Here, we introduce a method to evaluate transpedicular screws by using an in vivo porcine lumbar spine model.
Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle screws after spinal surgery, which is a serious concern. To address this problem, diverse types of pedicle screws have been examined to identify those with good fixation strength and osseointegration in spine bone. The porcine spine is a good alternative for the human spine in the evaluation of pedicle screws due to the anatomical size, mechanical characteristics, and cost. Although several studies have reported that pedicle screws are efficient in the porcine model, no study has described detailed protocols for the evaluation of a pedicle screw using the porcine model. Here, we describe a detailed method for evaluating transpedicular screws using an in vivo porcine lumbar spine model. The technical details for anesthesia, spine surgery, and harvest provided here will facilitate with the evaluation of the transpedicular screw fixation model.
Transpedicular screw fixation is a gold-standard treatment for degenerative lumbar spine and bursting fracture because it involves three columns of the spine and achieves stabilization1,2. However, most patients who undergo such surgery also have osteoporosis3,4. Many studies have evaluated the fixation strength and the osseointegration status of transpedicular screws, because the loosening of pedicle screws currently in use has been reported in patients with osteoporosis5,6.
The porcine spine is similar to the human spine in terms of size. It is less expensive compared to a primate model7. Furthermore, an in vivo mechanical study has demonstrated that the quadruped porcine spine is essentially loaded in the same way as that of the human spine8, which is why many researchers use porcine spines for studies on the prevention of pedicle screw loosening. However, it takes several months to study pedicle screws in the porcine spine because identifying the long-term stability of pedicle screws takes times. In order to compare different types of screws in the vertebral body, it is necessary to insert the screws in similar positions. Therefore, researchers should be well-acquainted with proper anesthesia techniques, standardized surgical protocols, and harvest procedures before performing any experiments. Here, we describe a detailed method for anesthesia, surgery, and harvest for the evaluation of pedicle screw fixation using a porcine spine model, including ex vivo imaging, histology, and strength testing.
The Institutional Animal Care and Use Committee of Chonbuk National University approved this study. The treatment, use, and handling of animals followed all guidelines and policies. Maintain the operating room at 24 °C.
1. Anesthesia
2. Spine Surgery
3. Harvest Procedure
A detailed protocol for anesthesia, surgery, and harvest for the evaluation of transpedicular screws using an in vivo porcine lumbar spine model is described here. This protocol is suitable for a number of downstream analyses, including mechanical testing (Figure 1), quantitative micro-CT evaluation (Figure 2), and histology (Figure 3). Representative mechanical testing (Figure 1) shows the mean extraction torsional peak torque. It represents the bonding strength between the pedicle screw and the bone using a mechanical testing gauge. Three types of pedicle screws were evaluated: uncoated, hydroxyapatite (HA)-coated, and titanium-coated. Data were collected from 14 pedicle screws from each of the three groups. The mean extraction torsional peak torque was greater in the titanium-coated pedicle screw group.
Representative micro-CT images (Figure 2A) demonstrate that the region of interest (the inner space of the full length of the screw) can be evaluated by a micro-CT program for the analysis of the bone volume fraction (Figure 2B1), bone surface density (Figure 2B2), and specific bone surface (Figure 2B3). Data were collected from 4 pedicle screws from each of the three groups.
A representative histology image (Figure 3) was stained with Goldner trichrome. The interface between the pedicle screw and bone was observable. The red color indicates fibrous tissue, while the blue color indicates bone. In uncoated pedicle screws, fibrous tissue was observed at the interface between the pedicle screw and bone. New bone formation was found at the interface between the threads of both HA- and titanium-coated pedicle screws and bone. In the titanium-coated pedicle screw group, the space between threads of the screw and the bone were compacted with bone (Figure 3).
Figure 1: Mechanical Analyses of Pedicle Screws in the Porcine Lumbar Spine. The mean extraction torsional peak torque for the bonding strength between the pedicle screw and the bone was measured with a mechanical testing gauge. Modified from reference6. The values are presented as the mean ±SEM (n = 14). Please click here to view a larger version of this figure.
Figure 2. Histomorphological Analyses of Pedicle Screws in the Porcine Lumbar Spine. (A) Manual regions of interest (ROI) were set with the inner space of the full length of the screw. (B) The bone volume fraction, bone surface density, and specific bone surface area were measured with micro-CT. Modified from reference6. The values are presented as the mean ±SEM (n = 4). Please click here to view a larger version of this figure.
Figure 3: Histological Analyses of Pedicle Screws in the Porcine Lumbar Spine. Goldner trichrome staining (x1, x20, and x40) was performed to observe the interface between the pedicle screw surface and the bone. Modified from reference6. Scale bar (black) = 1 mm. Scale bar (white) = 500 µm. Please click here to view a larger version of this figure.
The evaluation of transpedicular screws in the porcine spine requires much time and effort. First, the miniature pig is a large animal. For animal care and anesthesia, the researcher needs a specialized protocol. Second, surgery should maintain an environment similar to that of human surgery. The purpose of evaluating pedicle screws in the porcine spine is to develop an efficient screw that can be applied to humans. Third, evaluating the long-term stability of transpedicular screws requires about three months after the spine surgery. Accordingly, researchers in the field of pedicle screw fixation need to standardize the protocol by performing accurate planning. Although many studies on pedicle screws in spine models have been performed9,10,11, no study has provided a detailed protocol for the evaluation of transpedicular screws using an in vivo porcine lumbar spine model.
Although an ideal model for the human spine does not exist, the porcine spine model is an alternative due to the nature of this experiment and to the anatomical size and mechanical characteristics of the spine. In addition, it is relatively inexpensive. McLain et al.12 have compared the morphometry of the fourth lumbar vertebra from human, porcine, ovine, sheep, and dog specimens and concluded that porcine specimens demonstrate several advantages over other animal models as an alternate to the human spine. Furthermore, the quadruped spine of the porcine model is essentially loaded in the same way as that of the human spine8. Therefore, the porcine spine is used as an alternate model to the human spine for experiments involving spinal fixation and instrumentation techniques.
In this study, we described the detailed method of anesthesia, surgery, and harvest in the porcine L3-L5 lumbar spine for the evaluation of pedicle screw fixation. Many studies have evaluated pedicle screws after multiple-level spine surgeries7,13,14. In many cases of human degenerative disease, spine surgeries are performed through one or two spine fusions. This means that two or three spine levels of pedicle screws are used for fixation15. Micro-CT for the evaluation of bone formation around pedicle screws has a limited measurement range. In this case, we used two control screws for L3 pedicles, two HA-coated screws for L4 pedicles, and two titanium-coated screws for L5 pedicles. Because the anatomical dimensions of the L3-L5 lumbar spines are almost the same16, a comparison between L3-L5 screws is more reliable than a comparison between multiple-level screws. As a result, a two- or three-level fixation of pedicle screws in the lumbar spine is more appropriate compared to multiple-level fixations.
To precisely evaluate and compare the transpedicular screws, one critical point should be kept in mind: each screw should be located at a similar position on the vertebral body. However, the majority of protocols for implanting transpedicular screws in animal models call for exposing and pre-drilling the pedicles of the spine before inserting the pedicle screws5,13,14. On the other hand, Upasani et al. proposed a surgical protocol that includes determining the position and size of the pedicle screws before surgery by using computed tomography images of the spine2. This protocol suggests the insertion of a guide pin at the entry point of the pedicle screw in the lumbar spine. Defining the position of the pedicle screw using C-arm or portable X-rays is also suggested in this protocol. Furthermore, by using a pedicle sounding device, the complete intraosseous trajectory can be confirmed. This protocol can be used to determine the proper positioning of the pedicle screws so that the misplacement of the pedicle can be prevented. This protocol is based on the human spine surgery technique used in our hospital.
This method has some limitations. First, the surgery was performed with a healthy porcine model. Because the purpose of evaluating pedicle screws is to reduce complications in patients with osteoporosis, this protocol should be applied to osteoporotic porcine spine models to demonstrate the effectiveness of the pedicle screws. Second, the porcine spine model requires the purchase and housing of, as well as the surgical equipment for, pigs. This can increase the cost, thus restricting the number of animals that can be used in each study group. Third, this study only included 12-month-old miniature pigs because they were easy to acquire and handle. In addition, although there are several types of devices for spine fixation, only a protocol using a rigid fixation system was used here because it is the most commonly used in spine surgery.
In conclusion, a porcine model of pedicle screw fixation provides a key clinical platform to investigate efficient fixation techniques that cause fewer complications in patients with osteoporosis. This protocol provides technical details for anesthesia, surgery, and harvest in a porcine lumbar spine model. This will facilitate the evaluation of transpedicular screw fixation using this model.
The authors have nothing to disclose.
This study was supported by a grant (CNUH-BRI-2012-02-005) funded by the Biomedical Research Institute of Chonbuk National University Hospital (CNUH-BRI), Republic of Korea.
Miniature pig | OrientBio | ||
Atropine | Jeil pharmaceutical | A04900241 | Anesthesia |
Over-the needle plastic catheter | BD | REF382412 | Maintenance of IV line |
Ketamine | Yuhan | A04502441 | Anesthesia |
Xylazine | Bayer Korea | A00800071 | Anesthesia |
Laryngoscope | Karl storz | Intubation | |
Endotracheal tube | Covidien | Intubation | |
Isoflurane | JW pharmaceutical Co | A02104781 | Anesthesia |
Eye ointment | Hanlim pharma | A37851721 | Protection of pig's eye |
Cefazolin | Donga pharma | A01503951 | Antibiotics |
Saline | JW pharmaceutical Co | A02151392 | Maintenance of fluid homeostasis |
Fentanyl | Hana pharm | C03200032 | Pain control |
Enrofloxacin | Bayer | 93106-60-6 | Antibiotics |
Morphine | Myungmoon pharma | C03700091 | Pain control |
Meloxicam | Boehringer Ingelheim | A07600711 | Antibiotics |
Povidone-iodine | Hyundai pharma | Wound dressing | |
Scalpel blade size 15 | Braun | I1 BB515 | Skin incision |
Cobb elevator | Codman | 65-2546 | Dissection of muscle |
Burr | Medtronic | Making of starting point of screw | |
Rongeur | Aesculap | FO515R | Making of starting point of screw |
Guide pin (K-wire) | CE | 01067803 | Guidance of screw trajectory |
C-arm | GE | OEC 9800 plus | Guidance of screw trajectory |
Portable X-ray | Siemens | Mobile XP hybrid | Guidance of screw trajectory |
Pedicle probe | OtisBiotech | SPI-02-01 | Guidance of screw trajectory |
Pedicle sounding device | OtisBiotech | SPI-03-01 | Guidance of screw trajectory |
Pedicle screw | OtisBiotech | MS-40025 | |
Posterior fixator systems | OtisBiotech | ||
Rod | OtisBiotech | ROD-60140 | Rigid fixation between screws |
Universal handle | OtisBiotech | SPI-08-01 | To fix the screws to the rod |
Straight socket wrench | OtisBiotech | SPI-06-01 | To fix the screws to the rod |
counter torque wrench | OtisBiotech | SPI-07-01 | To fix the screws to the rod |
Bulb irrigation syringe | Hyupsug medical | HS-IR-140 | Irrigation |
Silicone drain | Sewon medical | 2205-006 | To drain the fluid at the surgical site |
3.0 metric absorbable suture | Ethicon | BA1673H | Muscle suture |
2.0 metric nonabsorbable nylon suture | Ethicon | W1626T | Skin suture |
Gauze | Kingphar Korea | KP120-06 | |
Pentobarbital | Hanlim pharma | 645301221 | Euthanasia |
Oscillating saw | Zimmer | Harvest spine | |
Tower forceps | Aesculap | BF461R | Harvest spine |