mechanical testing were processed at the Biomedical Engineering Laboratory, College of Engineering, The University of Michigan, Ann Arbor, MI, USA. Three-point bending test was done to determine their stiffness using an MTS Alliance RT/30 Elite™ Controller testing machine (TestResources Inc, Minnesota, USA).

The specimens were thawed from -20°C to room temperature for 2 hours before mechanical testing. All specimens were maintained in a moist condition until the test was completed. Before mechanical testing, the mini plates and screws as well as most of the soft tissue around the mandible were removed. Due to the instability of the bone segments of the samples, a thin layer of muscle tissue enveloping the mandibles was preserved to help maintain the integrity of the reconstruction site. Bilateral mandibular coronoid processes and canine cusps were trimmed to prevent interference with the fixation jig during mechanical testing. Subsequently, each specimen was placed on the biomechanical 3-point bending testing fixtures in a reverse position with both condyles and mid-anterior lingual bone surface placed on the custom-made jig. A force with constant displacement rate of 25 mm/min was applied on the lower border of the mandibular until 111.20 N was reached. The load-displacement data were recorded at a frequency of 15 Hz to determine the elastic stiffness (N/mm) of the reconstructed mandibles without breaking

the specimens. After mechanical testing, all specimens were immersed in 10% formaldehyde for histological analysis.

Histological Analysis

The specimens were reduced in size, dehydrated in a graded series of ethanol, embedded in methyl methacrylate resin and polymerized. The tissue blocks were mounted in a modified inner circular saw microtome (Leica® RM 2165, Wetzlar Germany) and 10 µm thick sections were prepared. Serial bucco-lingual cross sections were stained with methylene blue and basic fuchsin for histological and histomorphological analysis. At least, seven bucco-lingual histological cross sections were made from the reconstructed mandible from each specimen i.e. one at the midline of the device’s body, three at the junction between device body and the stem and three at mid of the scaffold’s stem.

Histological sections were recorded using a light microscope equipped with a camera (Carl Zeiss, Oberkachen, Germany) and then evaluated with a digital image analysis system (Leica QWin Pro, Wetzlar, Germany). Histological and histomorphometrical analysis was done in three regions; (1) mid scaffold, (2) junctions between device body-stem and (3) mid posterior stem. Bone quality and quantity analysis was performed using a bone score scale for ‘bone presentation’ and ‘bone-device contact’ at each region (Table 1). The percentage bone–stem contact and the BV% were analyzed for the area around the posterior stem.

Table 1. Parameters and scoring for histology evaluation by light microscopy

Parameter Score

Bone Formation around device •

(Body of device, Body-stem Junction, stem)

2 : Completely surrounded with bone 1 : partially surrounded with bone 0 : No bone (fibrous formation)

Bone contact at device surface •

(Body of device, Body-stem Junction, stem)

2 : Completely device surface contact with bone

1 : partially surface contact with bone 0 : No bone contact (fibrous formation)

Percent bone-device surface •

contact (%) (stem)

0-100%

Bone volume and Percent Bone •

Volume (%) (stem)

Bone volume (mm3_{) and percent Bone}

volume (0-100%) at the area of bone at 2 mm from stem surface