Percutaneous vertebroplasty combined with interstitial implantation of ¹²⁵I seeds in banna mini-pigs

Brachytherapy seeds iodine-125 (BT-125-1) were purchased from Shanghai Xinke Medicine Ltd. (Shanghai, China). Apparent radioactivity was 1.00 mCi/seed and half time is 59.4 days. Before purchase, the I-125 seeds were randomly picked up for activity testing to confirm the seed container integrity and apparent activity of the seeds.

X-ray computed tomography (CT) was from Siemens AG., Munich, Germany; DSA (digital subtraction angiography) was from Philips Medical Systems, Best, The Netherlands; the treatment planning system (TPS) was from Hejie Medical Instruments, Tianjin, China. The CRC-15R calibrator was from CAPINTEC Inc., Pittsburgh, PA, USA.

Bone cement type II was purchased from the Tianjin Material Institute, Tianjin, China. All the small instruments including sterile common needles, hammers, and seed injection needles were purchased from Guan Long Ltd., Shandong, China.

All the animal experiments were approved by the Ethics Committee of Laboratory Animals of Tunor Hopital of Yunnan Province.

A total of 14 healthy adult female banna mini-pigs were selected for experiments. They were randomly divided into three groups, group A received the PVP with ¹²⁵I implantation (n = 6), group B received the ¹²⁵I implantation alone (n = 6), Group C were kept as age-matched normal control (n = 2). The animals were provided and raised by the Animal Center at Kunming Medical College. Their weights ranged from 20 to 25 kg (average 22.1 kg). The mini-pigs were raised and monitored for their food habits, excreta and activities for one week before the experiments. The protocol of the investigation was in accordance with the principles outlined in the China Practice for the Care and Use of Laboratory Animals. The pigs were raised for up to eight months (equal to four half-lives of ¹²⁵I).

After careful CT scans and consideration, the T13 level in the spine of the banna pig was selected as our ¹²⁵I implanting target because it was easier to control and facilitated the whole operation. The target of the T13 level is shown in Figure 1.

Based on the classical dosimetry calculation method cited by the Memorial Sloan-Kettering Cancer Center, the seed number was determined by the length of the three-dimensional axis, which is the distance from the seed to the target. The formula is as follows: seed number = ((length + width + thickness/3) ×5) ÷ (seed original activity), and according to the empirical experience, the actual dose density should be increased approximately 20 to 30% based on the theoretical value.

With the help of accurate CT scans, 1 cm was determined for the target length, width and thickness, the apparent activity for one seed was 1 mCi, all the data were put into formula, the predicted seed number was ((1 + 1 + 1)/3 × 5) ÷ 1 = 5, then 3 more seeds were added, so the total dosage was 8.0 mCi.

This study adopted Monte Carlo-aided dosimetry to measure the radiation dose received by the mini-pig spinal cord during the whole of the brachytherapy process. Briefly, at the beginning, we calculated the initial dose (termed as D₍₀₎) immediately after the ¹²⁵I particles were implanted into the spinal cord T13 level; the formula was D₍₀₎ = A₀ × 1.27 × Λ × g_(r) × F_{(r θ)}/r². A₀ is the particle initial radiation dose, which was tested by CRC-15R calculator on the day before implanting; Λ is constant parameter for ¹²⁵I, in our research the value is 1.06; r is the distance between the spinal surface to the ¹²⁵I particles, which was obtained from the magnetic resonance imaging (MRI) detecting data; g_(r) is radial dose functions; and F_{(r θ)} is anisotropy constant; the detailed data we calculated based on the published method[8, 9]. After D₀ had been confirmed, the radiation dose received by the spine was calculated by the formula D_(T) = D₍₀₎*T_1/2*1.443*(1- e^{-T*0.693/T1/2}). D_(T) means the total received dose within time interval T, e is natural constant.

For group A, briefly, DSA-guided vertebroplasty was performed under local anesthesia, and acrylic bone cement was injected into the vertebra through a bone trocar to the center of the lesion, with simultaneous interstitial implantation of ¹²⁵I seeds.

A TPS system was implemented to manage the whole procedure. Before the operation, the T13 CT scan and the physical parameters of the ¹²⁵I seeds were put into the TPS system to let the software create the detailed treatment plan automatically. We then followed the TPS procedure to do the operation, the mini-pigs were anesthetized with sodium pentobarbital through ear veins, and were then placed in prone positions, followed by skin preparation and sterilization. DSA was used to precisely localize the surface projection of the T13 vertebra body and vertebral pedicle. Following the template, a syringe needle, mounted with a 20 to 30 degree angle to coronal plate, went to the pedicle of the vertebral arch where it connects vertebra, and was placed into the T13 anterior spinal canal without damaging the dura mater. Meglumine diatrizoate was used to confirm the location of the needle. Then ¹²⁵I seeds were implanted into the vertebral body. Bone cement was made up with water and photographic developer, the mixture ratio was 3:2:1 respectively, and while the cement was in the toothpaste stage, it was injected into the vertebral body, with frequent changes of direction to make the cement distribute equally and to avoid leakage. The PVP dose was approximately 0.8 to 1.3 mL, the average was 0.9 mL. After the whole operation, a CT scan was performed immediately to show the distribution of PVP and the seed locations. Figure 2 shows the detailed implant position in the vertebral body.

The Tarlov scale is a five-point scale to assess upper and lower limb locomotion. This scale is widely accepted by the American Spinal Injury Association (ASIA) as the International Standards for assessing spinal impairment. In this present study, the Tarlov scale was used to measure the banna mini-pigs’ spinal impairment after the operation. Hematological analyses were also performed, the peripheral blood samples were collected at five time points, they were before the seed implantation, one week, one month, two months and eight months postoperation. The white cell number, hemoglobin, blood platelet and creatinine were examined as indexes.

After eight months, all the animals were anesthetized and executed, the T13 level was taken out to examine the filling and distribution of the bone cement and ¹²⁵I seeds. The spinal cord and vertebral body were put into 10% formaldehyde solution for fixation. After 48 hours in 10% formaldehyde solution, the spinal cord was cut into 4mm thick slice for hematoxylin and eosin (H & E) staining, and the vertebral body remained in 1% nitric acid for further decalcification. After another 72 hours of decalcification treatment, a pin was used to test the softness of the vertebral body, if was soft, it was stained with H & E as well.

A series of sections from the vertebral body and spinal cord from six banna mini-pigs were processed for electron microscopic double staining to reveal cellular and subcellular alteration in morphology. For this double-labeling procedure, sections were first fixed with 3.5% glutaraldehyde solution and 1% osmic acid solution, and then dehydrated with gradient ethanol and acetone. The last step was to double stain with uranyl acetate and lead citrate, then observe under JEM-1-11 transmission electron microscopy.

Standard statistical software (SPSS version11.0; SPSS, Inc, Chicago, IL, USA) was used for data analysis. Student’s t-test was used for variable data analysis. P <0.05 was considered statistically significant. Data were presented at mean ± SD.

A DSA scan recorded the whole operation procedure, and the operation was completed successfully. See Figure 3.

After complete the operation in 15 minutes, a CT scan was performed to confirm the bone cement distribution and ¹²⁵I seed locations. The CT scan proved the bone cement and seeds were distributed well in all the experimental animals, meeting the same requirements as human clinical standards. See Figure 4.

After the operation was completed, a CT scan was performed, with the help of the TPS; the postimplant dosimetry used the dose-volume histograms (DVH) to assess implant quality. After calculation, the minimum peripheral dose (mPD) was 80.1 Gy, D90 was 90.2 Gy, the mean dose for the target region was 120.2Gy, D₉₀ >mPD, suggested the seeds were successfully implanted into the correct position and the radiation dose met the requirements of brachytherapy.

With the CT scan, the radiation dose distribution through the axial, sagittal and coronal planes of the vertebral body was determined in this study. Based on the formula mentioned in the Methods section, the average radiation dose to the T13 level vertebral body for each group were obtained, see Table 1.

No paraplegia case was noted for eight months after ¹²⁵I implantation. Tail swaying and defecating and urinating were the same as normal. The Tarlov scale was five for all the treatment animals from starting to eight months. Hematological analysis is shown in Table 2. All the data suggest banna mini-pigs had stable physical conditions within the eight months.

Observation results showed the bone cement was well distributed into the vertebral body, it was almost merged together, it was so hard to separate. Under the light microscope, the bone cement was mainly distributed among bone trabeculas, stained with blue by H & E; the peripheral bone trabecula surrounding the bone cement and seeds were normal in structure; no necrosis and denature were noted for bone cells; and the morphological structure remained in normal condition as well. See Figure 5.

H & E staining suggested the spine surface structure was intact; significant necrosis was not noted under the microscope; white matter was normal and grey matter had a clear structure; for neurons, shrinkage, denaturation and necrosis were not found; no swelling was observed in neuroglial cells, and microvessels in the bone were also normal. See Figure 6.

Electron microscopy (EM) observation was performed for subcellular identification. Under EM, the nerve fiber structure was intact and cellular organelle was clear, no obvious impairment was found for most organelles, and only light swelling was found for mitochondria. See Figure 7.